[go: up one dir, main page]

WO2025012118A2 - 5t4 binding agents and methods of use - Google Patents

5t4 binding agents and methods of use Download PDF

Info

Publication number
WO2025012118A2
WO2025012118A2 PCT/EP2024/069004 EP2024069004W WO2025012118A2 WO 2025012118 A2 WO2025012118 A2 WO 2025012118A2 EP 2024069004 W EP2024069004 W EP 2024069004W WO 2025012118 A2 WO2025012118 A2 WO 2025012118A2
Authority
WO
WIPO (PCT)
Prior art keywords
seq
antigen
amino acid
acid sequence
comprises seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2024/069004
Other languages
French (fr)
Other versions
WO2025012118A3 (en
Inventor
Paula Maria Wilhelmina Van Helden
Robertus Cornelis ROOVERS
David Lutje Hulsik
Johannes Jelle VAN DER VLIET
Anton Egbert Peter Adang
Milon DE JONG
Manfred Saller
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Lava Therapeutics NV
Original Assignee
Lava Therapeutics NV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Lava Therapeutics NV filed Critical Lava Therapeutics NV
Publication of WO2025012118A2 publication Critical patent/WO2025012118A2/en
Publication of WO2025012118A3 publication Critical patent/WO2025012118A3/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present disclosure further provides methods of treating cancer in a subject in need thereof comprising administration of the 5T4 binding agents and multi-specific binding agents comprising the same to a subject in need thereof.
  • 5T4 also known as Trophoblast glycoprotein (TPBG) and Wnt-Activated Inhibitory Factor 1 (WAIF1), is a single-pass transmembrane protein first identified in human placental tissues. 5T4 inhibits Wnt/ ⁇ -catenin signaling, a signaling system central to many developmental and pathological processes.5T4 is rarely expressed in normal adult tissues, but is present in placenta and in many common tumors.
  • 5T4 is expressed in more than 80% of kidney, breast, colon, prostate, and ovarian cancers.
  • MVA attenuated vaccinia virus modified vaccinia Ankara
  • TroVax tumor antigen 5T4
  • WO13041687 Amgen
  • WO16097408 Choome Bioscience
  • WO21048423 Genmab
  • Bispecific T-cell engaging antibodies have a tumor target binding specificity and a CD3-directed T-cell binding specificity and thus boost tumor immunity by re-directing T-cell cytotoxicity to malignant cells, see e.g., 338699-2168 Huehls et al. (2015) Immunol Cell Biol 93:290; Ellerman (2019) Methods, 154:102.
  • reported results with CD3-binding bispecific antibodies vary significantly.
  • bispecific antibodies targeting the different tumor targets showed strong variation in their capacity to induce target cell cytotoxicity and that the cytotoxicity did not correlate with antigen expression levels.
  • CD3-based bispecific antibodies targeting HLA-DR or CD138 were not able to induce cytotoxicity, in spite of intermediate to high HLA-DR and CD138 expression levels (Engelberts et al. (2020) Ebiomedicine 52:102625).
  • T-cell redirecting therapies have reached late-stage clinical development, possibly due to significant toxicity, manufacturing problems, immunogenicity, a narrow therapeutic window, and low response rates.
  • toxicity has been observed to occur when the T-cell engager includes a CD3 binding arm and treatment results in uncontrolled and exaggerated immune activation and cytokine release.
  • the first bispecific 5T4-CD3 antibodies were available already in 2012 (WO13041687), none of these antibodies have made significant progress through clinical development. Some have even been discontinued due to a narrow or absent therapeutic window. See for example ClinicalTrials.gov ID NCT04424641.
  • the present disclosure provides binding agents comprising a 5T4 antigen-binding domain.
  • the present disclosure further provides multi-specific binding agents comprising a 5T4 antigen-binding domain and a ⁇ -T cell antigen-binding domain. In some embodiments, such multi-specific binding agents target tumor cells to activated ⁇ -T cells.
  • multi-specific binding agents comprising a first antigen-binding domain capable of binding human 5T4 and a second antigen-binding domain capable of binding a human V ⁇ 9V ⁇ 2 T cell receptor.
  • binding agents comprising a first antigen- binding region capable of binding human 5T4.
  • a single domain antibody that binds 5T4 comprising: (a) a complementarity determining region (CDR)1 comprising an amino acid sequence selected from SEQ ID NOs: 88, 98, 105, 111, 115, 122, 123, 133, 137, 141, 145, 149, 153, 157, 161, 165, and 169; (b) a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: : 89, 99, 106, 112, 116, 124, 125, 134, 138, 142, 146, 150, 154, 158, 162, 338699-2168 166 and 170; and (c) a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 90, 100, 107, 113, 117, 126, 127, 135, 139, 143, 147, 151, 155, 159, 163, 137 and 171.
  • CDR complementarity determining region
  • the CDR1, CDR2 and CDR3 sequences are selected from the combination disclosed in TABLE 4A.
  • the single domain antibody comprises an amino acid sequence that is at least 90%, or at least 95%, identical to any one of SEQ ID NOs: 92, 93, 94, 95, 96, 97, 101, 102, 103, 104, 108, 109, 110, 114, 118, 119, 120, 121, 128, 129, 130, 131, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, and 172.
  • the single domain antibody comprises SEQ ID NOs: 92, 93, 94, 95, 96, 97, 101, 102, 103, 104, 108, 109, 110, 114, 118, 119, 120, 121, 128, 129, 130, 131, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, and 172.
  • the single domain antibody consists of SEQ ID NOs: 92, 93, 94, 95, 96, 97, 101, 102, 103, 104, 108, 109, 110, 114, 118, 119, 120, 121, 128, 129, 130, 131, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, and 172.
  • the CDR1 comprises SEQ ID NO: 88
  • the CDR2 comprises SEQ ID NO: 89
  • the CDR3 comprises SEQ ID NO: 90.
  • the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to any one of SEQ ID NOs: 92, 93, 94, 95 and 96.
  • the CDR1 comprises SEQ ID NO: 88
  • the CDR2 comprises SEQ ID NO: 89
  • the CDR3 comprises SEQ ID NO: 91.
  • the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 97.
  • the CDR1 comprises SEQ ID NO: 98
  • the CDR2 comprises SEQ ID NO: 99
  • the CDR3 comprises SEQ ID NO: 100.
  • the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to any one of SEQ ID NOs: 101, 102, 103, and 104.
  • the CDR1 comprises SEQ ID NO: 105
  • the CDR2 comprises SEQ ID NO: 106
  • the CDR3 comprises SEQ ID NO: 107.
  • the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to any one of SEQ ID NOs: 108, 109, and 110.
  • the CDR1 comprises SEQ ID NO: 111
  • the CDR2 comprises SEQ ID NO: 112
  • the CDR3 comprises SEQ ID NO: 113.
  • the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 114.
  • the CDR1 comprises SEQ ID NO: 115
  • the CDR2 comprises SEQ ID NO: 116
  • the CDR3 comprises SEQ ID NO: 117.
  • the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to any one of SEQ ID NOs: 118, 119, 120, and 121. 338699-2168
  • the CDR1 comprises SEQ ID NO: 122
  • the CDR2 comprises SEQ ID NO: 124
  • the CDR3 comprises SEQ ID NO: 126.
  • the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 128.
  • the CDR1 comprises SEQ ID NO: 123
  • the CDR2 comprises SEQ ID NO: 124
  • the CDR3 comprises SEQ ID NO: 126.
  • the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 129.
  • the CDR1 comprises SEQ ID NO: 122
  • the CDR2 comprises SEQ ID NO: 125
  • the CDR3 comprises SEQ ID NO: 126.
  • the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 130.
  • the CDR1 comprises SEQ ID NO: 122
  • the CDR2 comprises SEQ ID NO: 124
  • the CDR3 comprises SEQ ID NO: 127.
  • the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 131.
  • the CDR1 comprises SEQ ID NO: 123
  • the CDR2 comprises SEQ ID NO: 125
  • the CDR3 comprises SEQ ID NO: 127.
  • the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 132.
  • the CDR1 comprises SEQ ID NO: 133
  • the CDR2 comprises SEQ ID NO: 134
  • the CDR3 comprises SEQ ID NO: 135.
  • the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 136.
  • the CDR1 comprises SEQ ID NO: 137
  • the CDR2 comprises SEQ ID NO: 138
  • the CDR3 comprises SEQ ID NO: 139.
  • the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 140.
  • the CDR1 comprises SEQ ID NO: 141
  • the CDR2 comprises SEQ ID NO: 142
  • the CDR3 comprises SEQ ID NO: 143.
  • the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 144.
  • the CDR1 comprises SEQ ID NO: 145
  • the CDR2 comprises SEQ ID NO: 146
  • the CDR3 comprises SEQ ID NO: 147.
  • the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 148. 338699-2168
  • the CDR1 comprises SEQ ID NO: 149
  • the CDR2 comprises SEQ ID NO: 150
  • the CDR3 comprises SEQ ID NO: 151.
  • the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 152.
  • the CDR1 comprises SEQ ID NO: 153
  • the CDR2 comprises SEQ ID NO: 154
  • the CDR3 comprises SEQ ID NO: 155.
  • the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 156.
  • the CDR1 comprises SEQ ID NO: 157
  • the CDR2 comprises SEQ ID NO: 158
  • the CDR3 comprises SEQ ID NO: 159.
  • the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 160.
  • the CDR1 comprises SEQ ID NO: 161
  • the CDR2 comprises SEQ ID NO: 162
  • the CDR3 comprises SEQ ID NO: 163.
  • the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 164.
  • the CDR1 comprises SEQ ID NO: 165
  • the CDR2 comprises SEQ ID NO: 166
  • the CDR3 comprises SEQ ID NO: 167.
  • the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 168.
  • the CDR1 comprises SEQ ID NO: 169
  • the CDR2 comprises SEQ ID NO: 170
  • the CDR3 comprises SEQ ID NO: 171.
  • the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 172.
  • the CDR1 comprises SEQ ID NO: 88
  • the CDR2 comprises SEQ ID NO: 89
  • the CDR3 comprises SEQ ID NO: 90.
  • the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 93.
  • the single domain antibody comprises or consists of the amino acid sequence of SEQ ID NO: 93.
  • disclosed herein is a polynucleotide encoding the single domain of any of the above aspects or embodiments.
  • a vector comprising the polynucleotide of any of the above aspects or embodiments.
  • a host comprising the polynucleotide or vector of any of the above aspects or embodiments. 338699-2168
  • a method of producing the single domain antibody of any one of the above aspects or embodiments comprising: (a) introducing the vector of any of the above aspects or embodiments into a host cell under conditions permitting expression of the polynucleotide encoding the single domain antibody; and (b) isolating the single domain antibody from the culture supernatant.
  • a pharmaceutical composition comprising the single domain antibody of any one of the above aspects or embodiments and a pharmaceutically acceptable carrier.
  • a binding agent comprising the single domain antibody of any one of the above aspects or embodiments.
  • a multi-specific binding agent comprising: (a) a first antigen-binding domain that specifically binds to human 5T4 comprising the single domain antibody of any one of the above aspects or embodiments; and (b) a second antigen-binding domain that specifically binds to an antigen expressed on an immune cell.
  • the antigen expressed on the immune cell is selected from CD3, CD2, an ⁇ T cell receptor, a ⁇ T cell receptor (TCR).
  • the ⁇ T cell receptor is a V ⁇ 2 TCR.
  • the V ⁇ 2 TCR is a human V ⁇ 2 T cell receptor.
  • the human V ⁇ 2 T cell receptor is a V ⁇ 9V ⁇ 2 T cell receptor.
  • the first and second antigen-binding domains are present in the same polypeptide chain. In some embodiments, the first antigen-binding domain and second antigen-binding domain are covalently linked via a peptide linker.
  • the multi-specific binding agent comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 362 or 363. In some embodiments, the multi-specific binding agent comprises or consists of the amino acid sequence of SEQ ID NOs: 362 or 363. In some embodiments, the first and second antigen-binding domains are present on different polypeptide chains. In some embodiments, the multi-specific binding agent comprises an Fc domain comprising a first and a second Fc domain monomer.
  • the multi-specific binding agent comprises (a) a first polypeptide comprising the first antigen-binding domain that binds to a human 5T4 protein and the first Fc domain monomer; and (b) a second polypeptide comprising the second antigen-binding domain that binds to a human V ⁇ 2 T cell receptor and the second Fc domain monomer.
  • the human V ⁇ 2 T cell receptor is a V ⁇ 9V ⁇ 2 T cell receptor.
  • the antigen-binding domain that binds to the V ⁇ 9V ⁇ 2 T cell receptor comprises SEQ ID NO: 22.
  • the antigen-binding domain that binds to the V ⁇ 9V ⁇ 2 T cell receptor consists of SEQ ID NO: 22.
  • the multi-specific binding agent induces proliferation of V ⁇ 9V ⁇ 2 T cells.
  • the first and second Fc domain monomers comprise a mutation at 338699-2168 position 234 and/or 235 according to the EU numbering system.
  • the first and second Fc domain monomers comprise an L234F and an L235E substitution.
  • the first Fc domain monomer comprises a T366W substitution and the second Fc polypeptide comprises T366S, L368A and Y407V substitutions, or vice versa, according to the EU numbering system; and/or (b) the cysteine residue at position 220 according to the EU numbering system in the first or second Fc domain monomers have been deleted or substituted.
  • a multi-specific binding agent comprising: (a) a first antigen-binding domain that binds to human 5T4, where the first antigen-binding domain comprises a complementarity determining region (CDR)1 comprising SEQ ID NO: 88, a CDR2 comprising SEQ ID NO: 89, and a CDR3 comprising SEQ ID NO: 90; and (b) a second antigen- binding domain that binds to V ⁇ 2, where the second antigen-binding domain comprises a CDR1 comprising SEQ ID NO: 18, a CDR2 comprising SEQ ID NO: 19, and a CDR3 comprising SEQ ID NO: 20.
  • CDR complementarity determining region
  • the first antigen-binding domain comprises SEQ ID NO: 93 and the second antigen-binding domain comprises SEQ ID NO: 22. In some embodiments, the first and second antigen-binding domains are comprised in the same polypeptide chain.
  • a multi-specific binding agent comprising (a) a first polypeptide comprising (i) a first antigen-binding domain that binds to a human 5T4 protein and comprises a complementarity determining region (CDR)1 comprising SEQ ID NO: 88, a CDR2 comprising SEQ ID NO: 89, and a CDR3 comprising SEQ ID NO: 90; and (ii) a first Fc domain monomer; (b) a second polypeptide comprising (i) a second antigen-binding domain that binds to a human V ⁇ 2 T cell receptor and comprises a CDR1 comprising SEQ ID NO: 18, a CDR2 comprising SEQ ID NO: 19, and a CDR3 comprising SEQ ID NO: 20; and (ii) a second Fc domain monomer.
  • CDR complementarity determining region
  • the first Fc domain monomer comprises SEQ ID NO: 358 and the second Fc domain monomer comprises SEQ ID NO: 359. In some embodiments, the first Fc domain monomer comprises SEQ ID NO: 359 and the second Fc domain monomer comprises SEQ ID NO: 358. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 369. In some embodiments, the second antigen-binding domain comprises SEQ ID NO: 381. In some embodiments, (a) the first polypeptide comprises SEQ ID NO: 369; and (b) the second polypeptide comprises SEQ ID NO: 381.
  • a polynucleotide encoding the multi-specific binding agent of any one of the above aspects or embodiments.
  • an expression vector comprising the polynucleotide of any of the above aspects or embodiments. 338699-2168
  • a host cell comprising the polynucleotide or the expression vector of any one of the above aspects or embodiments. In some embodiments, the cell is a CHO cell.
  • a method of producing the multi-specific binding agent of any one of the above aspects or embodiments comprising culturing the host cell of any one of the above aspects or embodiments, under conditions sufficient to express the multi-specific binding agent from the polynucleotide or expression vector therein and purifying the expressed multi-specific binding agent from the culture supernatant.
  • a pharmaceutical composition comprising the single domain antibody, the binding agent, or the multi-specific binding agent according to any one the above aspects or embodiments, and a pharmaceutically-acceptable excipient.
  • a method of treatment a cancer in a subject in need thereof comprising administration to the subject the single domain antibody, the binding agent, the multi-specific binding agent, or the pharmaceutical composition of any one of the above aspects or embodiments.
  • the subject is human.
  • the cancer expresses 5T4.
  • the subject is suffering from a cervical, colorectal, head and neck, glioblastoma, melanoma, urothelial, gastric, renal cell carcinoma, lung cancer, pancreatic cancer, mesothelioma, pre-B acute lymphoblastic leukemia, or hepatocellular cancer.
  • FIG.1A-FIG.1B show the expression of 5T4 on tumor cells (FIG.1A) and percentage of V ⁇ 9 + V ⁇ 2 + T cells out of the total CD3 + T cells in different patient cancer samples (FIG.1B) .
  • FIG.2 shows the expression of 5T4 on non-malignant urothelial cells.
  • FIG.3A-FIG.3C show the binding profile of 5T4 binding agents to recombinant human 5T4 as determined by ELISA.
  • FIG.3A shows results for LV1138, LV1139, LV1140, LV1141, LV1142, LV1143, LV1144, and LV1145.
  • FIG.3B shows results for LV1145, LV1146, LV1147, LV1148, LV1149, LV1150, LV1151, and LV1152.
  • FIG.3C shows results for LV1292.
  • FIG.4A-FIG.4C show the binding profile of multi-specific binding agents comprising LV1138, LV1139, LV1140, or LV1142 with a V ⁇ 2-binding arm to CHO-K1 cells that were 338699-2168 transfected with human 5T4 (FIG. 4A), murine 5T4 (FIG. 4B), or cynomolgus monkey 5T4 (FIG.4C).
  • FIG.5A-FIG.5B show the binding profile of multi-specific binding agents comprising LV1138, LV1139, LV1140, or LV1142 with a V ⁇ 2-binding arm to U-251 (FIG.5A) or A-431 (FIG.5B) cancer cells as determined by flow cytometry.
  • FIG.6 shows the binding profile of multi-specific binding agents comprising LV1138, LV1139, LV1140, or LV1142 with a V ⁇ 2-binding arm to human V ⁇ 9V ⁇ 2 T cells as determined by flow cytometry.
  • FIG.7A-FIG.7H show the V ⁇ 9V ⁇ 2 T cell degranulation (FIGs.7A, 7C, 7E, and 7G) and cytotoxicity (FIGs.7B, 7D, 7F, and 7H) against U-251 cells expressing 5T4 induced by increasing concentrations of multi-specific binding agents comprising LV1138, LV1139, LV1140, or LV1142 with a V ⁇ 2-binding arm in the presence or absence of primary V ⁇ 9V ⁇ 2 T cells.
  • multi-specific binding agents comprising LV1138, LV1139, LV1140, or LV1142 with a V ⁇ 2-binding arm in the presence or absence of primary V ⁇ 9V ⁇ 2 T cells.
  • FIG.8A-FIG.8H show the V ⁇ 9V ⁇ 2 T cell degranulation (FIGs.7A, 7C, 7E, and 7G) and cytotoxicity (FIGs.7B, 7D, 7F, and 7H) against A-431 cells expressing 5T4 induced by increasing concentrations of multi-specific binding agents comprising LV1138, LV1139, LV1140, or LV1142 with a V ⁇ 2-binding arm in the presence or absence (control) of primary V ⁇ 9V ⁇ 2 T cells.
  • FIG. 9D show V ⁇ 9V ⁇ 2 T cell degranulation as measured by CD107a expression induced by 5T4-multi-specific binding agents in patient-derived cervical squamous cell carcinoma (FIG. 9A), cervical adenocarcinoma (FIG. 9B), metastatic colorectal cancer (FIG.9C), and glioblastoma (FIG.9D).
  • FIG.10A-FIG.10E show tumor cell lysis induced by 5T4-multi-specific binding agents in patient-derived squamous cell carcinoma at 18 hours (FIG.10A) and 7 days (FIG.10B), cervical adenocarcinoma at 18 hours (FIG.10C) and 7 days (FIG.10D), and in glioblastoma at 18 hours (FIG.10E).
  • FIG.11A-FIG.11B show V ⁇ 9V ⁇ 2 T cell degranulation (FIG.11A) and percent tumor lysis (FIG.11B) in head and neck, melanoma, glioblastoma, cervical, colorectal, renal, and urothelial cell cancers incubated with V ⁇ 9V ⁇ 2 T cells and with or without LAVA-378.
  • FIG.12A-FIG.12B show increased tumor cell lysis when LAVA-378 and V ⁇ 9V ⁇ 2 T cells are incubated with urothelial cancer cells (FIG.12A) and absence of an increase in cell lysis when LAVA-378 and V ⁇ 9V ⁇ 2 T cells are incubated with non-malignant urothelial cells (FIG.12B).
  • FIG.13 shows the increase in tumor lysis when LAVA-378 is incubated with urothelial cancer cells in the absence of additional expanded (allogeneic) V ⁇ 9V ⁇ 2-T cells. 338699-2168
  • FIG.14A-FIG.14B show 5T4 expression on patient colorectal cancer samples (FIG.
  • FIG. 15A-FIG. 15B show chromatographs illustrating retention of pyroglutamate- containing (LAVA-1395) (FIG. 15A) and non-pyroglutamate containing (LAVA-1433) (FIG. 15B) multi-specific binding agents in reverse-phase HPLC.
  • FIG.16 illustrates the different CDR numbering systems (Kabat, Chothia, IMGT, and Combined) for the 6H4 VHH.
  • TPBG trophoblast glycoprotein
  • V ⁇ 2 when used herein, refers to the rearranged ⁇ 2 chain of the human V ⁇ 9V ⁇ 2-T cell receptor (TCR).
  • UniProtKB - A0JD36 (A0JD36_HUMAN) gives an example of a variable TRDV2 sequence.
  • the sequence of human V ⁇ 2 is set forth in SEQ ID NO:383.
  • human V ⁇ 9 when used herein, refers to the rearranged ⁇ 9 chain of the V ⁇ 9V ⁇ 2-T cell receptor (TCR).
  • UniProtKB – Q99603_HUMAN gives an example of a variable TRGV9 sequence.
  • antibody and “binding agent” are used interchangeably and refer to a polypeptide or complex of polypeptides comprising at least one antigen-binding domain that specifically binds to a target antigen and includes intact immunoglobulin molecules, antigen- binding fragments thereof, and derivatives of either. Binding agents, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies and humanized antibodies, and antibody fragments provided by any known technique, such as enzymatic cleavage, peptide synthesis and recombinant techniques.
  • mAbs monoclonal antibodies
  • humanized antibodies and antibody fragments provided by any known technique, such as enzymatic cleavage, peptide synthesis and recombinant techniques.
  • a binding agent has the ability to specifically bind to an antigen under typical physiological conditions with a half-life of significant periods of time, such as at least about 30 minutes, at least about one hour, at least about two hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functionally-defined period (such as a time sufficient to induce, promote, enhance, and/or modulate a physiological response associated with antibody binding to the antigen and/or time sufficient for the antibody to recruit an effector activity).
  • a half-life of significant periods of time such as at least about 30 minutes, at least about one hour, at least about two hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functionally-defined period (such as a time sufficient to induce, promote, enhance, and/or modulate a physiological response associated with antibody binding to the antigen and/
  • antigen-binding domain or “antigen-binding region” refer to a portion of a binding agent that specifically binds a target antigen or target epitope. Antigen- binding domains may comprise variable regions of both the heavy and light chains of an antibody or antigen-binding fragments thereof, including single domain antibodies.
  • the terms “single domain antibody,” “Nanobody®,” “VHH,” “VHH antibody,” “single domain binding agent,” or “VHH domain” as used herein refer to an antigen-binding fragment comprising a single monomeric variable domain. Single-domain antibodies are well known to the skilled person, see e.g., Hamers-Casterman et al.
  • Single-domain antibodies may contain only the variable domain of an immunoglobulin chain, i.e., CDR1, CDR2, and CDR3 and framework regions.
  • Examples of single-domain antibodies are variable fragments of heavy-chain-only antibodies, antibodies that naturally do not comprise light chains, single-domain antibodies derived from conventional antibodies and engineered antibodies.
  • Single-domain antibodies may be derived from any species.
  • naturally occurring single-domain antibodies can be derived from antibodies raised in Camelidae species, for example in camel, dromedary, llama, alpaca, and guanaco. Like an intact immunoglobulin, a single-domain antibody is able to bind selectively to a single specific antigen.
  • the term “hinge region” as used herein is intended to refer to the hinge region of an immunoglobulin heavy chain.
  • the hinge region of a human IgG1 antibody corresponds to amino acids 216-230 according to the EU numbering. 338699-2168
  • CH2 region or “CH2 domain” as used herein is intended to refer to the CH2 region of an immunoglobulin heavy chain.
  • the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to the EU numbering.
  • the CH2 region may also be any of the other subtypes as described herein.
  • the term “CH3 region” or “CH3 domain” as used herein is intended to refer to the CH3 region of an immunoglobulin heavy chain.
  • the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the EU numbering.
  • the CH3 region may also be any of the other subtypes as described herein.
  • the terms “complementarity determining region” or “CDR,” as used herein, refer to the amino acid sequences within antibody variable regions which confer antigen specificity and binding affinity.
  • each heavy chain variable region e.g., HCDR1, HCDR2, and HCDR3
  • CDR1, LCDR2, and LCDR3 three CDRs in each heavy chain variable region
  • the precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.
  • the constant regions of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells and T cells) and components of the complement system such as C1q, the first component in the classical pathway of complement activation.
  • the term “antigen” refers to a molecule or a portion of a molecule capable of being bound by an antibody or an antigen-binding fragment thereof and additionally capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen.
  • an antigen-binding fragment refers to a polypeptide comprising at least one complementarity determining region (CDR) that binds to at least one epitope of an antigen of interest (e.g., 5T4).
  • an antigen-binding fragment may comprise 1, 2, 3, 4, 5, or all 6 CDRs of a variable heavy chain (VH) and/or variable light chain (VL) sequence.
  • Antigen-binding fragments include proteins that comprise a portion of a full length immunoglobulin, generally the antigen-binding or variable region thereof, such as Fab, F(ab’)2, Fab’, Fd, Fv fragments, minibodies, diabodies, single domain antibodies (dAb, also known as VHH, camelid antibodies, or nanobodies), single-chain variable fragments (scFv), rIgG, 338699-2168 antibody mimetics, and any other modified configuration of the immunoglobulin molecule that comprises an antigen-binding fragment of the required specificity.
  • antigen-binding domain or “antigen-binding region” refer to a portion of a binding agent that specifically binds a target antigen or target epitope. Antigen- binding domains may comprise variable regions of both the heavy and light chains of an antibody or antigen-binding fragments thereof, including single domain antibodies.
  • immunoglobulin refers to a class of structurally related glycoproteins typically consisting of two pairs of polypeptide chains, one pair of light (L) chains and one pair of heavy (H) chains, all four potentially inter-connected by disulfide bonds, although some mammalian species also produce heavy-chain only antibodies.
  • immunoglobulin heavy chain refers to one of the chains of an immunoglobulin.
  • a heavy chain typically comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH) which defines the isotype of the immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, or IgE).
  • the heavy chain constant region typically comprises three domains, CH1, CH2, and CH3.
  • the heavy chain constant region further comprises a hinge region.
  • each light chain is typically comprised of several regions: a light chain variable region (VL) and a light chain constant region (CL).
  • VL light chain variable region
  • CL light chain constant region
  • the VH and VL regions may be subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the term “isotype” as used herein refers to the immunoglobulin (sub)class (for instance IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) or any allotype thereof, such as IgG1m(za) and IgG1m(f) that is encoded by heavy chain constant region genes.
  • Each heavy chain isotype can be combined with either a kappa ( ⁇ ) or lambda ( ⁇ ) light chain.
  • An immunoglobulin disclosed herein can possess any isotype.
  • the terms “parent antibody” or “parent binding agent,” is to be understood as a binding agent which is identical to a binding agent disclosed herein, but wherein the parent binding agent does not have one or more of the specified mutations.
  • a “variant binding agent,” “variant antibody,” “binding agent variant,” “antibody variant,” or a “variant of a parent antibody” is a binding agent which comprises one or more mutations 338699-2168 (substitutions, deletions, or insertions) as compared to a binding agent or parent binding agent described here.
  • Amino acid substitutions may exchange a native amino acid for another naturally-occurring amino acid, or for a non-naturally-occurring amino acid derivative.
  • the amino acid substitution may be conservative or non-conservative.
  • one or more amino acid substitutions are introduced into a 5T4 binding agent described herein.
  • the one or more amino acid substitutions is a conservative amino acid substitution.
  • the phrases "conservative amino acid substitution” or "conservative mutation" refer to the replacement of one amino acid by another amino acid with a common property.
  • a functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and Schirmer, R.
  • groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz, G. E. and Schirmer, R. H., supra).
  • conservative mutations include amino acid substitutions of amino acids within the sub-groups above, for example, lysine for arginine and vice versa such that a positive charge may be maintained; glutamic acid for aspartic acid and vice versa such that a negative charge may be maintained; serine for threonine such that a free -OH can be maintained; and glutamine for asparagine such that a free -NH2 can be maintained.
  • amino acid classes are provided in Table 1, Table 2, and Table 3 below.
  • Table 1 Amino Acid Residue Classes for Conservative Substitutions Acidic Residues Asp (D) and Glu (E) Basic Residues Lys (K), Arg (R), and His (H) Hydrophilic Uncharged Residues Ser (S), Thr (T), Asn (N), and Gln (Q) Aliphatic Uncharged Residues Gly (G), Ala (A), Val (V), Leu (L), and Ile (I) Non-polar Uncharged Residues Cys (C), Met (M), and Pro (P) Aromatic Residues Phe (F), Tyr (Y), and Trp (W) 338699-2168 Table 2: Alternative Conservative Amino Acid Residue Substitution Classes 1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y W Table 3: Alternative Physical and Functional Classifications of Amino Acid Residues Alcohol group-containing residues S and T Aliphatic residues I, L, V, and
  • Non-conservative substitutions involve amino acid substitutions between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc. In this case, it is preferable for the non-conservative amino acid substitution to not interfere with or inhibit the biological activity of the binding agent (i.e., does not interfere with binding to the target antigen).
  • the non-conservative amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent binding agent.
  • a substitution in a variant is indicated as: Original amino acid – position – substituted amino acid;
  • the three-letter code, or one letter code, are used, including the codes Xaa and X to indicate amino acid residue.
  • the notation “T366W” means that the variant comprises a substitution of threonine with tryptophan in the variant amino acid position corresponding to the amino acid in position 366 in the parent antibody.
  • the term “a substitution” includes a substitution into any one of the other nineteen natural amino acids, or into other amino acids, such as non-natural amino acids.
  • a substitution of amino acid T in position 366 includes each of the following substitutions: 366A, 366C, 366D, 366G, 366H, 366F, 366I, 366K, 366L, 366M, 366N, 366P, 366Q, 366R, 366S, 366E, 366V, 366W, and 366Y. 338699-2168 [0094]
  • chimeric antibody or “chimeric binding agent” refer to a binding agent wherein the variable region is derived from a non-human species (e.g., derived from rodents) and the constant region is derived from a different species, such as human. Chimeric antibodies may be generated by genetic engineering. Chimeric monoclonal antibodies for therapeutic applications are developed to reduce antibody immunogenicity.
  • the binding agents described herein are humanized immunoglobulins or antigen-binding fragments thereof.
  • a “humanized” immunoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a non- human (such as a mouse, rat, or synthetic) immunoglobulin.
  • the non-human immunoglobulin providing the CDRs is termed a “donor,” and the human immunoglobulin providing the framework is termed an “acceptor.”
  • Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical.
  • all parts of a humanized immunoglobulin, except possibly the CDRs are substantially identical to corresponding parts of natural human immunoglobulin sequences.
  • Humanized immunoglobulins can be constructed by means of genetic engineering (for example, see U.S. Patent No.5,585,089).
  • humanized antibody refers to a genetically engineered non-human binding agent, which contains a humanized light chain and/or a humanized heavy chain.
  • a humanized antibody binds to the same antigen as the donor antibody that provides the CDRs.
  • the acceptor framework of a humanized antibody may have a limited number of substitutions by amino acids taken from the donor framework.
  • Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen-binding or other immunoglobulin functions.
  • multi-specific refers to the capability of a binding agent described herein to specifically bind two or more different target antigens or to specifically bind two or more different epitopes on a target antigen.
  • multi-specific antibody and multi-specific binding agent are used interchangeably and refer to a binding agent comprising two or more antigen-binding domains (e.g., two, three, four, or more), each binding to a different target antigen or to a different epitope on the same target antigen.
  • bispecific antibody and “bispecific binding agent” are used interchangeably and refer to a binding agent comprising not more than two antigen-binding domains, each binding to a different target antigen or to a different epitope on the same target antigen.
  • bispecific and multi-specific binding agents include but are not limited to (i) IgG-like molecules with complementary CH3 domains to force heterodimerization; (ii) recombinant IgG-like dual targeting molecules, wherein the two sides of the molecule each contain the Fab fragment or part of the Fab fragment of at least two different binding agents; (iii) IgG fusion molecules, wherein full length IgG antibodies are fused to extra Fab fragment or parts of Fab fragment; (iv) Fc fusion molecules, wherein single chain Fv molecules or stabilized diabodies are fused to heavy-chain constant- domains, Fc-regions or parts thereof; (v) Fab fusion molecules, wherein different Fab fragments are fused together, fused to heavy-chain constant-domains, Fc-regions or parts thereof; and (vi) scFv-and diabody-based and heavy chain-based antibodies (e.g., domain antibodies, Nanobodies®)
  • IgG-like molecules with complementary CH3 domains molecules include but are not limited to the Triomab® (Trion Pharma/Fresenius Biotech), the Knobs-into-Holes (Genentech), CrossMAbs (Roche) and the electrostatically-matched (Amgen, Chugai, Oncomed), the LUZ-Y (Genentech, Wranik et al. J. Biol. Chem.2012, 287(52): 43331-9, doi: 10.1074/jbc.M112.397869.
  • IgG-like dual targeting molecules include, but are not limited to, Dual Targeting (DT)-Ig (GSK/Domantis, WO2009058383), Two-in-one Antibody (Genentech, Bostrom, et al 2009. Science 323, 1610–1614), Cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star), ZybodiesTM (Zyngenia, LaFleur et al. MAbs.
  • DT Dual Targeting
  • GSK/Domantis WO2009058383
  • Two-in-one Antibody Genentech, Bostrom, et al 2009. Science 323, 1610–1614
  • Cross-linked Mabs Karmanos Cancer Center
  • mAb2 F-Star
  • ZybodiesTM Zyngenia, LaFleur et al. MAbs.
  • IgG fusion molecules include but are not limited to Dual Variable Domain (DVD)-Ig (Abbott), Dual domain double head antibodies (Unilever; Sanofi Aventis), IgG-like Bispecific (ImClone/Eli Lilly, Lewis et al. Nat Biotechnol.
  • Ts2Ab MedImmune/AZ, Dimasi et al. J Mol Biol. 2009 Oct 30;393(3):672-92
  • BsAb 338699-2168 Zymogenetics, WO2010111625
  • HERCULES Biogen Idec
  • scFv fusion Novartis
  • scFv fusion Changzhou Adam Biotech Inc
  • TvAb Roche
  • Fc fusion molecules include but are not limited to scFv/Fc Fusions (Academic Institution, Pearce et al Biochem Mol Biol Int.1997 Sep;42(6):1179), SCORPION (Emergent BioSolutions/Trubion, Blankenship JW, et al. AACR 100th Annual meeting 2009 (Abstract #5465); Zymogenetics/BMS, WO2010111625), Dual Affinity Retargeting Technology (Fc-DARTTM) (MacroGenics) and Dual(ScFv)2-Fab (National Research Center for Antibody Medicine – China).
  • Fab fusion bispecific antibodies include but are not limited to F(ab)2 (Medarex/AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock® (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol) and Fab-Fv (UCB-Celltech).
  • scFv-, diabody-based and domain antibodies include but are not limited to Bispecific T Cell Engager (BiTE®) (Micromet, Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DARTTM) (MacroGenics), Single-chain Diabody (Academic, Lawrence FEBS Lett.1998 Apr 3;425(3):479-84), TCR-like Antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack, WO2010059315) and COMBODY molecules (Epigen Biotech, Zhu et al. Immunol Cell Biol.
  • BiTE® Bispecific T Cell Engager
  • Tandab Tandem Diabody
  • DARTTM Dual Affinity Retargeting Technology
  • Single-chain Diabody Academic, Lawrence FEBS Lett.1998 Apr 3;425(3):479-84
  • TCR-like Antibodies AIT, Receptor
  • K D dissociation constant
  • K D dissociation constant
  • K D varies for different complexes of antibody and antigen and depends on both k on and k off .
  • the dissociation constant K D for a binding agent provided herein can be determined using any method provided herein or any other method well known to those skilled in the art. [0109] In the context of antibody binding to an antigen, the terms “binds” or “specifically binds” refer to the binding of a binding agent to a predetermined antigen or target (e.g.
  • K D values can be determined using surface plasmon resonance (SPR) technology in a BIAcore T200 or bio-layer interferometry (BLI) in an Octet RED96 instrument using the antigen as the ligand and the binding domain or binding agent as the analyte.
  • SPR surface plasmon resonance
  • BBI bio-layer interferometry
  • Specific binding 338699-2168 means that the binding agent binds to the predetermined antigen with an affinity corresponding to a K D that is at least ten-fold lower, such as at least 100-fold lower, for instance at least 1,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen.
  • a non-specific antigen e.g., BSA, casein
  • the degree with which the affinity is lower is dependent on the K D of the binding moiety or binding molecule, so that when the K D of the binding moiety or binding molecule is very low (that is, the binding moiety or binding molecule is highly specific), then the degree with which the affinity for the antigen is lower than the affinity for a non-specific antigen may be at least 10,000-fold.
  • Polynucleotide is used interchangeably with “oligonucleotide,” and “nucleic acid” herein, and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide.
  • the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions. However, it may be suitable in some instances, as discussed herein, for the nucleic acid to comprise one or more insertions, deletions, inversions, and/or substitutions.
  • the term “host cell” refers to any type of cell that comprises a vector or polynucleotide described herein and is typically used in the manufacturing of proteins (e.g., in the manufacturing of a binding agent).
  • the term “cancer” refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells.
  • subject refers to a vertebrate or an invertebrate, and includes mammals, birds, fish, reptiles, and amphibians.
  • Subjects include humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species.
  • Subjects include farm animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats, and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like; and aquatic animals such as fish, shrimp, and crustaceans.
  • first and second antigen-binding domains when used herein do not refer to their orientation/position in the antibody, i.e., they have no meaning with regard to the N- or C-terminus.
  • first and second only serve to correctly and consistently refer to the two different antigen-binding domains in the claims and the description.
  • 5T4 Binding agents [0116] 5T4 is an antagonist of the Wnt/ ⁇ -catenin pathway. Human 5T4 is a 420 amino acid protein. The amino acid sequence of wild type human 5T4 is provided as SEQ ID NO: 382.
  • Representative 5T4 polynucleotide sequences include NM_001166392.2 among others, each of which are available at the 5T4 gene page on the NCBI website.
  • the present disclosure provides binding agents comprising at least one antigen-binding domain that specifically binds to human 5T4 – referred to herein as 5T4 binding agents.
  • the 5T4 binding agent is an immunoglobulin or an antigen-binding fragment thereof.
  • the 5T4 binding agent is a single domain antibody.
  • the 5T4 binding agent comprises a first antigen-binding domain comprising a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 88, 98, 105, 111, 115, 122, 123, 133, 137, 141, 145, 149, 153, 157, 161, 165, and 169; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 89, 99, 106, 112, 116, 124, 125, 134, 138, 142, 146, 150, 154, 158, 162, 166 and 170; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs 90, 100, 107, 113, 117, 126, 127, 135, 139, 143, 147, 151, 155, 159, 163, 137 and 171.
  • the 5T4 binding agent comprises first antigen-binding domain comprising a CDR1, CDR2, and CDR3 combination selected from Table 4A, 4B, 4C, or 4D.
  • 5T4 binding agent comprises first antigen-binding domain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 92, 93, 94, 95, 96, 97, 101, 102, 103, 104, 108, 109, 110, 114, 118, 119, 120, 121, 128, 129, 130, 131, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, and 172.
  • the 5T4 binding agent comprises first antigen-binding domain comprising an amino acid sequence selected from SEQ ID NOs: 92, 93, 94, 95, 96, 97, 101, 102, 103, 104, 108, 109, 110, 114, 118, 119, 120, 121, 128, 129, 130, 131, 132, 136, 140, 144, 148, 338699-2168 152, 156, 160, 164, 168, and 172.
  • the 5T4 binding agent comprises a first antigen-binding domain consisting of an amino acid sequence selected from SEQ ID NOs: 92, 93, 94, 95, 96, 97, 101, 102, 103, 104, 108, 109, 110, 114, 118, 119, 120, 121, 128, 129, 130, 131, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, and 172.
  • Exemplary CDRs of 5T4 binding agents are shown in Table 4A – Table 4D.
  • Table 4A Exemplary 5T4-specific CDR (Kabat) and VHH Sequences Reference Component Sequence SEQ ID L V1139 CDR1 NYRMK 88 CDR2 TISHRVGRTYYADSVKG 89 CDR3 VAMGGSDYAPHDYDY 90 CDR3_var VDMGGSDYAPHDYDY 91 VHH EVQLVESGGGLVQPGGSLRLSCAANGFAFSNYRMKWVRQ 92 APGKGLEWVSTISHRVGRTYYADSVKGRFTISRDNAKNT VYLQMNSLKPEDTARYFCTRVAMGGSDYAPHDYDYTGQG TQVTVSS L V1139 (D) VHH DVQLVESGGGLVQPGGSLRLSCAANGFAFSNYRMKWVRQ 93 APGKGLEWVSTISHRVGRTYYADSVKGRFTISRDNAKNT VYLQMNSLKPEDTARYFCTRVAMGGSDYAPHDYDYTGQG TQVTVSS L V1139 (D) VHH
  • CDR sequences of a given binding agent as determined by multiple numbering systems are compiled into a single sequence that encompasses the entirety of each of the CDR amino acid ranges in the variable region.
  • CDRs for the 6H4 VHH described in Tables 5A, 5B, and 5C are shown in Fig.16.
  • the N- and C- terminal ranges for each CDR region are indicated by dashed lines.
  • the combined CDR sequences covering the entirety of these ranges are shown in the last row.
  • Illustrative combined CDRs for 5T4 binding agents are shown in Table 4D.
  • a 6H4 VHH comprising a CDR1 amino acid sequence comprising SEQ ID NO: 27, a CDR2 amino acid sequence comprising SEQ ID NO: 28, and a CDR3 amino acid sequence comprising SEQ ID NO: 29 as defined by the Kabat numbering 338699-2168 system will be understood by one of skill in the art to be the equivalent of (1) a 6H4 VHH comprising a CDR1 amino acid sequence comprising SEQ ID NO: 46, a CDR2 amino acid sequence comprising SEQ ID NO: 47, and a CDR3 amino acid sequence comprising SEQ ID NO: 48 as defined by the IMGT numbering system; (2) a 6H4 VHH comprising a CDR1 amino acid sequence comprising SEQ ID NO: 64, a CDR2 amino acid sequence comprising SEQ ID NO: 65, and a CDR3 amino acid sequence comprising SEQ ID NO: 66
  • the first antigen-binding domain comprise one or more framework regions (FR).
  • the binding agent disclosed herein are of the format FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Exemplary framework regions are shown in Table 5 below.
  • the first antigen-binding domain comprises an FR1 amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 317, 321, 325, 329, and 333.
  • the first antigen-binding domain comprises an FR1 amino acid sequence that comprises or consists of SEQ ID NOs: 317, 321, 325, 329, and 333. In some embodiments, the first antigen-binding domain comprises an FR2 amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 318, 322, 326, 330, and 334. In some embodiments, the first antigen-binding domain comprises an FR2 amino acid sequence that comprises or consists of SEQ ID NOs: 318, 322, 326, 330, and 334.
  • the first antigen-binding domain comprises an FR3 amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 319, 323, 327, 331, and 335. In some embodiments, the first antigen-binding domain comprises an FR3 amino acid sequence that comprises or consists of SEQ ID NOs: 319, 323, 327, 331, and 335.
  • the first antigen-binding domain comprises an FR4 amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 320, 324, 328, 332, and 336.
  • the first antigen-binding domain comprises an FR4 amino acid sequence that comprises or consists of SEQ ID NOs: 320, 324, 328, 332, and 336.
  • the first antigen-binding domain comprises or consists of, from N’ to C’ terminus, SEQ ID NOs: 317, 88, 318, 89, 319, 90, and 320.
  • the first antigen-binding domain comprises or consists of, from N’ to C’ terminus, SEQ ID NOs: 321, 98, 322, 99, 323, 100, and 324.
  • the first 338699-2168 antigen-binding domain comprises or consists of, from N’ to C’ terminus, SEQ ID NOs: 325, 105, 326, 106, 327, 107, and 328.
  • the first antigen-binding domain comprises or consists of, from N’ to C’ terminus, SEQ ID NOs: 329, 111, 330, 112, 331, 113, and 332.
  • the first antigen-binding domain comprises or consists of, from N’ to C’ terminus, SEQ ID NOs: 333, 115, 334, 116, 335, 117, and 336.
  • Table 5 Exemplary Framework Regions Ref Domain Sequence SEQ ID L V1139 FR1 EVQLVESGGGLVQPGGSLRLSCAANGFAFS 317 FR2 WVRQAPGKGLEWVS 318 FR3 RFTISRDNAKNTVYLQMNSLKPEDTARYFCTR 319 FR4 TGQGTQVTVSS 320 LV1138 FR1 EVQLVESGGGLVQAGGSLRLSCAASGDTLD 321 FR2 WFRQAPGKEREGVS 322 FR3 RFTISRDSAKNTVYLQMVSLKPEDTAVYYCAA 323 FR4 WGQGTQVTVSS 324 LV1140 FR1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS 325 FR
  • one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion).
  • CDR1 comprises the amino acid sequence of SEQ ID NO: 88, wherein one or two amino acids are modified.
  • CDR2 comprises the amino acid sequence of SEQ ID NO: 89, wherein one or two amino acids are modified.
  • CDR3 comprises the amino acid sequence of SEQ ID NO: 90, wherein one or two amino acids are modified.
  • the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 88, a CDR2 amino acid 338699-2168 sequence consisting of SEQ ID NO: 89, and a CDR3 amino acid sequence consisting of SEQ ID NO: 90.
  • the CDR3 sequence differs from the CDR3 sequence set forth in SEQ ID NO: 90 in one position, preferably position 2, wherein the substitution preferably is to D.
  • the CDR3 sequence comprises or consists of SEQ ID NO: 91.
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 92. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 92. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 92. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 93.
  • the first antigen-binding domain comprises SEQ ID NO: 93. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 93. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 94. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 94. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 94.
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 95. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 95. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 95. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 96. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 96.
  • the first antigen-binding domain consists of SEQ ID NO: 96. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 97. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 97. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 97.
  • the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 98, a CDR2 amino acid sequence comprising SEQ ID NO: 99, and a CDR3 amino acid sequence comprising SEQ ID NO: 100.
  • one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion).
  • CDR1 comprises the amino acid sequence of SEQ ID NO: 98, wherein one or two amino acids are modified.
  • CDR2 comprises the amino acid 338699-2168 sequence of SEQ ID NO: 99, wherein one or two amino acids are modified.
  • CDR3 comprises the amino acid sequence of SEQ ID NO: 100, wherein one or two amino acids are modified.
  • the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 98, a CDR2 amino acid sequence consisting of SEQ ID NO: 99, and a CDR3 amino acid sequence consisting of SEQ ID NO: 100.
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 101.
  • the first antigen-binding domain comprises SEQ ID NO: 101.
  • the first antigen-binding domain consists of SEQ ID NO: 101. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 102. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 102. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 102. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 103.
  • the first antigen-binding domain comprises SEQ ID NO: 103. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 103. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 104. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 104. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 104.
  • the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 105, a CDR2 amino acid sequence comprising SEQ ID NO: 106, and a CDR3 amino acid sequence comprising SEQ ID NO: 107.
  • one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion).
  • CDR1 comprises the amino acid sequence of SEQ ID NO: 105, wherein one or two amino acids are modified.
  • CDR2 comprises the amino acid sequence of SEQ ID NO: 106, wherein one or two amino acids are modified.
  • CDR3 comprises the amino acid sequence of SEQ ID NO: 107, wherein one or two amino acids are modified.
  • the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 105, a CDR2 amino acid sequence consisting of SEQ ID NO: 106, and a CDR3 amino acid sequence consisting of SEQ ID NO: 107. 338699-2168 [0129]
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 108.
  • the first antigen-binding domain comprises SEQ ID NO: 108. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 108. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 109. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 109. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 109.
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 110.
  • the first antigen-binding domain comprises SEQ ID NO: 110.
  • the first antigen-binding domain consists of SEQ ID NO: 110.
  • the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 111, a CDR2 amino acid sequence comprising SEQ ID NO: 112, and a CDR3 amino acid sequence comprising SEQ ID NO: 113.
  • one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion).
  • CDR1 comprises the amino acid sequence of SEQ ID NO: 111, wherein one or two amino acids are modified.
  • CDR2 comprises the amino acid sequence of SEQ ID NO: 112, wherein one or two amino acids are modified.
  • CDR3 comprises the amino acid sequence of SEQ ID NO: 113, wherein one or two amino acids are modified.
  • the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 111, a CDR2 amino acid sequence consisting of SEQ ID NO: 112, and a CDR3 amino acid sequence consisting of SEQ ID NO: 113.
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 114.
  • the first antigen-binding domain comprises SEQ ID NO: 114.
  • the first antigen-binding domain consists of SEQ ID NO: 114.
  • the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 115, a CDR2 amino acid sequence comprising SEQ ID NO: 116, and a CDR3 amino acid sequence comprising SEQ ID NO: 117.
  • one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion).
  • CDR1 comprises the amino acid sequence of SEQ ID NO: 115, wherein one or two amino acids are modified.
  • CDR2 comprises the amino acid sequence of SEQ ID NO: 116, wherein one or two amino acids are modified.
  • CDR3 comprises the amino acid sequence of SEQ ID NO: 117, wherein one or two amino acids are modified.
  • the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 115, a CDR2 amino acid sequence consisting of SEQ ID NO: 116, and a CDR3 amino acid sequence consisting of SEQ ID NO: 117.
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 118.
  • the first antigen-binding domain comprises SEQ ID NO: 118.
  • the first antigen-binding domain consists of SEQ ID NO: 118. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 119 In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 119. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 119.
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 120 In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 120. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 120. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 121 In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 121.
  • the first antigen-binding domain consists of SEQ ID NO: 121.
  • the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 122, a CDR2 amino acid sequence comprising SEQ ID NO: 124, and a CDR3 amino acid sequence comprising SEQ ID NO: 125.
  • one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion).
  • CDR1 comprises the amino acid sequence of SEQ ID NO: 122, wherein one or two amino acids are modified.
  • CDR2 comprises the amino acid sequence of SEQ ID NO: 124, wherein one or two amino acids are modified.
  • CDR3 comprises the amino acid sequence of SEQ ID NO: 126, wherein one or two amino acids are modified.
  • the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 122, a CDR2 amino acid 338699-2168 sequence consisting of SEQ ID NO: 124, and a CDR3 amino acid sequence consisting of SEQ ID NO: 126.
  • the CDR1 sequence differs from the CDR1 sequence set forth in SEQ ID NO: 122 in one position, preferably position 1, wherein the substitution preferably is to S.
  • the CDR1 sequence comprises or consists of SEQ ID NO: 123.
  • the CDR2 sequence differs from the CDR2 sequence set forth in SEQ ID NO: 124 in one, two, three, four, five or six positions, preferably positions 3, 4, 5, 7, 11 and/or 12 wherein the substitutions preferably are to T, S, G, D, T and A, respectively.
  • the CDR2 sequence comprises or consists of SEQ ID NO: 125.
  • the CDR3 sequence differs from the CDR3 sequence set forth in SEQ ID NO: 126 in one position, preferably position 3, wherein the substitution preferably is to D.
  • the CDR3 sequence comprises or consists of SEQ ID NO: 127.
  • the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 123, a CDR2 amino acid sequence comprising SEQ ID NO: 125, and a CDR3 amino acid sequence comprising SEQ ID NO: 127.
  • one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion).
  • CDR1 comprises the amino acid sequence of SEQ ID NO: 123, wherein one or two amino acids are modified.
  • CDR2 comprises the amino acid sequence of SEQ ID NO: 125, wherein one or two amino acids are modified.
  • CDR3 comprises the amino acid sequence of SEQ ID NO: 127, wherein one or two amino acids are modified.
  • the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 123, a CDR2 amino acid sequence consisting of SEQ ID NO: 125, and a CDR3 amino acid sequence consisting of SEQ ID NO: 127.
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 128.
  • the first antigen-binding domain comprises SEQ ID NO: 128.
  • the first antigen-binding domain consists of SEQ ID NO: 128. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 129. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 129. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 129. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 130.
  • the first antigen-binding domain comprises SEQ ID NO: 130. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 338699-2168 130. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 131. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 131. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 131.
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 132. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 132. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 132. [0137] In some embodiments, the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 133, a CDR2 amino acid sequence comprising SEQ ID NO: 134, and a CDR3 amino acid sequence comprising SEQ ID NO: 135.
  • one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion).
  • CDR1 comprises the amino acid sequence of SEQ ID NO: 133, wherein one or two amino acids are modified.
  • CDR2 comprises the amino acid sequence of SEQ ID NO: 134, wherein one or two amino acids are modified.
  • CDR3 comprises the amino acid sequence of SEQ ID NO: 135, wherein one or two amino acids are modified.
  • the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 133, a CDR2 amino acid sequence consisting of SEQ ID NO: 134, and a CDR3 amino acid sequence consisting of SEQ ID NO: 135. [0138] In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 136. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 136. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 136.
  • the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 137, a CDR2 amino acid sequence comprising SEQ ID NO: 138, and a CDR3 amino acid sequence comprising SEQ ID NO: 139.
  • one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion).
  • CDR1 comprises the amino acid sequence of SEQ ID NO: 137, wherein one or two amino acids are modified.
  • CDR2 comprises the amino acid sequence of SEQ ID NO: 338699-2168 138, wherein one or two amino acids are modified.
  • CDR3 comprises the amino acid sequence of SEQ ID NO: 139, wherein one or two amino acids are modified.
  • the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 137, a CDR2 amino acid sequence consisting of SEQ ID NO: 138, and a CDR3 amino acid sequence consisting of SEQ ID NO: 139.
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 140.
  • the first antigen-binding domain comprises SEQ ID NO: 140.
  • the first antigen-binding domain consists of SEQ ID NO: 140.
  • the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 141, a CDR2 amino acid sequence comprising SEQ ID NO: 142, and a CDR3 amino acid sequence comprising SEQ ID NO: 143.
  • one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion).
  • CDR1 comprises the amino acid sequence of SEQ ID NO: 141, wherein one or two amino acids are modified.
  • CDR2 comprises the amino acid sequence of SEQ ID NO: 142, wherein one or two amino acids are modified.
  • CDR3 comprises the amino acid sequence of SEQ ID NO: 143, wherein one or two amino acids are modified.
  • the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 141, a CDR2 amino acid sequence consisting of SEQ ID NO: 142, and a CDR3 amino acid sequence consisting of SEQ ID NO: 143.
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 144. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 144. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 144. [0143] In some embodiments, the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 145, a CDR2 amino acid sequence comprising SEQ ID NO: 146, and a CDR3 amino acid sequence comprising SEQ ID NO: 147.
  • one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion).
  • CDR1 comprises the amino acid sequence of SEQ ID NO: 145, wherein one or two amino acids are modified.
  • CDR2 comprises the amino acid sequence of SEQ ID NO: 146, wherein one or two amino acids are modified.
  • CDR3 comprises the amino acid sequence of SEQ ID NO: 147, wherein one or two amino acids are modified.
  • the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 145, a CDR2 amino acid sequence consisting of SEQ ID NO: 146, and a CDR3 amino acid sequence consisting of SEQ ID NO: 147. [0144] In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 148. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 148. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 148.
  • the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 149, a CDR2 amino acid sequence comprising SEQ ID NO: 150, and a CDR3 amino acid sequence comprising SEQ ID NO: 151.
  • one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion).
  • CDR1 comprises the amino acid sequence of SEQ ID NO: 149, wherein one or two amino acids are modified.
  • CDR2 comprises the amino acid sequence of SEQ ID NO: 150, wherein one or two amino acids are modified.
  • CDR3 comprises the amino acid sequence of SEQ ID NO: 151, wherein one or two amino acids are modified.
  • the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 149, a CDR2 amino acid sequence consisting of SEQ ID NO: 150, and a CDR3 amino acid sequence consisting of SEQ ID NO: 151.
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 152.
  • the first antigen-binding domain comprises SEQ ID NO: 152.
  • the first antigen-binding domain consists of SEQ ID NO: 152.
  • the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 153, a CDR2 amino acid sequence comprising SEQ ID NO: 154, and a CDR3 amino acid sequence comprising SEQ ID NO: 155.
  • one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion).
  • CDR1 comprises the amino acid sequence of SEQ ID NO: 153, wherein one or two amino acids are modified.
  • CDR2 comprises the amino acid sequence of SEQ ID NO: 154, wherein one or two amino acids are modified.
  • CDR3 comprises the amino acid sequence of SEQ ID NO: 155, wherein one or two amino acids are modified.
  • the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 153, a CDR2 amino acid sequence consisting of SEQ ID NO: 154, and a CDR3 amino acid sequence consisting of SEQ ID NO: 155.
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 156.
  • the first antigen-binding domain comprises SEQ ID NO: 156.
  • the first antigen-binding domain consists of SEQ ID NO: 156.
  • the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 157, a CDR2 amino acid sequence comprising SEQ ID NO: 158, and a CDR3 amino acid sequence comprising SEQ ID NO: 159.
  • one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion).
  • CDR1 comprises the amino acid sequence of SEQ ID NO: 157, wherein one or two amino acids are modified.
  • CDR2 comprises the amino acid sequence of SEQ ID NO: 158, wherein one or two amino acids are modified.
  • CDR3 comprises the amino acid sequence of SEQ ID NO: 159, wherein one or two amino acids are modified.
  • the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 157, a CDR2 amino acid sequence consisting of SEQ ID NO: 158, and a CDR3 amino acid sequence consisting of SEQ ID NO: 159. [0150] In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 160. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 160. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 160.
  • the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 161, a CDR2 amino acid sequence comprising SEQ ID NO: 162, and a CDR3 amino acid sequence comprising SEQ ID NO: 163.
  • one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion).
  • CDR1 comprises the amino acid sequence of SEQ ID NO: 161, wherein one or two amino acids are modified.
  • CDR2 comprises the amino acid sequence of SEQ ID NO: 162, wherein one or two amino acids are modified.
  • CDR3 comprises the amino acid sequence of SEQ ID NO: 163, wherein one or two amino acids are modified.
  • the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 161, a CDR2 amino acid sequence consisting of SEQ ID NO: 162, and a CDR3 amino acid sequence consisting of SEQ ID NO: 163.
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 164.
  • the first antigen-binding domain comprises SEQ ID NO: 164.
  • the first antigen-binding domain consists of SEQ ID NO:164.
  • the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 165, a CDR2 amino acid sequence comprising SEQ ID NO: 166, and a CDR3 amino acid sequence comprising SEQ ID NO: 167.
  • one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion).
  • CDR1 comprises the amino acid sequence of SEQ ID NO: 165, wherein one or two amino acids are modified.
  • CDR2 comprises the amino acid sequence of SEQ ID NO: 166, wherein one or two amino acids are modified.
  • CDR3 comprises the amino acid sequence of SEQ ID NO: 167, wherein one or two amino acids are modified.
  • the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 165, a CDR2 amino acid sequence consisting of SEQ ID NO: 166, and a CDR3 amino acid sequence consisting of SEQ ID NO: 167.
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 168. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 168. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 168. [0155] In some embodiments, the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 169, a CDR2 amino acid sequence comprising SEQ ID NO: 170, and a CDR3 amino acid sequence comprising SEQ ID NO: 171.
  • one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion).
  • CDR1 comprises the amino acid sequence of SEQ ID NO: 169, wherein one or two amino acids are modified.
  • CDR2 comprises the amino acid sequence of SEQ ID NO: 170, wherein one or two amino acids are modified.
  • CDR3 comprises the amino acid sequence of SEQ ID NO: 171, wherein one or two amino acids are modified.
  • the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 169, a CDR2 amino acid sequence consisting of SEQ ID NO: 170, and a CDR3 amino acid sequence consisting of SEQ ID NO: 171.
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 172.
  • the first antigen-binding domain comprises SEQ ID NO: 172.
  • the first antigen-binding domain consists of SEQ ID NO: 172.
  • the 5T4 binding agents described herein have a binding affinity (K D ) for human 5T4 of less than 10 ⁇ 7 M, such as approximately less than 10 ⁇ 8 M, less than 10 ⁇ 9 M, or less than 10 ⁇ 10 M when determined by surface plasmon resonance (SPR) using recombinant human 5T4 as the analyte and the 5T4 binding agent as the ligand.
  • K D binding affinity
  • the binding agent binds to human 5T4 with an affinity that is at least 1.1-, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2.0-, 2.5-, 3.0-, 3.5-, 4.0-, 4.5-, 5.0-, 6.0-, 7.0-, 8.0-, 9.0-, or 10.0-fold or greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein).
  • a non-specific antigen e.g., BSA, casein
  • Methods of testing binding agents for the ability to bind a target antigen include any antibody-antigen binding assay, such as, for example, radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assays (see, e.g., Janeway et al., infra, U.S. Patent Application Publication No. 2002/0197266 Al, and U.S. Patent No.7,338,929).
  • RIA radioimmunoassay
  • ELISA ELISA
  • Western blot Western blot
  • immunoprecipitation immunoprecipitation
  • competitive inhibition assays see, e.g., Janeway et al., infra, U.S. Patent Application Publication No. 2002/0197266 Al, and U.S. Patent No.7,338,929.
  • the 5T4 binding agents and multi-specific binding agents comprising the same described herein are capable of binding to recombinant 5T4 protein with a K D of less than 10 nM, for example less than 5 nM, less than 2 nM, less than 1 nM or less than 0.5 nM, as determined by Bio-Layer Interferometry as used in Example 2 herein.
  • the 5T4 binding agents or multi-specific binding agents described herein bind to CHO-K1 cells transfected with human 5T4 with an EC 50 of less than 10 nM, less than 8 nM, less than 6 nM, less than 4 nM or less than 2 nM, when tested as described in Example 5 herein.
  • the 5T4 binding agents or multi-specific binding agents described herein bind to 5T4-expressing U-251 cells with an EC 50 of less than 5 nM, less than 4 nM, or less than 2 nM, when tested as described in Example 6 herein.
  • the 5T4 binding agents or multi-specific binding agents described herein bind to 5T4-expressing A-431 cells with an EC 50 of less than 5 nM, less than 4 nM, or less than 2 nM when tested as described in Example 6 herein. 338699-2168 [0163] In some embodiments, the 5T4 binding agents or multi-specific binding agents described herein bind to V ⁇ 9V ⁇ 2 T cells with an EC 50 of less than 5 nM, such as less than 2 nM, when tested as described in Example 8 herein. [0164] In some embodiments, the 5T4 binding agents or multi-specific binding agents described herein are not capable of binding cynomolgus 5T4 or murine 5T4.
  • the 5T4 binding agents or multi-specific binding agents described herein are capable of mediating killing of 5T4-expressing tumor cells, such as U- 251 or A-431, by V ⁇ 9V ⁇ 2 T cells.
  • the 5T4 binding agents or multi-specific binding agents described herein are capable of inducing killing of U-251 cells through activation of V ⁇ 9V ⁇ 2 T cells with an EC 50 value of 100 pM or less, such as 20 pM or less, e.g., 10 pM or less, or even 7 pM or less, or 5 pM or less when tested as described in Example 9 herein.
  • the 5T4 binding agents or multi-specific binding agents described herein are capable of inducing killing of A-431 cells through activation of V ⁇ 9V ⁇ 2 T cells with an EC 50 value of 100 pM or less, such as 20 pM or less, e.g., 10 pM or less, or even 7 pM or less, or 5 pM or less when tested as described in Example 9 herein.
  • the 5T4 binding agents or multi-specific binding agents described herein are able to induce tumor lysis in cervical squamous cell carcinoma, cervical adenocarcinoma, metastatic colorectal cancer, and/or glioblastoma when tested as described in Example 10 herein.
  • the 5T4 binding agents or multi-specific binding agents described herein are not capable of enhancing killing of 5T4-negative cells, such as 5T4 negative human cells, by V ⁇ 9V ⁇ 2 T cells.
  • the 5T4 binding agents or multi-specific binding agents described herein are capable of enhancing killing of 5T4-positive cells, such as 5T4 positive tumor cells, by V ⁇ 9V ⁇ 2 T cells.
  • the 5T4 binding agents and multi-specific binding agents comprising the same described herein are: (a) capable of binding to recombinant human 5T4 with a K D of less than 10 nM, for example less than 5 nM, less than 2 nM, less than 1 nM, or less than 0.5 nM, as determined by Bio-Layer Interferometry according to the methods described in Example 2 herein; and (b) capable of inducing killing of A-431 cells through activation of V ⁇ 9V ⁇ 2 T cells with an EC 50 value of 10 pM or less, or even 7 pM or less, or 5 pM or less when tested as described in Example 9 herein.
  • the 5T4 binding agents and multi-specific binding agents comprising the same described herein are: 338699-2168 (a) capable of binding to recombinant human 5T4 with a K D of less than 10 nM, for example less than 5 nM, less than 2 nM, less than 1 nM, or less than 0.5 nM, as determined by Bio-Layer Interferometry according to the methods described in Example 2 herein; and (b) capable of inducing killing of U-251 cells through activation of V ⁇ 9V ⁇ 2 T cells with an EC 50 value of 10 pM or less, or even 7 pM or less, or 5 pM or less when tested as described in Example 9 herein.
  • the 5T4 binding agents and multi-specific binding agents comprising the same described herein have all of the following properties: (a) bind recombinant human 5T4 with a K D of less than 50 nM, for example less than 15 nM, less than 5 nM, less than 4 nM, less than 3 nM, or less than 2 nM, as determined by Bio-Layer Interferometry according to the method described in Example 2 herein; (b) bind 5T4-expressing U-251 cells with an EC 50 of less than 5 nM, less than 4 nM, or less than 2 nM, when tested for example as described in Example 6 herein; (c) binds 5T4-expressing A-431 cells with an EC 50 of less than 5 nM, less than 4 nM, or less than 2 nM, when tested for example as described in Example 6 herein; (d) bind V ⁇ 9V ⁇ 2 T cells with an EC 50 of less than 5 nM,
  • “capable of competing” or “able to compete” or “competes” refers to any detectably significant reduction in the propensity for a particular binding molecule (e.g., a 5T4 antibody) to bind a particular binding partner (e.g., 5T4) in the presence of another molecule (e.g., a different 5T4 antibody) that binds the binding partner.
  • competition means that saturating binding of the first binding agent to the antigen (5T4) blocks or prevents binding of the second binding agent, as determined by, e.g., biolayer interferometry (BLI) as described in Example 3 herein using sufficient amounts of the two (or more) competing binding agents.
  • binding agents comprising an antigen- binding domain capable of binding human 5T4 wherein the binding agent is capable of competing for binding to human 5T4 with a binding agent having the sequence set forth in SEQ ID NOs: 92, 93, 94, 95, 96, 97, 101, 102, 103, 104, 108, 109, 110, 114, 118, 119, 120, 121, 128, 129, 130, 131, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, and 172, e.g. when tested as described in Example 3 herein.
  • the binding agent is capable of competing for binding to human 5T4 with a binding agent having the sequence set forth in SEQ ID NO:91 and with a binding agent having the sequence set forth in SEQ ID NO:96.
  • the binding agent is capable of competing for binding to human 5T4 with a binding agent having the sequence set forth in SEQ ID NO:91 and with a binding agent having the sequence set forth in SEQ ID NO:140.
  • the binding agent is capable of competing for binding to human 5T4 with a binding agent having the sequence set forth in SEQ ID NO:96 and with a binding agent having the sequence set forth in SEQ ID NO:140.
  • the binding agent is capable of competing for binding to human 5T4 with a binding agent having the sequence set forth in SEQ ID NO:91, with a binding agent having the sequence set forth in SEQ ID NO:96, and with a binding agent having the sequence set forth in SEQ ID NO:140.
  • the binding agent is capable of competing for binding to human 5T4 with a binding agent having the sequence set forth in SEQ ID NO:108 and with a binding agent having the sequence set forth in SEQ ID NO:114.
  • the binding agent is capable of competing for binding to human 5T4 with a binding agent having the sequence set forth in SEQ ID NO:118 and with a binding agent having the sequence set forth in SEQ ID NO:128.
  • the binding agent is capable of competing for binding to human 5T4 with a binding agent having the sequence set forth in SEQ ID NO:118 and with a binding agent having the sequence set forth in SEQ ID NO:136.
  • the binding agent is capable of competing for binding to human 5T4 with a binding agent having the sequence set forth in SEQ ID NO:128 and with a binding agent having the sequence set forth in SEQ ID NO:136.
  • the binding agent is capable of competing for binding to human 5T4 with a binding agent having the sequence set forth in SEQ ID NO:118, with a binding 338699-2168 agent having the sequence set forth in SEQ ID NO:128, and with a binding agent having the sequence set forth in SEQ ID NO:136.
  • the binding agent of the present disclosure binds to the same epitope on 5T4 as binding agent LV1138, LV1139, LV1140, LV1141, LV1142, LV1143, LV1144, LV1145, LV1146, LV1147, LV1148, LV1149, LV1150, LV1151, LVA1152, LV1292 or any of the variants of these binding agents set forth in SEQ ID NOs: 93, 94, 95, 96, 97, 102, 103, 104, 109, 110, 119, 120, 121, 129, 130, 131, or 132 disclosed herein and/or to the same epitope on V ⁇ 2 as binding agent 5C8 or 6H4 (WO2015156673) or any of the variants of 5C8 set forth in SEQ ID NOs: 8, 9, 13, 17, 21, 22, or 26.
  • mapping binding agent binding epitopes on target antigens including but not limited to: crosslinking coupled mass spectrometry, allowing identification of peptides that are part of the epitope, and X-ray crystallography identifying individual residues on the antigen that form the epitope.
  • Epitope residues can be determined as being all amino acid residues with at least one atom less than or equal to 5 ⁇ from the binding agent.5 ⁇ was chosen as the epitope cutoff distance to allow for atoms within a van der Waals radius plus a possible water-mediated hydrogen bond.
  • epitope residues can be determined as being all amino acid residues with at least one atom less than or equal to 8 ⁇ .
  • Crosslinking coupled mass spectrometry begins by binding the binding agent and the antigen with a mass labeled chemical crosslinker. Next the presence of the complex is confirmed using high mass MALDI detection. Because after crosslinking chemistry the Ab/Ag complex is extremely stable, many various enzymes and digestion conditions can be applied to the complex to provide many different overlapping peptides. Identification of these peptides is performed using high resolution mass spectrometry and MS/MS techniques. Identification of the crosslinked peptides is determined using mass tag linked to the cross-linking reagents.
  • peptides that are crosslinked and are derived from the antigen are part of the epitope, while peptides derived from the antibody are part of the paratope. All residues between the most N- and C-terminal crosslinked residue from the individual crosslinked peptides found are considered to be part of the epitope or paratope.
  • the binding agent binds to the same epitope on 5T4 as a binding agent having the sequence set forth in any one of SEQ ID NOs: 92, 93, 94, 95, 96, 97, 101, 102, 103, 104, 108, 109, 110, 114, 118, 119, 120, 121, 128, 129, 130, 131, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, and 172.
  • the binding agent binds to the same epitope on 5T4 as a binding agent having the sequence set forth in SEQ ID NO:91 and with a binding agent having the sequence set forth in SEQ ID NO:96. 338699-2168 [0188] In some embodiments, the binding agent binds to the same epitope on 5T4 as a binding agent having the sequence set forth in SEQ ID NO:91 and with a binding agent having the sequence set forth in SEQ ID NO:140. [0189] In some embodiments, the binding agent binds to the same epitope on 5T4 as a binding agent having the sequence set forth in SEQ ID NO:96 and with a binding agent having the sequence set forth in SEQ ID NO:140.
  • the binding agent binds to the same epitope on 5T4 as a binding agent having the sequence set forth in SEQ ID NO:91, with a binding agent having the sequence set forth in SEQ ID NO:96, and with a binding agent having the sequence set forth in SEQ ID NO:140. [0191] In some embodiments, the binding agent binds to the same epitope on 5T4 as a binding agent having the sequence set forth in SEQ ID NO:108 and with a binding agent having the sequence set forth in SEQ ID NO:114.
  • the binding agent binds to the same epitope on 5T4 as a binding agent having the sequence set forth in SEQ ID NO:118 and with a binding agent having the sequence set forth in SEQ ID NO:128. [0193] In some embodiments, the binding agent binds to the same epitope on 5T4 as a binding agent having the sequence set forth in SEQ ID NO:118 and with a binding agent having the sequence set forth in SEQ ID NO:136. [0194] In some embodiments, the binding agent binds to the same epitope on 5T4 as a binding agent having the sequence set forth in SEQ ID NO:128 and with a binding agent having the sequence set forth in SEQ ID NO:136.
  • the binding agent binds to the same epitope on 5T4 as a binding agent having the sequence set forth in SEQ ID NO:118, with a binding agent having the sequence set forth in SEQ ID NO:128, and with a binding agent having the sequence set forth in SEQ ID NO:136.
  • Multi-specific Binding Agents [0196]
  • the 5T4 binding agent is a multi-specific binding agent.
  • the multi-specific binding agent comprises a first antigen-binding domain that specifically binds to human 5T4 described above and a second antigen-binding domain that specifically binds to a second target antigen or epitope (e.g., a bispecific binding agent).
  • the multi-specific binding agent comprises a first antigen-binding domain that specifically binds to human 5T4 described above, a second antigen-binding domain that 338699-2168 specifically binds to a second target antigen or epitope, and a third antigen-binding domain that specifically binds to a third target antigen or epitope.
  • the first, second, and/or third antigen-binding domain is a single-domain antibody.
  • the first, second, and/or third antigen-binding region is humanized.
  • the multi-specific binding agent is a bispecific binding agent, wherein the first antigen-binding domain is a VHH and the second antigen- binding region is a VHH.
  • the multi-specific binding agent is in a VHH-Fc format, i.e., the binding agent comprises two or more single-domain antigen-binding domains that are linked to each other via a human Fc domain dimer. In this format, each single-domain antigen-binding domain is fused to an Fc region polypeptide and the two fusion polypeptides form a dimeric bispecific binding agent via disulfide bridges in the hinge region.
  • Such constructs typically do not contain full, or any, CH1 or light chain sequences.
  • the multi-specific binding agents provided herein comprise a first antigen-binding domain that specifically binds to human 5T4 and a second antigen-binding domain that specifically binds to an antigen expressed on an immune cell.
  • the term “immune cell” refers to cells involved in mounting innate and adaptive immune responses, including but not limited to lymphocytes (such as T-cells and B-cells), natural killer (NK) cells, NKT cells, macrophages, monocytes, eosinophils, basophils, neutrophils, dendritic cells, and mast cells.
  • the immune cell is a T cell, such as a CD4+ T cell, a CD8+ T cell (also referred to as a cytotoxic T cell or CTL), or a ⁇ T cell. In some embodiments, the immune cell is a ⁇ T cell.
  • the second antigen-binding domain specifically binds to a target antigen selected from CD3, CD2, ⁇ T cell receptors, T cell receptors (e.g., V ⁇ 2 TCR, V ⁇ 1 TCR, V ⁇ 9, or V ⁇ 3 TCR), CD56, CD16, CD19, and CD20.
  • the V ⁇ 2 TCR is a human V ⁇ 2 T cell receptor.
  • the human V ⁇ 2 T cell receptor is a V ⁇ 9V ⁇ 2 T cell receptor.
  • the second antigen-binding domain specifically binds to a ⁇ T cell receptor.
  • Antigen-binding domains that specifically bind to V ⁇ 1+ T cells are known in the art. See e.g., WO 2021/032963.
  • Antigen-binding domains that specifically bind to V ⁇ 2+ T cell receptors are known in the art.
  • the second antigen-binding domain specifically binds to human V ⁇ 2.
  • the multi-specific binding agents provided herein are capable of 338699-2168 binding the V ⁇ 2 chain of a V ⁇ 9V ⁇ 2-TCR.
  • “Capable of binding the V ⁇ 2 chain of a V ⁇ 9V ⁇ 2-TCR” means that the binding agent disclosed herein or multi-specific binding agent comprising the same can bind the V ⁇ 2 chain as a separate molecule and/or as part of a V ⁇ 9V ⁇ 2-TCR. This can be referred to as a V ⁇ 2 binding agent. However, the binding agent or multi-specific binding agent comprising the same will not bind to the V ⁇ 9 chain as a separate molecule. Exemplary V ⁇ 9V ⁇ 2 TCR antigen-binding domain sequences are shown in Table 6A, Table 6B, Table 6C, and Table 6D.
  • Table 6A Exemplary V ⁇ 2 TCR-specific CDR (Kabat) and VHH Sequences Reference Component Sequence SEQ ID 5 C8 CDR1 NYAMG 1 CDR2 AISWSGGSTSYADSVKG 2 CDR3 QFSGADYGFGRLGIRGYEYDY 3 VHH EVQLVESGGGLVQAGGSLRLSCAASGRPFSNYAMGWF 4 RQAPGKEREFVAAISWSGGSTSYADSVKGRFTISRDN AKNTVYLQMNSPKPEDTAIYYCAAQFSGADYGFGRLG IRGYEYDYWGQGTQVTVSS 5C8 var1 CDR1 NYAMS 5 5C8 var2 CDR2 AISWSGGSTSYADSVKG 6 CDR3 QFSGADYGFGRLGIRGYEYDY 7 VHH (var1) EVQLLESGGGSVQPGGSLRLSCAASGRPFSNYAMSWF 8 RQAPGKEREFVSAISWSGGSTSYADSVKGRFTISRDN SKNT
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 92, 93, 94, 95, 96, and 97
  • the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain comprises a sequence selected from SEQ ID NOs: 92, 93, 94, 95, 96, and 97
  • the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain consists of a sequence 338699-2168 selected from SEQ ID NOs: 92, 93, 94, 95, 96, and 97
  • the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 98, a CDR2 of SEQ ID NO: 99, and a CDR3 of SEQ ID NO: 100; and (ii) a second antigen-binding domain that binds to V ⁇ 2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs).
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 101, 102, 103, and 104
  • the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain comprises any one of SEQ ID NOs: 101, 102, 103, and 104
  • the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30
  • the first antigen-binding domain consists of any one of SEQ ID NOs: 101, 102, 103, and 104
  • the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 105, a CDR2 of SEQ ID NO: 106, and a CDR3 of SEQ ID NO: 107; and (ii) a second antigen-binding domain that binds to V ⁇ 2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs).
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 108, 109, and 110
  • the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain comprises any one of SEQ ID NOs: 108, 109, and 110
  • the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain consists of any one of SEQ ID NOs: 108, 109, and 110
  • the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 111, a CDR2 of SEQ ID NO: 112, and a CDR3 of SEQ ID NO: 113; and (ii) a second antigen-binding domain that binds to V ⁇ 2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs).
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 114
  • the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain comprises SEQ ID NO: 114
  • the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain consists of SEQ ID NO: 114
  • the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 115, a CDR2 of SEQ ID NO: 116, and a CDR3 of SEQ ID NO: 117; and (ii) a second antigen-binding domain that binds to V ⁇ 2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs).
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 118, 119, 120, and 121
  • the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain comprises any one of SEQ ID NOs: 118, 119, 120, and 121
  • the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain consists of any one of SEQ ID NOs: 118, 119, 120, and 121
  • the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 122 or 123, a CDR2 of SEQ ID NO: 124 or 125, and a CDR3 of SEQ ID NO: 126 or 127; and (ii) a second antigen- 338699-2168 binding domain that binds to V ⁇ 2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs).
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128, 129, 130, 131, and 132
  • the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen- binding domain comprises any one of SEQ ID NOs: 128, 129, 130, 131, and 132
  • the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain consists of any one of SEQ ID NOs: 128, 129, 130, 131, and 132
  • the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 133, a CDR2 of SEQ ID NO: 134, and a CDR3 of SEQ ID NO: 135; and (ii) a second antigen-binding domain that binds to V ⁇ 2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs).
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 136
  • the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain comprises SEQ ID NO: 136
  • the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain consists of SEQ ID NO: 136
  • the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 137, a CDR2 of SEQ ID NO: 138, and a CDR3 of SEQ ID NO: 139; and (ii) a second antigen-binding domain that binds to V ⁇ 2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs).
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, 338699-2168 at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 140
  • the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain comprises SEQ ID NO: 140
  • the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain consists of SEQ ID NO: 140
  • the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 141, a CDR2 of SEQ ID NO: 142, and a CDR3 of SEQ ID NO: 143; and (ii) a second antigen-binding domain that binds to V ⁇ 2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs).
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 144
  • the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain comprises SEQ ID NO: 144
  • the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain consists of SEQ ID NO: 144
  • the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 145, a CDR2 of SEQ ID NO: 146, and a CDR3 of SEQ ID NO: 147; and (ii) a second antigen-binding domain that binds to V ⁇ 2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs).
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 148
  • the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% 338699-2168 identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain comprises SEQ ID NO: 148
  • the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain consists of SEQ ID NO: 148
  • the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 149, a CDR2 of SEQ ID NO: 150, and a CDR3 of SEQ ID NO: 151; and (ii) a second antigen-binding domain that binds to V ⁇ 2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs).
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 152
  • the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain comprises SEQ ID NO: 152
  • the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain consists of SEQ ID NO: 152
  • the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 153, a CDR2 of SEQ ID NO: 154, and a CDR3 of SEQ ID NO: 155; and (ii) a second antigen-binding domain that binds to V ⁇ 2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs).
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 156
  • the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain comprises SEQ ID NO: 156
  • the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain consists of SEQ ID 338699-2168 NO: 156
  • the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 157, a CDR2 of SEQ ID NO: 158, and a CDR3 of SEQ ID NO: 159; and (ii) a second antigen-binding domain that binds to V ⁇ 2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs).
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 160
  • the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain comprises SEQ ID NO: 160
  • the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain consists of SEQ ID NO: 160
  • the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 161, a CDR2 of SEQ ID NO: 162, and a CDR3 of SEQ ID NO: 163; and (ii) a second antigen-binding domain that binds to V ⁇ 2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs).
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 164
  • the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain comprises SEQ ID NO: 164
  • the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain consists of SEQ ID NO: 164
  • the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 165, a CDR2 of SEQ 338699-2168 ID NO: 166, and a CDR3 of SEQ ID NO: 167; and (ii) a second antigen-binding domain that binds to V ⁇ 2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs).
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 168
  • the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain comprises SEQ ID NO: 168
  • the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain consists of SEQ ID NO: 168
  • the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 169, a CDR2 of SEQ ID NO: 170, and a CDR3 of SEQ ID NO: 171; and (ii) a second antigen-binding domain that binds to V ⁇ 2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs).
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 172
  • the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain comprises SEQ ID NO: 172
  • the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • the first antigen-binding domain consists of SEQ ID NO: 172
  • the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30.
  • any of the multi-specific binding agents disclosed herein are modified to reduce or eliminate the formation of pyroglutamic acid at the N-terminus of the modified multi-specific binding agents as compared to the unmodified multi-specific binding agents of this disclosure.
  • the glutamate residue (E) or glutamine (Q) residue at the first position is modified to an aspartic acid residue (D).
  • any of the multi-specific binding agents of the present disclosure may comprise an aspartic acid (D) residue in the first position.
  • the 5T4 binding agent, or multi-specific binding agent comprising the same comprises an aspartic acid or a glutamic acid residue at the N-terminal amino acid position and elutes earlier in reversed-phase HPLC than a 5T4 binding agent or multi-specific binding agent comprising the same that comprises a pyroglutamate residue at the N-terminal amino acid position.
  • the V ⁇ 2 binding agent, or multi-specific binding agent comprising the same comprises an aspartic acid or a glutamic acid residue at the N-terminal amino acid position and elutes earlier in reversed-phase HPLC than a V ⁇ 2 binding agent or multi-specific binding agent comprising the same that comprises a pyroglutamate residue at the N-terminal amino acid position.
  • the second antigen-binding domain is capable of binding the V ⁇ 9 chain of a human V ⁇ 9V ⁇ 2 T cell receptor.
  • the multi-specific binding agents provided herein are capable of binding the V ⁇ 9 chain of a V ⁇ 9V ⁇ 2-TCR.
  • “Capable of binding the V ⁇ 9 chain of a V ⁇ 9V ⁇ 2-TCR” means that the binding agent disclosed herein or multi-specific binding agent comprising the same can bind the V ⁇ 9 chain as a separate molecule and/or as part of a V ⁇ 9V ⁇ 2-TCR. This can be referred to as a V ⁇ 9 binding agent. However, the binding agent or multi-specific binding agent comprising the same will not bind to the V ⁇ 2 chain as a separate molecule. Exemplary V ⁇ 9V ⁇ 2 TCR antigen-binding domain sequences are known in the art, see e.g., WO 2015/156673. [0223] In some embodiments, the multi-specific binding agents provided herein are able to activate V ⁇ 9V ⁇ 2-T cells.
  • “Able to activate V ⁇ 9V ⁇ 2-T cells” in the context of the present disclosure refers to activation of V ⁇ 9V ⁇ 2-T cells in the presence of a multi-specific binding agent, such as a bispecific binding agent, disclosed herein in the presence of a target cell expressing 5T4.
  • a multi-specific binding agent such as a bispecific binding agent
  • the activation of the V ⁇ 9V ⁇ 2-T cells is measurable through gene- expression and/or (surface) marker expression (e.g., activation markers, such as CD25, CD69, or CD107a) and/or secretory protein (e.g., cytokines or chemokines) profiles.
  • the binding agent is able to induce activation (e.g., upregulation of CD69 and/or CD25 expression) resulting in degranulation (marked by an increase in CD107a expression; see Examples 9, 10, and 13 herein) and cytokine production (e.g., TNF, IFN- ⁇ ) by V ⁇ 9V ⁇ 2-T cells.
  • the multi-specific binding agent that is used is able to increase CD107a expression on V ⁇ 9V ⁇ 2 T cells to at least 10%, more preferably at least 20%, more preferably at least 40%, most preferably at least 90%, when used in an assay as described in Example 9 herein, wherein e.g., 10% means that 10% of the total number of V ⁇ 9V ⁇ 2 T cells is positive for CD107a.
  • the number of V ⁇ 9V ⁇ 2 T cells positive for CD107a is 338699-2168 increased 1.5-fold, such as 2-fold, e.g., 5-fold, in the presence of an multi-specific binding agent of the disclosure.
  • the multi-specific binding agents of the disclosure have an EC 50 value for increasing the percentage of CD107a positive V ⁇ 9V ⁇ 2 T cells of 100 pM or less, such as 25 pM or less, e.g., 12 pM or less, when tested using V ⁇ 9V ⁇ 2 T cells and 5T4 expressing U-251 target cells as described herein in Example 9.
  • the multi-specific binding agent is not capable of inducing degranulation of V ⁇ 9V ⁇ 2 T cells marked by an increase in CD107a in the absence of 5T4-expressing cells.
  • the binding agents disclosed herein are able to induce cytotoxicity in the presence of V ⁇ 9V ⁇ 2-T cells.
  • V ⁇ 9V ⁇ 2-T cells are activated in the presence of a multi-specific binding agent, such as a bispecific binding agent, of the disclosure in the presence of a target cell expressing 5T4, and induce the death of the target cell through any means of cellular cytotoxicity, such as but not limited to apoptosis, necroptosis and pyroptosis.
  • a multi-specific binding agent such as a bispecific binding agent
  • the multi-specific binding agents disclosed herein comprise one or more linkers.
  • linker refers to a short stretch of amino acids used to connect two functional domains (e.g., antigen-binding domains or Fc domains) together in a polypeptide chain.
  • the antigen-binding domains are linked together on a polypeptide chain via one or more peptide linkers.
  • Peptide linkers can also be used to attach other domains (such as half-life extension domains) to the multi-specific binding agents of the disclosure.
  • the first and second antigen-binding regions in the multi-specific binding agents may be arranged in various ways.
  • antigen-binding domains are connected to each other via a linker, such as a covalent linker.
  • the first antigen-binding domain and the second antigen-binding domain are covalently linked to each other via a peptide linker, e.g. a linker having a length of from 1 to 20 amino acids, e.g. from 1 to 10 amino acids, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
  • Suitable linkers are known in the art and include, for example, peptide linkers containing flexible amino acid residues such as glycine, alanine, and serine.
  • a linker can contain motifs, e.g., multiple or repeating motifs, of GA, GS, GG, GGA, GGS, GGG, GGGA (SEQ ID NO: 337), GGGS (SEQ ID NO: 338), GGGG (SEQ ID NO: 339), GGGGA (SEQ ID NO: 340), GGGGS (SEQ ID NO: 341), GGGGG (SEQ ID NO: 342), GGAG (SEQ ID NO: 343), GGSG (SEQ ID NO: 344), AGGG (SEQ ID NO: 345), or SGGG (SEQ ID NO: 346).
  • motifs e.g., multiple or repeating motifs, of GA, GS, GG, GGA, GGS, GGG, GGGA (SEQ ID NO: 337), GGGS (SEQ ID NO: 338), GGGG (SEQ ID NO: 339), GGGGA (SEQ ID NO: 340), GGGGS (SEQ ID NO: 341)
  • the peptide linker comprises or consists of GGGGS 338699-2168 (SEQ ID NO: 341). In some embodiments, the linker comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 repeating motifs. Fc domains [0228] Multi-specific binding agents disclosed herein, such as bispecific binding agent, may contain additional molecules, domains, or polypeptide sequences beyond the first and second antigen-binding domains. [0229] In some embodiments, the present disclosure provides a 5T4 binding agent or multi- specific binding agent comprising the same, further comprising an Fc domain. In some embodiments, the Fc domain comprises a first and a second Fc domain monomer.
  • Fc domain or “Fc region” describes the minimum region (in the context of a larger polypeptide) or smallest protein folded structure (in the context of an isolated protein) that can bind to or be bound by an Fc receptor (FcR).
  • the Fc domain is the fragment of an antibody which would be typically generated after digestion with papain, and generally includes the two CH2-CH3 regions of an immunoglobulin and a hinge region.
  • Fc domain monomer describes the single chain protein that, when associated with another Fc domain monomer, forms a functional Fc domain. The association of two Fc domain monomers creates one Fc domain. When two Fc domain monomers associate, the resulting Fc domain has Fc receptor binding activity.
  • an Fc domain is a dimeric structure that can bind an Fc receptor.
  • Fc domain includes the polypeptides comprising the constant region of an antibody excluding the first constant region immunoglobulin domain.
  • Fc domain refers to the last two constant region immunoglobulin domains (CH2, CH3) and optionally the flexible hinge N-terminal to these domains.
  • the Fc domain may be derived from any of a variety of different antibody isotypes, including but not limited to, a wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA, IgE, or IgM. In some embodiments, the Fc domain is derived from a human IgG1. [0233] There are many known polymorphs for the IgG1 Fc domain, including the “DEL” polymorph and the “EEM” polymorph. The DEL polymorph comprises the amino acids D-E-L at positions 356, 357, and 358, respectively (e.g., SEQ ID NO: 348).
  • the EEM polymorph comprises the amino acids E-E-M at positions 356, 357, and 358, respectively (e.g., SEQ ID NO: 347).
  • Two binding agents that are otherwise identical except for the presence of a DEL Fc domain or an EEM Fc domain are expected to demonstrate similar properties in terms of ligand binding and therapeutic efficacy.
  • the Fc domain is a DEL Fc domain.
  • the Fc domain is an EEM Fc domain.
  • the Fc domain is a variant Fc domain that forms a variant Fc domain with a desirable property, such as increased half-life, compared to naturally occurring (wild-type) Fc sequences.
  • a “variant Fc domain” refers to a non-naturally occurring Fc domain, for example an Fc domain comprising one or more non-naturally occurring amino acid residues, one or more amino acid substitutions relative to a wild-type human constant domain, or one or more amino acid deletion, addition and/or modification.
  • the multi-specific binding agent further comprises a half-life extension domain, i.e., a domain which prolongs the half-life of the molecules in the circulation of a human patient.
  • the multi-specific binding agent has a terminal half- life that is longer than about 168 hours when administered to a human subject. Most preferably the terminal half-life is 336 hours or longer.
  • the “terminal half-life” of a binding agent when used herein refers to the time taken for the serum concentration of the polypeptide to be reduced by 50%, in vivo in the final phase of elimination.
  • the serum half-life of binding agents comprising Fc domains may be increased by increasing the binding affinity of the Fc domain for FcRn.
  • the Fc domain variant has enhanced serum half-life relative to a comparable molecule.
  • the Fc domain variant comprises at least one amino acid substitution at one or more positions selected from the group consisting of M252Y, S254T and T256E (referred to herein as “YTE”; e.g., SEQ ID NO: 349 and 350).
  • the Fc domain variant comprises a Y at position 252 (referred to herein as “Y”, e.g., SEQ ID NO: 351 and 352).
  • the Fc domain variant may comprise a non-naturally occurring amino acid residue at additional and/or alternative positions known to one skilled in the art (see, e.g., U.S. Pat. Nos.
  • the group of peptides, proteins or protein domains includes peptides binding to other proteins with preferred pharmacokinetic profile in the human body such as serum albumin (see WO 2009/127691).
  • serum albumin refers to the albumin protein present in human blood plasma. Human serum albumin is the most abundant protein in the blood. It constitutes about half of the blood serum protein. In some embodiments, a human serum albumin has the sequence of UniProt ID NO: P02768.
  • An alternative concept of such half-life extending peptides includes peptides binding to the neonatal Fc receptor (FcRn, see WO 2007/098420), which can also be used in the polypeptides and binding agents of the present disclosure.
  • the concept of attaching larger domains of proteins or complete proteins includes the fusion of human serum albumin, variants, or mutants of human serum albumin (see WO 2011/051489, WO 2012/059486, WO 2012/150319, WO 2013/135896, WO 2014/072481, WO 2013/075066) or domains thereof as well as the fusion of constant region of immunoglobulins (Fc domains) and variants thereof, as described herein.
  • Such variants of Fc domains may be optimized/modified in order to allow the desired pairing of dimers or multimers, to abolish Fc receptor binding (e.g., the Fc ⁇ receptor), to enhance binding to FcRn, or for other reasons.
  • the Fc domain monomer allows assembly of two or more polypeptide chains in a covalent manner, for example by disulfide linking between cysteine residues. In this way the Fc domain monomer acts as a dimerization domain, allowing assembly of two polypeptide chains to form a dimer.
  • such dimers comprise two polypeptides, each polypeptide including an antigen-binding domain described herein linked to an Fc domain monomer, thereby forming a bivalent binding agent.
  • such dimers comprise two polypeptides, one polypeptide comprising an antigen-binding domain described herein linked to an Fc domain monomer, and one polypeptide comprising an Fc domain monomer, thereby forming a monovalent binding agent.
  • the Fc domain may be a heterodimer comprising two Fc monomers, wherein the first antigen-binding domain is fused to the first Fc monomer and the second antigen-binding domain is fused to the second Fc monomer and wherein the first and second Fc monomers comprise asymmetric amino acid mutations that favor the formation of heterodimers over the formation of homodimers (see e.g. Ridgway et al.
  • the CH3 regions of the Fc monomers may comprise said asymmetric amino acid 338699-2168 mutations, for example the first Fc monomer may comprise a T366W substitution and the second Fc monomer may comprise T366S, L368A and Y407V substitutions, or vice versa, wherein the amino acid positions correspond to human IgG1 according to the EU numbering system.
  • the cysteine residues at position 220 in the first and second Fc monomers may have been deleted or substituted, wherein the amino acid position corresponds to human IgG1 according to the EU numbering system.
  • the first and/or second Fc monomers may contain mutations that render the binding agent inert, i.e., unable to, or having reduced ability to, mediate Fc effector functions (e.g., Fc-mediated cross-linking of FcRs).
  • the inert Fc domain may in addition not be able to bind complement component (e.g., C1q) and/or one or more Fc receptors.
  • the first and second Fc monomers may comprise a mutation at position 234 and/or 235, for example the first and second Fc monomers may comprise an L234F and an L235E substitution, wherein the amino acid positions correspond to human IgG1 according to the EU numbering system.
  • the first and second Fc monomers may comprise an L234A and L235A substitution. In some embodiments, the first and second Fc domain monomers may further comprise a P329G substitution and/or a D265A substitution.
  • Exemplary Fc domain monomer sequences are shown in TABLE 7. Hinge sequences are shown in bold and italicized text; DEL and EEM polymorphisms are shown in boxed text; mutations relative to WT IgG1 are shown in bold and underlined text.
  • Fc domain sequences Fc domain AA Sequence SEQ ID: W T IgG1 (EEM) EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC 347 VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G W T IgG1 (DEL) EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC 348 VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKT
  • the Fc domains described in Table 7 comprise a modified IgG1 hinge (e.g., SEQ ID NO: 361 – AAASDKTHTCPPCP).
  • the Fc domain sequences may further comprise a lysine (K) residue at the C terminus.
  • Exemplary Multi-specific Binding Agents [0244]
  • the present disclosure provides a multi-specific binding agent comprising a first antigen-binding domain that binds to 5T4 and a second antigen-binding domain that binds to V ⁇ 2.
  • the first and second antigen-binding domains are comprised in the same polypeptide chain.
  • the multi-specific binding agent comprises the following format, from N’ to C’ terminus: first antigen-binding domain – linker – second antigen-binding domain. In some embodiments, the multi-specific binding agent comprises the following format, from N’ to C’ terminus: second antigen-binding domain – linker – first antigen-binding domain.
  • the linker is a glycine-serine linker. In some embodiments, the linker is G 4 S and comprises SEQ ID NO: 341.
  • the multi-specific binding agent comprises (i) a first antigen- binding domain comprising a CDR1 of SEQ ID NO: 88, a CDR2 of SEQ ID NO: 89 and a CDR3 of SEQ ID NO: 90 and (ii) a second antigen-binding domain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2 and a CDR3 of SEQ ID NO: 3.
  • the first antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 92 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4.
  • the first antigen-binding domain comprises SEQ ID NO: 92 and the second antigen-binding domain comprises SEQ ID NO: 4.
  • the first antigen-binding domain consists of SEQ ID NO: 92 and the second antigen-binding domain consists of SEQ ID NO: 4.
  • the multi-specific binding agent comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at 338699-2168 least 99% identical to SEQ ID NO: 362.
  • the multi-specific binding agent comprises SEQ ID NO: 362.
  • the multi-specific binding agent consists of SEQ ID NO: 362. Such a multi-specific binding agent is referred to herein as LAVA-378.
  • the multi-specific binding agent comprises (i) a first antigen- binding domain comprising a CDR1 of SEQ ID NO: 115, a CDR2 of SEQ ID NO: 116 and a CDR3 of SEQ ID NO: 117 and (ii) a second antigen-binding domain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2 and a CDR3 of SEQ ID NO: 3.
  • the first antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 118 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4.
  • the first antigen-binding domain comprises SEQ ID NO: 118 and the second antigen-binding domain comprises SEQ ID NO: 4.
  • the first antigen-binding domain consists of SEQ ID NO: 118 and the second antigen-binding domain consists of SEQ ID NO: 4.
  • the multi-specific binding agent comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 363.
  • the multi- specific binding agent comprises SEQ ID NO: 363.
  • the multi-specific binding agent consists of SEQ ID NO: 363. Such a multi-specific binding agent is referred to herein as LAVA-380.
  • the multi-specific binding agent comprises (i) a first antigen- binding domain comprising a CDR1 of SEQ ID NO: 98, a CDR2 of SEQ ID NO: 99 and a CDR3 of SEQ ID NO: 100 and (ii) a second antigen-binding domain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2 and a CDR3 of SEQ ID NO: 3.
  • the first antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 101 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4.
  • the first antigen-binding domain comprises SEQ ID NO: 101 and the second antigen-binding domain comprises SEQ ID NO: 4.
  • the first antigen-binding domain consists of SEQ ID NO: 101 and the second antigen-binding domain consists of SEQ ID NO: 4.
  • the multi-specific binding agent comprises an amino acid sequence that is at least 90%, at least 91%, at least 338699-2168 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 364.
  • the multi-specific binding agent comprises SEQ ID NO: 364.
  • the multi-specific binding agent consists of SEQ ID NO: 364.
  • the multi-specific binding agent comprises (i) a first antigen- binding domain comprising a CDR1 of SEQ ID NO: 105, a CDR2 of SEQ ID NO: 106 and a CDR3 of SEQ ID NO: 107 and (ii) a second antigen-binding domain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2 and a CDR3 of SEQ ID NO: 3.
  • the first antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 108 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4.
  • the first antigen-binding domain comprises SEQ ID NO: 108 and the second antigen-binding domain comprises SEQ ID NO: 4.
  • the first antigen-binding domain consists of SEQ ID NO: 108 and the second antigen-binding domain consists of SEQ ID NO: 4.
  • the multi-specific binding agent comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 365.
  • the multi- specific binding agent comprises SEQ ID NO: 365.
  • the multi-specific binding agent consists of SEQ ID NO: 365. [0249] Exemplary embodiments of such single chain multi-specific binding agents are provided in TABLE 8. Linkers are shown in bold.
  • Table 8 Exemplary single chain multi-specific binding agents.
  • Bispecific Format Amino acid sequence SEQ agent ID LV1139 x V ⁇ 2 LV1139- EVQLVESGGGLVQPGGSLRLSCAANGFAFSNYRMKWVRQAPGK 362 (LAVA-378) G 4 S-5C8 GLEWVSTISHRVGRTYYADSVKGRFTISRDNAKNTVYLQMNSL KPEDTARYFCTRVAMGGSDYAPHDYDYTGQGTQVTVSSGGGGS EVQLVESGGGLVQAGGSLRLSCAASGRPFSNYAMGWFRQAPGK EREFVAAISWSGGSTSYADSVKGRFTISRDNAKNTVYLQMNSP KPEDTAIYYCAAQFSGADYGFGRLGIRGYEYDYWGQGTQVTVS S LV1142 x V ⁇ 2 LV1142- EVQLVESGGGLVQAGGSLRLSCAASGSIFSMGVIDWHRQAPGK 363 (LAVA-380) G 4 S
  • the multi-specific binding agent comprises a first and a second polypeptide, wherein the first polypeptide comprises a first antigen-binding domain that binds to 5T4 and a first Fc domain monomer, and wherein the second polypeptide comprises a second antigen-binding domain and a second Fc domain monomer.
  • the Fc domain monomers associate to form a dimeric multi-specific binding agent.
  • the Fc domain monomers comprise one or more mutations to facilitate dimerization and/or reduce effector function.
  • Fc domains are shown in regular text.
  • the Fc domain corresponds to SEQ ID NO: 357 or 359 (i.e., effector mutations + hole mutations, w/ or w/o a C-terminal lysine).
  • the Fc domain corresponds to SEQ ID NO: 356 or 358 (i.e., effector mutations + knob mutations, w/ or w/o a C-terminal lysine).
  • VHH-Fc Binding Agents Reference AA Sequence SEQ ID: LV1139 EVQLVESGGGLVQPGGSLRLSCAANGFAFSNYRMKWVRQAPGKGLEWVSTISHR 366 IgG1 VGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTARYFCTRVAMGGSDYAP (Effector+ HDYDYTGQGTQVTVSSAAASDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMIS Hole) RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSPG LV1139
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 92 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 21.
  • the first antigen-binding domain comprises SEQ ID NO: 92 and the second antigen-binding domain comprises SEQ ID NO: 21.
  • the first antigen-binding domain consists of SEQ ID NO: 92 and the second antigen-binding domain consists of SEQ ID NO: 21.
  • the first Fc domain monomer comprises SEQ ID NO: 356 or 358 and the second Fc domain monomer comprises SEQ ID NO: 357 or 359.
  • the first Fc domain monomer comprises SEQ ID NO: 357 or 359 and the second Fc domain monomer comprises SEQ ID NO: 356 or 358.
  • the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 367 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 379.
  • the first polypeptide comprises SEQ ID NO: 367 and the second polypeptide comprises SEQ ID NO: 379.
  • the first polypeptide consists of SEQ ID NO: 367, referred to herein as LAVA-395, and the second polypeptide consists of SEQ ID NO: 379, referred to herein as LAVA-328.
  • LAVA-1395 Such a multi-specific binding agent is referred to herein as LAVA-1395.
  • the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 366 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 378.
  • the first polypeptide comprises SEQ ID NO: 366 and the second polypeptide comprises SEQ ID NO: 378.
  • the first polypeptide consists of SEQ ID NO: 366 and the second polypeptide consists of SEQ ID NO: 378. 338699-2168 [0253]
  • the multi-specific binding agent comprises (i) a first polypeptide comprising a first antigen-binding domain that binds 5T4 comprising a CDR1 of SEQ ID NO: 88, a CDR2 of SEQ ID NO: 89 and a CDR3 of SEQ ID NO: 90 and a first Fc domain monomer; and (ii) a second polypeptide comprising a second antigen-binding domain that binds to V ⁇ 2 and comprises a CDR1 of SEQ ID NO: 18, a CDR2 of SEQ ID NO: 19 and a CDR3 of SEQ ID NO: 20 and a second Fc domain monomer.
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 93 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 22.
  • the first antigen-binding domain comprises SEQ ID NO: 93 and the second antigen-binding domain comprises SEQ ID NO: 22.
  • the first antigen-binding domain consists of SEQ ID NO: 93 and the second antigen-binding domain consists of SEQ ID NO: 22.
  • the first Fc domain monomer comprises SEQ ID NO: 356 or 358 and the second Fc domain monomer comprises SEQ ID NO: 357 or 359.
  • the first Fc domain monomer comprises SEQ ID NO: 357 or 359 and the second Fc domain monomer comprises SEQ ID NO: 356 or 358.
  • the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 369 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 381.
  • the first polypeptide comprises SEQ ID NO: 369 and the second polypeptide comprises SEQ ID NO: 381.
  • the first polypeptide consists of SEQ ID NO: 369, referred to herein as LAVA-433, and the second polypeptide consists of SEQ ID NO: 381, referred to herein as LAVA-424.
  • LAVA-1433 Such a multi-specific binding agent is referred to herein as LAVA-1433.
  • the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 368 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 380.
  • the first polypeptide comprises SEQ ID NO: 368 and the second polypeptide comprises SEQ ID NO: 380.
  • the first polypeptide consists of SEQ ID NO: 368 and the second polypeptide consists of SEQ ID NO: 380. 338699-2168 [0254]
  • the multi-specific binding agent comprises (i) a first polypeptide comprising a first antigen-binding domain that binds 5T4 comprising a CDR1 of SEQ ID NO: 98, a CDR2 of SEQ ID NO: 99, and a CDR3 of SEQ ID NO: 100 and a first Fc domain monomer; and (ii) a second polypeptide comprising a second antigen-binding domain that binds to V ⁇ 2 and comprises a CDR1 of SEQ ID NO: 18, a CDR2 of SEQ ID NO: 19 and a CDR3 of SEQ ID NO: 20 and a second Fc domain monomer.
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 101 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 21.
  • the first antigen-binding domain comprises SEQ ID NO: 101 and the second antigen-binding domain comprises SEQ ID NO: 21.
  • the first antigen-binding domain consists of SEQ ID NO: 101 and the second antigen-binding domain consists of SEQ ID NO: 21.
  • the first Fc domain monomer comprises SEQ ID NO: 356 or 358 and the second Fc domain monomer comprises SEQ ID NO: 357 or 359.
  • the first Fc domain monomer comprises SEQ ID NO: 357 or 359 and the second Fc domain monomer comprises SEQ ID NO: 356 or 358.
  • the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 371 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 379.
  • the first polypeptide comprises SEQ ID NO: 371 and the second polypeptide comprises SEQ ID NO: 379.
  • the first polypeptide consists of SEQ ID NO: 371, and the second polypeptide consists of SEQ ID NO: 379.
  • the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 370 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 378.
  • the first polypeptide comprises SEQ ID NO: 370 and the second polypeptide comprises SEQ ID NO: 378. In some embodiments, the first polypeptide consists of SEQ ID NO: 370 and the second polypeptide consists of SEQ ID NO: 378.
  • the multi-specific binding agent comprises (i) a first polypeptide comprising a first antigen-binding domain that binds 5T4 comprising a CDR1 of SEQ ID NO: 98, a CDR2 of SEQ ID NO: 99 and a CDR3 of SEQ ID NO: 100 and a first Fc domain monomer; and (ii) a second polypeptide comprising a second antigen-binding domain that binds to V ⁇ 2 and comprises a CDR1 of SEQ ID NO: 18, a CDR2 of SEQ ID NO: 19 and a CDR3 of SEQ ID NO: 20 and a second Fc domain monomer.
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 101 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 22.
  • the first antigen-binding domain comprises SEQ ID NO: 101 and the second antigen-binding domain comprises SEQ ID NO: 22.
  • the first antigen-binding domain consists of SEQ ID NO: 101 and the second antigen-binding domain consists of SEQ ID NO: 22.
  • the first Fc domain monomer comprises SEQ ID NO: 356 or 358 and the second Fc domain monomer comprises SEQ ID NO: 357 or 359.
  • the first Fc domain monomer comprises SEQ ID NO: 357 or 359 and the second Fc domain monomer comprises SEQ ID NO: 356 or 358.
  • the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 371 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 381.
  • the first polypeptide comprises SEQ ID NO: 371 and the second polypeptide comprises SEQ ID NO: 381.
  • the first polypeptide consists of SEQ ID NO: 371, and the second polypeptide consists of SEQ ID NO: 381.
  • the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 370 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 380.
  • the first polypeptide comprises SEQ ID NO: 370 and the second polypeptide comprises SEQ ID NO: 380. In some embodiments, the first polypeptide consists of SEQ ID NO: 370 and the second polypeptide consists of SEQ ID NO: 380.
  • the multi-specific binding agent comprises (i) a first polypeptide comprising a first antigen-binding domain that binds 5T4 comprising a CDR1 of SEQ ID NO: 105, a CDR2 of SEQ ID NO: 106, and a CDR3 of SEQ ID NO: 107 and a first Fc domain monomer; and (ii) a second polypeptide comprising a second antigen-binding domain that binds to V ⁇ 2 and comprises a CDR1 of SEQ ID NO: 18, a CDR2 of SEQ ID NO: 19 and a CDR3 of SEQ ID NO: 20 and a second Fc domain monomer.
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 108 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 21.
  • the first antigen-binding domain comprises SEQ ID NO: 108 and the second antigen-binding domain comprises SEQ ID NO: 21.
  • the first antigen-binding domain consists of SEQ ID NO: 108 and the second antigen-binding domain consists of SEQ ID NO: 21.
  • the first Fc domain monomer comprises SEQ ID NO: 356 or 358 and the second Fc domain monomer comprises SEQ ID NO: 357 or 359.
  • the first Fc domain monomer comprises SEQ ID NO: 357 or 359 and the second Fc domain monomer comprises SEQ ID NO: 356 or 358.
  • the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 373 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 379.
  • the first polypeptide comprises SEQ ID NO: 373 and the second polypeptide comprises SEQ ID NO: 379.
  • the first polypeptide consists of SEQ ID NO: 373
  • the second polypeptide consists of SEQ ID NO: 379.
  • the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 372
  • the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 378.
  • the first polypeptide comprises SEQ ID NO: 372 and the second polypeptide comprises SEQ ID NO: 378. In some embodiments, the first polypeptide consists of SEQ ID NO: 372 and the second polypeptide consists of SEQ ID NO: 378.
  • the multi-specific binding agent comprises (i) a first polypeptide comprising a first antigen-binding domain that binds 5T4 comprising a CDR1 of SEQ ID NO: 105, a CDR2 of SEQ ID NO: 106, and a CDR3 of SEQ ID NO: 107 and a first Fc domain monomer; and (ii) a second polypeptide comprising a second antigen-binding domain that binds to V ⁇ 2 and comprises a CDR1 of SEQ ID NO: 18, a CDR2 of SEQ ID NO: 19 and a CDR3 of SEQ ID NO: 20 and a second Fc domain monomer.
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 108 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 22.
  • the first antigen-binding domain comprises SEQ ID NO: 108 and the second antigen-binding domain comprises SEQ ID NO: 22.
  • the first antigen-binding domain consists of SEQ ID NO: 108 and the second antigen-binding domain consists of SEQ ID NO: 22.
  • the first Fc domain monomer comprises SEQ ID NO: 356 or 358 and the second Fc domain monomer comprises SEQ ID NO: 357 or 359.
  • the first Fc domain monomer comprises SEQ ID NO: 357 or 359 and the second Fc domain monomer comprises SEQ ID NO: 356 or 358.
  • the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 373 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 381.
  • the first polypeptide comprises SEQ ID NO: 373 and the second polypeptide comprises SEQ ID NO: 381.
  • the first polypeptide consists of SEQ ID NO: 373
  • the second polypeptide consists of SEQ ID NO: 381.
  • the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 372
  • the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 380.
  • the first polypeptide comprises SEQ ID NO: 372 and the second polypeptide comprises SEQ ID NO: 380. In some embodiments, the first polypeptide consists of SEQ ID NO: 372 and the second polypeptide consists of SEQ ID NO: 380.
  • the multi-specific binding agent comprises (i) a first polypeptide comprising a first antigen-binding domain that binds 5T4 comprising a CDR1 of SEQ ID NO: 111, a CDR2 of SEQ ID NO: 112, and a CDR3 of SEQ ID NO: 113 and a first Fc domain monomer; and (ii) a second polypeptide comprising a second antigen-binding domain that binds to V ⁇ 2 and comprises a CDR1 of SEQ ID NO: 18, a CDR2 of SEQ ID NO: 19 and a CDR3 of SEQ ID NO: 20 and a second Fc domain monomer.
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 114 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 21.
  • the first antigen-binding domain comprises SEQ ID NO: 114 and the second antigen-binding domain comprises SEQ ID NO: 21.
  • the first antigen-binding domain consists of SEQ ID NO: 114 and the second antigen-binding domain consists of SEQ ID NO: 21.
  • the first Fc domain monomer comprises SEQ ID NO: 356 or 358 and the second Fc domain monomer comprises SEQ ID NO: 357 or 359.
  • the first Fc domain monomer comprises SEQ ID NO: 357 or 359 and the second Fc domain monomer comprises SEQ ID NO: 356 or 358.
  • the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 375 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 379.
  • the first polypeptide comprises SEQ ID NO: 375 and the second polypeptide comprises SEQ ID NO: 379.
  • the first polypeptide consists of SEQ ID NO: 375
  • the second polypeptide consists of SEQ ID NO: 379.
  • the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 374
  • the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 378.
  • the first polypeptide comprises SEQ ID NO: 374 and the second polypeptide comprises SEQ ID NO: 378. In some embodiments, the first polypeptide consists of SEQ ID NO: 374 and the second polypeptide consists of SEQ ID NO: 378.
  • the multi-specific binding agent comprises (i) a first polypeptide comprising a first antigen-binding domain that binds 5T4 comprising a CDR1 of SEQ ID NO: 111, a CDR2 of SEQ ID NO: 112, and a CDR3 of SEQ ID NO: 113 and a first Fc domain monomer; and (ii) a second polypeptide comprising a second antigen-binding domain that binds to V ⁇ 2 and comprises a CDR1 of SEQ ID NO: 18, a CDR2 of SEQ ID NO: 19 and a CDR3 of SEQ ID NO: 20 and a second Fc domain monomer.
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 114 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 22.
  • the first antigen-binding domain comprises SEQ ID NO: 114 and the second antigen-binding domain comprises SEQ ID NO: 22.
  • the first antigen-binding domain consists of SEQ ID NO: 114 and the second antigen-binding domain consists of SEQ ID NO: 22.
  • the first Fc domain monomer comprises SEQ ID NO: 356 or 358 and the second Fc domain monomer comprises SEQ ID NO: 357 or 359.
  • the first Fc domain monomer comprises SEQ ID NO: 357 or 359 and the second Fc domain monomer comprises SEQ ID NO: 356 or 358.
  • the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 375 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 381.
  • the first polypeptide comprises SEQ ID NO: 375 and the second polypeptide comprises SEQ ID NO: 381.
  • the first polypeptide consists of SEQ ID NO: 375
  • the second polypeptide consists of SEQ ID NO: 381.
  • the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 374
  • the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 380.
  • the first polypeptide comprises SEQ ID NO: 374 and the second polypeptide comprises SEQ ID NO: 380. In some embodiments, the first polypeptide consists of SEQ ID NO: 374 and the second polypeptide consists of SEQ ID NO: 380.
  • the multi-specific binding agent comprises (i) a first polypeptide comprising a first antigen-binding domain that binds 5T4 comprising a CDR1 of SEQ ID NO: 115, a CDR2 of SEQ ID NO: 116, and a CDR3 of SEQ ID NO: 117 and a first Fc domain monomer; and (ii) a second polypeptide comprising a second antigen-binding domain that binds to V ⁇ 2 and comprises a CDR1 of SEQ ID NO: 18, a CDR2 of SEQ ID NO: 19 and a CDR3 of SEQ ID NO: 20 and a second Fc domain monomer.
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 118 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 21.
  • the first antigen-binding domain comprises SEQ ID NO: 118 and the second antigen-binding domain comprises SEQ ID NO: 21.
  • the first antigen-binding domain consists of SEQ ID NO: 118 and the second antigen-binding domain consists of SEQ ID NO: 21.
  • the first Fc domain monomer comprises SEQ ID NO: 356 or 358 and the second Fc domain monomer comprises SEQ ID NO: 357 or 359.
  • the first Fc domain monomer comprises SEQ ID NO: 357 or 359 and the second Fc domain monomer comprises SEQ ID NO: 356 or 358.
  • the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 377 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 379.
  • the first polypeptide comprises SEQ ID NO: 377 and the second polypeptide comprises SEQ ID NO: 379.
  • the first polypeptide consists of SEQ ID NO: 377
  • the second polypeptide consists of SEQ ID NO: 379.
  • the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 376
  • the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 378.
  • the first polypeptide comprises SEQ ID NO: 376 and the second polypeptide comprises SEQ ID NO: 378. In some embodiments, the first polypeptide consists of SEQ ID NO: 376 and the second polypeptide consists of SEQ ID NO: 378.
  • the multi-specific binding agent comprises (i) a first polypeptide comprising a first antigen-binding domain that binds 5T4 comprising a CDR1 of SEQ ID NO: 115, a CDR2 of SEQ ID NO: 116, and a CDR3 of SEQ ID NO: 117 and a first Fc domain monomer; and (ii) a second polypeptide comprising a second antigen-binding domain that binds to V ⁇ 2 and comprises a CDR1 of SEQ ID NO: 18, a CDR2 of SEQ ID NO: 19 and a CDR3 of SEQ ID NO: 20 and a second Fc domain monomer.
  • the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 118 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 22.
  • the first antigen-binding domain comprises SEQ ID NO: 118 and the second antigen-binding domain comprises SEQ ID NO: 22.
  • the first antigen-binding domain consists of SEQ ID NO: 118 and the second antigen-binding domain consists of SEQ ID NO: 22.
  • the first Fc domain monomer comprises SEQ ID NO: 356 or 358 and the second Fc domain monomer comprises SEQ ID NO: 357 or 359.
  • the first Fc domain monomer comprises SEQ ID NO: 357 or 359 and the second Fc domain monomer comprises SEQ ID NO: 356 or 358.
  • the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 377 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 381.
  • the first polypeptide comprises SEQ ID NO: 377 and the second polypeptide comprises SEQ ID NO: 381.
  • the first polypeptide consists of SEQ ID NO: 377
  • the second polypeptide consists of SEQ ID NO: 381.
  • the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 376
  • the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 380.
  • the first polypeptide comprises SEQ ID NO: 376 and the second polypeptide comprises SEQ ID NO: 380. In some embodiments, the first polypeptide consists of SEQ ID NO: 376 and the second polypeptide consists of SEQ ID NO: 380. 338699-2168 Polynucleotides [0262] In some embodiments, the present disclosure provides a polynucleotide encoding any one of the 5T4 binding agents described herein.
  • the polynucleotide encodes a polypeptide sequence having at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOs: 92, 93, 94, 95, 96, 97, 101, 102, 103, 104, 108, 109, 110, 114, 118, 119, 120, 121, 128, 129, 130, 131, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, and 172.
  • sequence similarity or identity may be determined using the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math.2, 482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch J Mol. Biol. 48, 443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci.
  • WU-BLAST-2 uses several search parameters, which are optionally set to the default values.
  • the parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.
  • an additional useful algorithm is gapped BLAST as reported by Altschul et al, (1997) Nucleic Acids Res.25, 3389-3402. Unless otherwise indicated, percent identity is determined herein using the algorithm available at the internet address: blast.ncbi.nlm.nih.gov/Blast.cgi.
  • polynucleotide sequences described herein have been codon- optimized for improved expression. Without wishing to be bound by theory, it is believed that codon optimization of the nucleotide sequence increases the translation efficiency of the mRNA transcripts. Codon optimization of the nucleotide sequence may involve substituting a native codon for another codon that encodes the same amino acid, but can be translated by tRNA that is more readily available within a cell, thus increasing translation efficiency. 338699-2168 Optimization of the nucleotide sequence may also reduce secondary mRNA structures that would interfere with translation, thus increasing translation efficiency.
  • the polynucleotides provided by the present disclosure may be not naturally occurring or may have a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques, such as those described in Sambrook et al., supra.
  • the nucleic acids can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. See, for example, Sambrook et al., supra, and Ausubel et al. supra.
  • a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides).
  • modified nucleotides that can be used to generate the nucleic acids include, but are not limited to, 5-flurouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopenenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueo
  • the polynucleotides described herein comprise a nucleic acid sequence encoding a signal peptide at the 5’ end of the nucleic acid sequence encoding the binding agents described herein.
  • the signal peptide facilitates secretion of the encoded protein from a host cell (e.g., secretion of the 5T4 binding agents or multi- specific binding agents comprising the same).
  • a host cell e.g., secretion of the 5T4 binding agents or multi- specific binding agents comprising the same.
  • Signal peptides suitable for use according to the present disclosure are known in the art, for example signal peptides derived from GMCSF, the Igk -chain, CD8 ⁇ , CD33, or TPA. 338699-2168 Vectors [0268]
  • the present disclosure provides a vector comprising a polynucleotide encoding a 5T4 binding agent or multi-specific binding agent comprising the same.
  • the vector comprising a polynucleotide encoding a 5T4 binding agent or multi-specific binding agent comprising the same is a viral vector.
  • Illustrative viral vectors include, but are not limited to, adeno- associated virus (AAV), retrovirus (e.g., lentivirus), herpes simplex virus, adenovirus, and vaccinia virus.
  • AAV (rAAV) vectors are typically composed of, at a minimum, a transgene and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs).
  • the rAAV comprises ITRs and capsid sequences isolated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.
  • the ITR sequences of one AAV serotype and the capsid sequences of a different AAV serotype may be used to create a chimeric rAAV.
  • engineering and selection methods can be applied to AAV capsids to make them more likely to transduce cells of interest. Construction of rAAV vectors, production, and purification thereof have been disclosed, e.g., in U.S. Patent Nos. 9,169,494; 9,169,492; 9,012,224; 8,889,641; 8,809,058; and 8,784,799, each of which is incorporated by reference herein in its entirety.
  • Illustrative retroviruses suitable for use in particular embodiments include, but are not limited to: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV) and lentivirus.
  • M-MuLV Moloney murine leukemia virus
  • MoMSV Moloney murine sarcoma virus
  • HaMuSV Harvey murine sarcoma virus
  • MuMTV murine mammary tumor virus
  • GaLV gibbon ape leukemia virus
  • FLV feline leukemia virus
  • RSV Rous Sarcoma Virus
  • Illustrative lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2); visna-maedi virus (VMV) virus; the caprine arthritis- encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).
  • HIV human immunodeficiency virus
  • VMV visna-maedi virus
  • CAEV caprine arthritis- encephalitis virus
  • EIAV equine infectious anemia virus
  • FV feline immunodeficiency virus
  • BIV bovine immune deficiency virus
  • SIV simian immunodeficiency virus
  • a lentiviral vector contemplated herein comprises one or more LTRs, and one or more of the following accessory elements: a cPPT/FLAP, a Psi packaging signal, an export element, polyA sequences, and may optionally comprise a WPRE or HPRE, an insulator element, a selectable marker, and a cell suicide gene.
  • lentiviral vectors contemplated herein may be integrative or non-integrating or integration defective lentivirus (e.g., having an integrase that lacks the capacity to integrate the viral genome into the genome of the host cells. Integration-incompetent viral vectors have been described in patent application WO 2006/010834, which is herein incorporated by reference in its entirety.
  • expression of heterologous sequences in viral vectors is increased by incorporating post-transcriptional regulatory elements, efficient polyadenylation sites, and optionally, transcription termination signals into the vectors.
  • posttranscriptional regulatory elements can increase expression of a heterologous polynucleotide at the protein, e.g., woodchuck hepatitis vims posttranscriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); the posttranscriptional regulatory element present in hepatitis B vims (HPRE) (Huang et al., Mol. Cell.
  • the vector comprising a polynucleotide encoding a 5T4 binding agent or multi-specific binding agent comprising the same described herein is a plasmid.
  • plasmid Numerous suitable plasmid expression vectors are known to those of skill in the art, and many are commercially available. The following vectors are provided by way of example; for eukaryotic host cells: pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia).
  • any other plasmid vector may be used so long as it is compatible with the host cell.
  • any of a number of suitable transcription and translation control elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544).
  • a polynucleotide sequence encoding a 5T4 binding agent or multi-specific binding agent comprising the same is operably linked to a control element, e.g., a transcriptional control element, such as a promoter.
  • the transcriptional control element may be functional in either a eukaryotic cell (e.g., a mammalian cell) or a prokaryotic cell (e.g., bacterial or archaeal cell).
  • a polynucleotide sequence encoding a 5T4 binding agent or multi-specific binding agent comprising the same is operably linked to multiple control elements that allow expression of the polynucleotide in both prokaryotic and eukaryotic cells.
  • any of a number of suitable transcription and translation control elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see e.g., Bitter et al.
  • eukaryotic promoters include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, and mouse metallothionein-l. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • the expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector may also include appropriate sequences for amplifying expression.
  • the expression 338699-2168 vector may also include nucleotide sequences encoding protein tags (e.g., 6xHis tag, hemagglutinin tag, green fluorescent protein, etc.) that are fused to the 5T4 binding agent or multi-specific binding agent comprising the same, thus resulting in a chimeric polypeptide.
  • protein tags e.g., 6xHis tag, hemagglutinin tag, green fluorescent protein, etc.
  • a polynucleotide sequence encoding a 5T4 binding agent or multi-specific binding agent comprising the same is operably linked to an inducible or a constitutive promoter.
  • Transfection methods include calcium phosphate co-precipitation (see, e.g., Graham et al., supra), direct micro injection into cultured cells (see, e.g., Capecchi, Cell, 22: 479-488 (1980)), electroporation (see, e.g., Shigekawa et al., BioTechniques, 6: 742-751 (1988)), liposome mediated gene transfer (see, e.g., Mannino et al., BioTechniques, 6: 682-690 (1988)), lipid mediated transduction (see, e.g., Feigner et al., Proc. Natl. Acad. Sci.
  • the vectors provided by the present disclosure can be prepared using standard techniques described in, for example, Sambrook et al., supra, and Ausubel et al., supra.
  • Constructs of expression vectors which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell, Replication systems can be derived, e.g., from ColEl, 2 ⁇ plasmid, ⁇ , SV40, bovine papilloma virus, and the like.
  • the vector may comprise regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host cell (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate, and taking into consideration whether the vector is DNA- or RNA-based.
  • the expression vector may comprise restriction sites to facilitate cloning.
  • the vector can include one or more marker genes, which allow for selection of transformed or transfected host cells. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like. Suitable marker genes for the inventive expression vectors include, for instance, neomycin/G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes. [0280] The vectors can be designed for either transient expression, for stable expression, or for both, of the polynucleotides comprised therein.
  • compositions comprising the 5T4 binding agents and/or multi-specific binding agents described herein.
  • pharmaceutical compositions comprising the 5T4 binding agents and/or multi-specific binding agents described herein and a pharmaceutically-acceptable excipient.
  • formulations include all physiologically acceptable compositions including derivatives and/or prodrugs, solvates, stereoisomers, racemates, or tautomers thereof with any physiologically acceptable carriers, diluents, and/or excipients.
  • a “therapeutic composition” or “pharmaceutical composition” is a composition capable of being administered to a subject for the treatment of a particular disease or disorder.
  • pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, and/or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans and/or domestic animals.
  • Exemplary pharmaceutically acceptable carriers include, but are not limited to, to sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffins, silicones, bentonites, silicic acid, zinc oxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water
  • “Pharmaceutically acceptable salt” includes both acid and base addition salts.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2- disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid,
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like.
  • Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methyl
  • organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.
  • Wetting agents, emulsifiers, and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl 338699-2168 palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl 338699-2168 palmitate, butylated hydroxyanisole (BHA), butylated hydroxy
  • the present disclosure provides a method of producing the 5T4 binding agents and multi-specific binding agents comprising the same comprising introducing a polynucleotide encoding the 5T4 binding agent or a vector comprising the same into a host cell, culturing the host cell under conditions suitable for expression of the 5T4 binding agent, and purifying the expressed 5T4 binding agent from the culture supernatant.
  • a host cell can be a eukaryotic cell, e.g., plant, animal, fungi, or algae, or can be a prokaryotic cell, e.g., bacteria or protozoa.
  • the host cell can be a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human.
  • the host cell can be an adherent cell or a suspended cell, i.e., a cell that grows in suspension.
  • Suitable host cells are known in the art and include, for instance, DK5 ⁇ E.
  • the host cell may be a prokaryotic cell, e.g., a DH5 ⁇ cell.
  • the host cell may be a mammalian cell.
  • the present disclosure provides a host cell comprising a polynucleotide described herein or vector comprising the same.
  • Antibodies can be produced by transgenic mice that are transgenic for specific heavy and light chain immunoglobulin genes. Such methods are known in the art and described in, for example U.S. Patents 5,545,806 and 5,569,825, and Janeway et al., supra.
  • Multi-specific binding agents disclosed herein are typically produced recombinantly, i.e., by expression of polynucleotides encoding the multi-specific binding agents in suitable host cells, followed by purification of the produced recombinant multi-specific binding agent from the cell culture.
  • Polynucleotides can be produced by standard molecular biological techniques well-known in the art.
  • the polynucleotides are typically introduced into the host cell using an expression vector. Suitable nucleic acid constructs and expression vectors are known in the art.
  • Host cells suitable for the recombinant expression of antibodies are well- known in the art, and include CHO (Chinese Hamster Ovary), HEK-293, Expi293F, PER-C6, NS/0 and Sp2/0 cells.
  • CHO Choinese Hamster Ovary
  • HEK-293 HEK-293
  • Expi293F HEK-293
  • Expi293F HEK-293
  • Expi293F HEK-293
  • Expi293F HEK-293
  • Expi293F HEK-293
  • Expi293F HEK-293
  • Expi293F HEK-293
  • Expi293F HEK-293
  • Expi293F HEK-293
  • Expi293F HEK-293
  • Expi293F HEK-293
  • Expi293F HEK-293
  • Expi293F
  • expression vectors comprising polynucleotides that encode multi-specific binding agents as disclosed herein.
  • the expression vector typically further comprises a promoter.
  • host cells such as a non-human host cell, for example a CHO cell, that comprise one or more polynucleotides that encode a multi- specific binding agent as disclosed herein or an expression vector comprising a polynucleotide encoding a multi-specific binding agent as disclosed herein.
  • the present disclosure provides a method of treating cancer in a subject in need thereof comprising administering a 5T4 binding agent described herein, a multi-specific binding agent comprising the same, or a pharmaceutical composition comprising the same.
  • treatment comprises delivering an effective amount of a 5T4 binding agent or multi-specific binding agent comprising the same, or a pharmaceutical composition thereof, to a subject in need thereof.
  • treating refers to the treatment of a cancer in a mammal, e.g., in a human, including (a) inhibiting the cancer, i.e., arresting cancer development or preventing cancer progression; (b) relieving the cancer, i.e., causing regression of the cancer state or relieving one or more symptoms of the cancer; and (c) curing the cancer, i.e., remission of one or more cancer symptoms.
  • treatment may refer to a short-term (e.g., temporary and/or acute) and/or a long-term (e.g., sustained) reduction in one or more cancer symptoms.
  • treatment results in an improvement or remediation of the symptoms of the cancer.
  • an “effective amount” or “an amount effective to treat” refers to a dose that is adequate to prevent or treat cancer in an individual. In some embodiments, an effective amount is also one in which any toxic or detrimental effects of the antibody are outweighed by the therapeutically beneficial effects.
  • the effective amount of a 5T4 binding agent or multi-specific binding agent, or a pharmaceutical composition comprising the same administered to a particular subject will depend on a variety of factors, several of which will differ from patient to patient including the disorder being treated and the severity of the disorder; activity of the specific agent(s) employed; the age, body weight, general health, sex and diet of the patient; the timing of administration, route of administration; the duration of the treatment; the ability of the binding agent or multi-specific binding agent comprising the same to elicit a desired response in the individual; drugs used in combination; the judgment of the prescribing physician; and like factors known in the medical arts.
  • the size of the dose will also be determined by the existence, nature, and extent of any adverse side- effects that might accompany the administration of a particular active, and the desired physiological effect. It will be appreciated by one of skill in the art that various diseases or disorders could require prolonged treatment involving multiple administrations, perhaps using various rounds of administration.
  • the cancer can be any cancer, including any of acute lymphocytic cancer, acute myeloid leukemia (AML), alveolar rhabdomyosarcoma, B-cell chronic lymphoproliferative disorders, bladder cancer (e.g., bladder carcinoma), blastic plasmacytoid dendritic cell neoplasm, bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, extrahepatic bile duct , cancer of the joints, cancer of the head and neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, 338699-2168 chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and
  • the cancer is colorectal cancer (CRC).
  • the colorectal cancer is metastatic colorectal cancer (mCRC).
  • the colorectal cancer is primary colorectal cancer (pCRC).
  • the cancer is clear cell renal cell cancer.
  • the cancer is glioblastoma multiforme (GBM).
  • the cancer is cervical cancer.
  • the cancer is gastric cancer.
  • the cancer is head and neck cancer.
  • the cancer is urothelial cell carcinoma.
  • the cancer is melanoma.
  • the cancer is characterized by the expression of 5T4.
  • the cancer is selected from colorectal cancer, primary colorectal cancer, metastatic colorectal cancer, clear cell renal cell cancer, glioblastoma multiforme (GBM), cervical cancer, cervical squamous cell carcinoma, cervical adenocarcinoma, gastric cancer, head and neck cancer, urothelial cell carcinoma, melanoma, kidney cancer, breast cancer, colon cancer, prostate cancer, non-small lung cancer, lung cancer, pancreatic cancer, mesothelioma, pre-B acute lymphoblastic leukemia, hepatocellular, and ovarian cancer.
  • GBM glioblastoma multiforme
  • cervical cancer cervical squamous cell carcinoma
  • cervical adenocarcinoma gastric cancer
  • head and neck cancer urothelial cell carcinoma
  • melanoma kidney cancer
  • breast cancer colon cancer
  • prostate cancer non-small lung cancer
  • lung cancer pancreatic cancer
  • “Treatment” or “treating” refers to the administration of an effective amount of a binding agent or multi-specific binding agent comprising the same according to the present disclosure with the purpose of easing, ameliorating, arresting, eradicating (curing) or preventing symptoms or disease states.
  • the subject may be a neonate, a juvenile, or an adult.
  • Mammalian species that may be treated with the present methods include canines and felines; equines; bovines; ovines; etc. and primates, 338699-2168 particularly humans.
  • the present disclosure provides methods of expanding V ⁇ 9V ⁇ 2 T cells comprising contacting the V ⁇ 9V ⁇ 2 T cells with a 5T4 binding agent described herein, a multi-specific binding agent comprising the same, or a pharmaceutical composition comprising the same.
  • the contacting is in vitro, in vivo, or ex vivo.
  • Expansion of V ⁇ 9V ⁇ 2 T cells may be measured by any method known in the art (e.g., labeling with a suitable agent such as carboxyfluorescein succinimidyl ester followed by quantification of fluorescence by e.g., flow cytometry).
  • a suitable agent such as carboxyfluorescein succinimidyl ester followed by quantification of fluorescence by e.g., flow cytometry.
  • Administration of the 5T4 binding agent or multi-specific binding agent, or of a pharmaceutical composition comprising the same can occur by injection, irrigation, inhalation, consumption, electro-osmosis, hemodialysis, iontophoresis, and other methods known in the art.
  • administration route is local or systemic.
  • administration route is intraarterial, intracranial, intradermal, intraduodenal, intramammary, intrameningeal, intraperitoneal, intrathecal, intratumoral, intravenous, intravitreal, ophthalmic, parenteral, spinal, subcutaneous, ureteral, urethral, vaginal, intramuscular, inhalation, oral, or intrauterine.
  • the administration will be parenteral.
  • the administration route is by infusion (e.g., continuous or bolus). Examples of methods for local administration, that is, delivery to the site of injury or disease, include through an Ommaya reservoir, e.g.
  • the administration route is by topical administration or direct injection.
  • the binding agents described herein may be provided to the subject alone or with a suitable substrate or matrix.
  • at least 0.01 ⁇ g/kg of the 5T4 binding agent or multi-specific binding agent, or of a pharmaceutical composition comprising the same is administered to a subject.
  • between about 0.01 ⁇ g/kg and about 100mg/kg, between about 0.1 ⁇ g/kg and about 50 mg/kg, between about 0.01 mg/kg and about 20 mg/kg, or between about 0.01 mg/kg and about 10 mg/kg of the 5T4 binding agent or multi-specific binding agent, or of a pharmaceutical composition comprising the same is administered to a subject.
  • At least about 0.01 mg/kg, at least about 0.03 mg/kg, at least about 0.05 mg/kg, at least about 0.1 mg/kg, about 0.3 mg/kg, about 0.5 mg/kg, about 1 mg/kg, 338699-2168 about 3 mg/kg, about 5 mg/kg, about 8 mg/kg, or about 10 mg/kg of the 5T4 binding agent or multi-specific binding agent, or of a pharmaceutical composition comprising the same, is administered to the subject.
  • administration to a subject may vary.
  • administration to the subject may be a one-time event.
  • such treatment may require an on-going series of repeated treatments.
  • multiple administrations may be required before an effect is observed.
  • the exact protocols depend upon the disease or condition, the stage of the disease and parameters of the individual subject being treated.
  • the effective amount of a 5T4 binding agent, a multi-specific binding agent comprising the same, or a pharmaceutical composition comprising the same may be the amount required to result in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more fold decrease in tumor mass or volume, decrease in the number of tumor cells, or decrease in the number of metastases.
  • the effective amount of a 5T4 binding agent, a multi-specific binding agent comprising the same, or a pharmaceutical composition comprising the same may be the amount required to achieve an increase in life expectancy, an increase in progression-free or disease-free survival, or amelioration of various physiological symptoms associated with the disease being treated.
  • the methods of treatment provided herein further comprise the administration of one or more additional therapeutic agents.
  • the one or more additional therapeutic agents can be administered prior to, simultaneously with, or after the 5T4 binding agent or multi-specific binding agent comprising the same, or composition comprising the same described herein.
  • the additional therapeutic agent is IL-2, IL-15, or variants thereof.
  • the additional therapeutic agent is IL-2.
  • the IL-2 is Aldesleukin/Proleukin.
  • IL-2 examples include NKTR-214 (Bempegaldesleukin/Nektar), ALK 4230 (nemvaleukin/Aalkermes-Reliant), SAR444245 – THOR 707/Synthorx/Sanofi) and XTX-202 (Xilio).
  • the IL-2 is a variant IL-2.
  • the variant IL-2 binds to the IL-2Rbeta and gamma chains, but 338699-2168 does not bind or demonstrates reduced/abrogated binding to the IL-2Ralpha chain (e.g., NL201 from Neoleukin, Silva et al.
  • the IL-2 and/or variants thereof is pegylated.
  • the additional therapeutic agent is IL-15. Suitable forms of IL- 15 are known in the art. See e.g., Int J Mol Sci.2022 Jul; 23(13): 7311, which is herein incorporated by reference in its entirety.
  • the IL-15 is a heterodimer of IL-15 and IL-15 receptor alpha (hetIL-15), which is also referred to as NIZ985.
  • the IL-15 is N-803, formerly known as ALT-803, which is an IL-15 variant complexed with a human IL-15R ⁇ sushi domain-Fc fusion protein.
  • the IL-15 is SOT101, also referred to as Nanrilkefusp alfa, which is a human fusion protein comprising the cytokine IL-15 and the high-affinity binding sushi+ domain of IL-15 receptor alpha (IL-15R ⁇ ).
  • the IL-15 is NKTR-255, which is a polyethylene glycol- conjugate of rhIL-15.
  • the IL-15 and/or variants thereof is pegylated.
  • the 5T4 binding agent or multi-specific binding agent comprising the same is administered as monotherapy.
  • 5T4 binding agents or multi-specific binding agents disclosed herein may also be administered in combination therapy, i.e., combined with other therapeutic agents relevant for the disease or condition to be treated.
  • the combination therapy comprises administration of a 5T4 binding agent or multi-specific binding agent comprising the same and an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor is an anti PD-1 antibody (e.g., Pembrolizumab (Keytruda®), nivolumab (Opdivo®)), an anti-PDL1 antibody (e.g., Atezolizumab (Tecentriq®), Avelumab (Bavencio®), Durvalumab (Imfinzi®)), or an anti- CTLA4 antibody (e.g., Ipilimumab (Yervoy®)).
  • PD-1 antibody e.g., Pembrolizumab (Keytruda®), nivolumab (Opdivo®)
  • an anti-PDL1 antibody e.g., Atezolizumab (Tecentriq®), Avelumab (Bavencio®), Durvalumab (Imfinzi®)
  • an anti- CTLA4 antibody e.g., Ipilimumab (Yervoy®
  • Lama glama were immunized with either MCF7 cells or A-431 cells followed by 2 injections of recombinant human 5T4.
  • Whole blood samples were collected from the 338699-2168 immunized animals for peripheral blood lymphocyte (PBL) isolation to generate phage libraries.
  • PBL peripheral blood lymphocyte
  • the purified PCR product was digested with appropriate restriction enzymes, after which the digested products were ligated in frame with geneIII into the pQ81 phagemid vector.
  • the ligated library was transformed into TG1 E. coli by electroporation for display on filamentous bacteriophage. Two consecutive rounds of selection were performed, followed by screening single clones for binding to plate-bound recombinant human 5T4. Those clones that scored positive for binding were sequenced and a selection of clones having different sequences were then tested for binding to recombinant human 5T4 by ELISA.
  • LV1138 (SEQ ID NO: 101), LV1139 (SEQ ID NO: 92), LV1140 (SEQ ID NO: 108), LV1141 (SEQ ID NO: 114), LV1142 (SEQ ID NO: 118), LV1143 (SEQ ID NO: 128), LV1144 (SEQ ID NO: 136), LV1145 (SEQ ID NO: 140), LV1146 (SEQ ID NO: 144), LV1147 (SEQ ID NO: 148), LV1148 (SEQ ID NO: 152), LV1149 (SEQ ID NO: 156), LV1150 (SEQ ID NO: 160), LV1151 (SEQ ID NO: 164), LV1152 (SEQ ID NO: 168), and LV1292 (SEQ ID NO: 172).
  • Example 2 Affinity determination of VHHs for binding to human 5T4 using biolayer interferometry (BLI) [0320]
  • biotinylated recombinant purified human 5T4 protein (Sino Biological) was loaded onto SAX biosensors (Sartorius) at a concentration of 2.5 ⁇ g/mL for an Octet Red96e (Sartorius) instrument.
  • SAX biosensors Sartorius
  • the sensors were then dipped in different concentrations of the various 5T4 binding agents diluted in 10x kinetic buffer (10xKB) provided by the supplier.
  • Table 10 shows the binding affinities (K D ) of the binding agents determined in two independent runs. The remainder of the binding agents did not show binding to (biotinylated) 5T4. 338699-2168 Table 10: Binding affinities (KD) of binding agents for human 5T4 determined in 2 independent BLI runs.
  • SAX biosensors were loaded with biotinylated human 5T4 protein as described in Example 2.
  • the eight different binding agents identified in the previous example (LV1138, LV1139, LV1140, LV1141, LV1142, LV1143, LV1144, LV1150) were used in concentrations based on the affinities as indicated above.
  • a fixed amount of the first binding agent was allowed to bind to the 5T4 loaded sensor, after which the sensor was exposed to a mixture of the same amount of the first binding agent and a fixed amount of a second binding agent, followed by dissociation in 10X KB.
  • Example 4 Synthetic gene synthesis, production, and purification of multi-specific binding agents [0324]
  • the sequences of four 5T4 binding agents (LV1138, LV1139, LV1140, and LV1142) were then re-formatted into multi-specific binding agents by linking the 5T4 binding agent to a V ⁇ 2 binding agent (5C8 – SEQ ID NO:5) in the following orientation, from N- to C-terminus: 5T4 binding agent – linker– V ⁇ 2 binding agent.
  • the linker between the two VHHs was a glycine(G)-serine(S) stretch with the sequence G 4 S (SEQ ID NO: 341).
  • the cDNAs encoding 338699-2168 these proteins were prepared by synthetic gene assembly and cloned into a proprietary expression vector. The proteins were then expressed by transient transfection of the modified expression vector into HEK293E-253 cells. The multi-specific binding agents were then purified from the conditioned cell culture supernatant using HiTrap Fibro PrismA columns according to the supplier’s protocol. After elution from the column, the multi-specific binding agents were buffer-exchanged to PBS. Purified multi-specific binding agents were always >95% pure as determined by SDS-PAGE analysis using Coomassie staining and contained very low levels of endotoxin ( ⁇ 0.05 EU/mg).
  • Example 5 Binding of 5T4xV ⁇ 2 multi-specific binding agents to human, cynomolgus monkey and mouse 5T4 expressing cells using cell-based ELISA
  • Protein sequences of human (Uniprot number Q13641), cynomolgus monkey (Uniprot number Q4R8Y9) and mouse (Uniprot number Q9Z0L0) 5T4 were reverse-translated into cDNA and then codon-optimized for expression in human cells.
  • Regulatory elements were added to each cDNA, namely an N-terminal Kozak sequence and C-terminal stop codon (including BamH1 and Age1 restriction sites for cloning) and the cDNA was made into a synthetic gene.
  • cDNAs were then cloned into a suitable vector and the sequences were verified. Expression of the proteins was performed by transient transfection of the resulting plasmids in CHO-K1 cells. Expression of human/cynomolgus 5T4 was confirmed using commercially available control antibody 5T4-AF647(R&D systems) in flow cytometry, and expression of mouse 5T4 was confirmed by expression of the eGFP-tag incorporated in the expression vector.
  • FIG. 4A-FIG. 4C show that all four 5T4xV ⁇ 2 multi-specific binding agents bound to CHO-K1 cells expressing human 5T4.
  • the EC 50 for each of the multi-specific binding agents to human 5T4 is shown in Table 11. None of the multi-specific VHHs specifically bound to non- transfected CHO-K1 cells (not shown), or to CHO-K1 cells expressing mouse or cynomolgus monkey 5T4.
  • Table 11 EC50 values for the indicated 5T4 multi-specific binding agents to human 5T4 Multi-specific binding agent EC 50 LV1138xV ⁇ 2 (SEQ ID NO: 364) 3.13 nM LV1139xV ⁇ 2 (SEQ ID NO: 362) 1.51 nM LV1140xV ⁇ 2 (SEQ ID NO: 365) 6.11 nM LV1142xV ⁇ 2 (SEQ ID NO: 363) 3.03 nM
  • Example 6 Binding of 5T4xV ⁇ 2 multi-specific binding agents to 5T4 expressing human tumor cell lines [0328] Based on the results in Example 5, the 5T4 binders were further characterized.
  • FIG.5A and FIG.5B show that LV1139xV ⁇ 2 and LV1142xV ⁇ 2 bound to both cell lines with high affinity as seen in Table 12. LV1138xV ⁇ 2 and LV1140xV ⁇ 2 also bound to the tumor cell lines, yet with lower affinity.
  • Table 12 EC 50 values for the LV1139xV ⁇ 2 and LV1142xV ⁇ 25T4 multi-specific binding agents to 5T4-expressing tumor cells
  • Tumor cell line EC 50 LV1139xV ⁇ 2
  • EC 50 LV1142xV ⁇ 2 U-251 2.5 nM 1.8 nM
  • A-431 1.9 nM 1.7 nM
  • Example 7 PBMC isolation and generation of human donor-derived V ⁇ 9V ⁇ 2-T cell cultures [0332] Buffy coats were obtained from blood supply service Sanquin and used for isolation of peripheral blood mononuclear cells (PBMC). PBMC were isolated using Lymphoprep TM density gradient centrifugation.
  • V ⁇ 9V ⁇ 2-T cells were then isolated from healthy donor-derived PBMC by magnetic-activated cell sorting (MACS) using a FITC-labeled anti-TCR V ⁇ 2 mouse 338699-2168 monoclonal antibody (Mab) in combination with goat anti-mouse IgG microbeads.
  • Purified V ⁇ 9V ⁇ 2-T cells were stimulated with a feeder cell mix consisting of irradiated PBMC from two (other) healthy donors and an Epstein Barr Virus transformed B cell line (JY) resuspended in Roswell Park Memorial Institute (RPMI) medium supplemented with 10 IU/mL IL-7, 10 ng/mL IL-15 and 50 ng/ml PHA.
  • RPMI Roswell Park Memorial Institute
  • V ⁇ 9V ⁇ 2-T cell cultures were tested for purity (and only used when >90% pure) before being used for experiments.
  • Example 8 Binding of 5T4xV ⁇ 2 multi-specific binding agents to V ⁇ 9V ⁇ 2 T cells [0333] Binding of the 5T4xV ⁇ 2 bispecific binding agents to human V ⁇ 9V ⁇ 2 T cells was determined by flow cytometry as described above. FIG. 6 shows that all 45T4xV ⁇ 2 multi- specific binding agents bound to V ⁇ 9V ⁇ 2 T cells with similar EC 50 s of 1-1.8 nM.
  • Example 9 V ⁇ 9V ⁇ 2-T cell degranulation and cytotoxicity of tumor cell lines induced by 5T4xV ⁇ 2 multi-specific binding agents [0334]
  • U-251 or A-431 tumor target cells and V ⁇ 9V ⁇ 2 T cells from normal donors were mixed in a 1:1 ratio in culture medium with labeled anti-CD107a antibody.
  • This mixture was seeded in 96-well culture plates (with 50,000 target/V ⁇ 9V ⁇ 2 T cells per well) and serial dilutions of 5T4xV ⁇ 2 multi-specific binding agents were added.
  • Tumor cells and V ⁇ 9V ⁇ 2-T cells alone with, and without, the highest concentration of 5T4xV ⁇ 2 multi-specific binding agents were also prepared as controls. After incubation for 24 hours at 37°C/5% CO 2 , cells were resuspended, washed, and transferred to 96-well U bottom FACS plates. Remaining adherent cells were detached using trypsin/0.5% EDTA, washed and transferred to the corresponding wells of the FACS plates. The plates were washed and the cells were resuspended in FACS buffer (PBS/1% BSA) and incubated with labeled anti-CD3 (Biolegend) and anti-V ⁇ 9 (Beckman Coulter) antibodies for 30 minutes at 4°C.
  • FACS buffer PBS/1% BSA
  • FIGs.7A-FIG. 8H show that all four 5T4xV ⁇ 2 multi-specific binding agents induced similar levels of V ⁇ 9V ⁇ 2 T cell degranulation as determined by CD107a expression in all of the donor V ⁇ 9V ⁇ 2-T cells. Surprisingly, despite differences in binding to tumor cells (See Example 6), all four 5T4xV ⁇ 2 bispecific VHHs induced a comparable level of lysis of tumor cells with similar range of EC50s (See Table 13 and Table 14).
  • Table 13 EC 50 values for V ⁇ 9V ⁇ 2-T cell degranulation and cytotoxicity of U-251 cells induced by 5T4 multi-specific binding agents (nM) LV1138xV ⁇ 2 LV1139xV ⁇ 2 LV1140xV ⁇ 2 LV1142xV ⁇ 2 Donor Degranulation 0.0046 0.0030 0.0115 0.0064 1 Cytotoxicity 0.0032 0.0039 0.0070 0.0059 Donor Degranulation 0.0050 0.0030 0.0095 0.0070 2 Cytotoxicity 0.0037 0.0019 0.0074 0.0055 Donor Degranulation 0.0056 0.0050 0.0162 0.0119 3 Cytotoxicity 0.0036 0.0033 0.0106 0.0045 Donor Degranulation 0.0037 0.0037 0.0124 0.0047 4 Cytotoxicity 0.0051 0.0060 0.0199 0.0059 Table 14: EC 50 values for V ⁇ 9V ⁇ 2-T cell degranulation and cytotoxicity of A-431 cells induced by 5T4 multi-specific binding agents LV1138
  • V ⁇ 9V ⁇ 2-T cells After enzymatic and mechanical dissociation, the presence of V ⁇ 9V ⁇ 2-T cells and the expression of 5T4 on tumor cells was determined by flow cytometry. All of the samples contained V ⁇ 9V ⁇ 2-T cells (varying between 0.18 to 2.42 of total T cells present in the sample) and 5T4-expressing tumor cells (data not shown).
  • V ⁇ 9V ⁇ 2-T cell activation and tumor cytotoxicity against patient-derived target cells dissociated tumor-suspensions were co- cultured with expanded allogeneic V ⁇ 9V ⁇ 2-T cells in a 1:2 effector:target (E:T) ratio for 18 hours or 7 days at 37°C in the absence or presence of 10 nM of the 5T4-multi-specific binding agent.
  • E:T effector:target
  • FIG. 9A-FIG. 9D show that V ⁇ 9V ⁇ 2 T cell degranulation as measured by CD107a expression was induced by all of the tested 5T4-multi-specific binding agents in patient- derived cervical squamous cell carcinoma (FIG. 9A), cervical adenocarcinoma (FIG. 9B), metastatic colorectal cancer (FIG.9C), and glioblastoma multiforme (FIG.9D).
  • FIG.10A-FIG.10E show that tumor cell lysis was induced by all of the tested 5T4- multi-specific binding agents in patient-derived cervical squamous cell carcinoma at 18 hours (FIG.10A) and 7 days (FIG.10B), cervical adenocarcinoma at 18 hours (FIG.10C) and 7 days (FIG.10D), and in glioblastoma multiforme at 18 hours (FIG.10E).
  • Example 11 Humanization of the anti-5T4 VHHs [0341]
  • the amino acid sequence of the llama-derived 5T4 binding agents (LV1138, LV1139, LV1140 and LV1142) were aligned to the human V gene database and the closest match to a human germline sequence were identified.
  • 5T4 Percentage identity Percentage identity Human germline VHH full sequence, including (framework only, IMGT CDR1/2) numbering
  • IGHV3-23*04 LV1140 86%
  • IGHV3-48*03 LV1142 74% 78% IGHV3-66*01
  • Two or three humanized variants of each of the 5T4 binding agents were designed based on sequence differences in the framework regions between the human and llama- derived sequence (SEQ ID NOs: 94, 95, 96, 102, 103, 104, 109, 110, 119, 120, and 121). Framework residues close to the CDRs were not changed.
  • FIG.1A shows the expression of 5T4 on freshly isolated patient tumor samples and FIG.2 shows the expression of 5T4 in non-malignant urothelial cells as measured by the mean fluorescence index (MFI) (geometric (g)MFI stained/gMFI isotype).
  • FIG. 1B shows the percentage of V ⁇ 9 + V ⁇ 2 + T cells out of total CD3 + T cells in different cancer tissues.
  • FIG. 11A-FIG. 11B there was an increase in both V ⁇ 9V ⁇ 2 T cell degranulation (as measured by percent increase in CD107a expression (FIG.11A)) and tumor cell lysis (FIG. 11B) in tumor samples incubated with allogeneic V ⁇ 9V ⁇ 2 T cells in the presence of LAVA-378 as opposed to the tumor samples that were only incubated with allogeneic V ⁇ 9V ⁇ 2 T cells.
  • a multiple paired T test was used to test for significance.
  • Non-cancerous and cancerous urothelial cells were incubated with V ⁇ 9V ⁇ 2 T cells and LAVA-378. As shown in FIG.12A and FIG.12B, the addition of LAVA-378 led to an increase in urothelial cancer cell lysis but did not affect non-malignant urothelial cell lysis. The multiple paired T test was used to test for significance.
  • FIG.14A shows the 5T4 expression on the patient samples.
  • FIG.14B shows that both LAVA-378 and LAVA-380 induced V ⁇ 9V ⁇ 2 T cell degranulation.
  • Example 15 In vivo testing of the 5T4xV ⁇ 2 multi-specific binding agents [0349] The anti-tumor effect of the multi-specific binding agents are tested in vivo. Mice are inoculated with 5T4 + solid tumor cells.
  • mice are then split into different treatment groups: 1) V ⁇ 9V ⁇ 2-T cells; 2) V ⁇ 9V ⁇ 2-T cells + 0.1 mg/kg LAVA-1433, comprising SEQ ID NOs: 367 and 381 (LV1139 (E1D) x 5C8 var1 (E1D-Y105F-R109A); 3) V ⁇ 9V ⁇ 2-T cells + 0.5 mg/kg LAVA-1433; 4) V ⁇ 9V ⁇ 2-T cells + 1 mg/kg LAVA-1433; 5) V ⁇ 9V ⁇ 2-T cells + 2 mg/kg LAVA- 1433; 6) 0.1 mg/kg LAVA-1433; 7) 0.5 mg/kg LAVA-1433; 8) 1 mg/kg LAVA-1433; 9) 2 mg/kg LAVA-1433; and 10) vehicle.
  • Example 16 Modifications to binding agents to prevent pyroglutamate formation
  • Pyroglutamate formation at the first amino acid position in proteins increases heterogeneity and can reduce the stability of the proteins at 2-8°C, therefore requiring storage at -20°C as opposed to storage at 2-8°C.
  • a multi-specific binding agent was generated wherein aspartic acid, which cannot form pyroglutamate, was substituted for glutamic acid at the first N-terminal amino acid position. Pyroglutamination increases the hydrophobicity of a protein.
  • reverse phase-HPLC was used to determine the presence of pyroglutamination, as reverse phase-HPLC separates proteins based on hydrophobicity. More hydrophilic protein species elute at shorter retention times and more hydrophobic species elute at longer retention times.
  • CHO Chinese hamster ovary
  • LAVA-1395 forms pyroglutamate
  • LAVA-1433 cannot form pyroglutamate
  • the proteins were then purified and analyzed by reversed phase-HPLC. Protein samples were loaded onto a liquid chromatography system equipped with a reversed-phase column. Organic solvents were used for binding and eluting of proteins.
  • results are shown in FIG.15A and FIG.15B.
  • the main species of LAVA-1395 and LAVA-1433 exhibit essentially identical hydrophobicities as demonstrated by their highly similar retention times.
  • the LAVA-1395 chromatogram (Fig.15A) displays a small peak that elutes around 30 seconds after the main species. This peak carries mainly the N-terminal modification pyroglutamate.
  • the N-terminal aspartate that is present in LAVA-1433 cannot undergo such a modification since ring formation is hampered by the shorter sidechains. Consequently, no hydrophobic pyroglutamation peak is detected for LAVA-1433 (FIG.15B).

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Public Health (AREA)
  • Cell Biology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Animal Behavior & Ethology (AREA)
  • Peptides Or Proteins (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The present disclosure relates 5T4 binding agents, multi-specific binding agents thereof, polynucleotides encoding the same, and pharmaceutical compositions comprising the same. The present disclosure further provides methods of treating cancer in a subject in need thereof comprising administration of the 5T4 binding agents and multi-specific binding agents comprising the same to a subject in need thereof.

Description

338699-2168 5T4 BINDING AGENTS AND METHODS OF USE CROSS- REFERENCE TO RELATED APPLICATIONS [0001] The present application claims priority to U.S. Provisional Application 63/525,435, filed July 7, 2023, the content of which is herein incorporated by reference in its entirety. REFERENCE TO SEQUENCE LISTING [0002] The contents of the electronic sequence listing (filename: LVAT_028_01WO_SeqList_ST26.XML; date of creation: July 3, 2024; file size 348,703 bytes) are herein incorporated by reference in its entirety. FIELD [0003] The present disclosure provides 5T4 binding agents and multi-specific binding agents comprising the same. The present disclosure further provides methods of treating cancer in a subject in need thereof comprising administration of the 5T4 binding agents and multi-specific binding agents comprising the same to a subject in need thereof. BACKGROUND [0004] 5T4, also known as Trophoblast glycoprotein (TPBG) and Wnt-Activated Inhibitory Factor 1 (WAIF1), is a single-pass transmembrane protein first identified in human placental tissues. 5T4 inhibits Wnt/β-catenin signaling, a signaling system central to many developmental and pathological processes.5T4 is rarely expressed in normal adult tissues, but is present in placenta and in many common tumors. For example, 5T4 is expressed in more than 80% of kidney, breast, colon, prostate, and ovarian cancers. [0005] There remains a need in the art for 5T4-targeting therapeutics for use in the treatment of a variety of cancers. SUMMARY [0006] 5T4 has been investigated as an antigen for therapeutic vaccination. The attenuated vaccinia virus modified vaccinia Ankara (MVA) was engineered to deliver the tumor antigen 5T4 (TroVax). The efficacy of the vaccine was tested in patients suffering from metastatic renal cancer, but no difference in survival was observed in the overall study population (Amato et al. (2010) Clin Cancer Res 16(22): 5539). [0007] 5T4-targeting antibodies have also been described previously. WO13041687 (Amgen), WO16097408 (Chiome Bioscience) and WO21048423 (Genmab) describe bispecific CD3-binding T-cell engaging antibodies that target 5T4. Bispecific T-cell engaging antibodies have a tumor target binding specificity and a CD3-directed T-cell binding specificity and thus boost tumor immunity by re-directing T-cell cytotoxicity to malignant cells, see e.g., 338699-2168 Huehls et al. (2015) Immunol Cell Biol 93:290; Ellerman (2019) Methods, 154:102. However, reported results with CD3-binding bispecific antibodies vary significantly. For example, in one study in which a CD3 binding moiety was combined with binding moieties against 8 different B-cell targets (CD20, CD22, CD24, CD37, CD70, CD79b, CD138 and HLA-DR), it was found that the bispecific antibodies targeting the different tumor targets showed strong variation in their capacity to induce target cell cytotoxicity and that the cytotoxicity did not correlate with antigen expression levels. For example, CD3-based bispecific antibodies targeting HLA-DR or CD138 were not able to induce cytotoxicity, in spite of intermediate to high HLA-DR and CD138 expression levels (Engelberts et al. (2020) Ebiomedicine 52:102625). Few T-cell redirecting therapies have reached late-stage clinical development, possibly due to significant toxicity, manufacturing problems, immunogenicity, a narrow therapeutic window, and low response rates. In particular, toxicity has been observed to occur when the T-cell engager includes a CD3 binding arm and treatment results in uncontrolled and exaggerated immune activation and cytokine release. Indeed, even though the first bispecific 5T4-CD3 antibodies were available already in 2012 (WO13041687), none of these antibodies have made significant progress through clinical development. Some have even been discontinued due to a narrow or absent therapeutic window. See for example ClinicalTrials.gov ID NCT04424641. [0008] Thus, while significant progress has been made, there is still a need for novel 5T4- targeting antibodies or binding agents that have improved therapeutic efficacy and specificity against a broad range of (target) tumor cells yet have acceptable toxicity, as well as good stability, developability and manufacturability properties. [0009] The present disclosure provides binding agents comprising a 5T4 antigen-binding domain. The present disclosure further provides multi-specific binding agents comprising a 5T4 antigen-binding domain and a γδ-T cell antigen-binding domain. In some embodiments, such multi-specific binding agents target tumor cells to activated γδ-T cells. [0010] Accordingly, in a first aspect, disclosed herein are multi-specific binding agents comprising a first antigen-binding domain capable of binding human 5T4 and a second antigen-binding domain capable of binding a human Vγ9Vδ2 T cell receptor. [0011] In a further main aspect, disclosed herein are binding agents comprising a first antigen- binding region capable of binding human 5T4. [0012] In some aspects disclosed herein is a single domain antibody that binds 5T4 comprising: (a) a complementarity determining region (CDR)1 comprising an amino acid sequence selected from SEQ ID NOs: 88, 98, 105, 111, 115, 122, 123, 133, 137, 141, 145, 149, 153, 157, 161, 165, and 169; (b) a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: : 89, 99, 106, 112, 116, 124, 125, 134, 138, 142, 146, 150, 154, 158, 162, 338699-2168 166 and 170; and (c) a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 90, 100, 107, 113, 117, 126, 127, 135, 139, 143, 147, 151, 155, 159, 163, 137 and 171. [0013] In some embodiments, the CDR1, CDR2 and CDR3 sequences are selected from the combination disclosed in TABLE 4A. In some embodiments, the single domain antibody comprises an amino acid sequence that is at least 90%, or at least 95%, identical to any one of SEQ ID NOs: 92, 93, 94, 95, 96, 97, 101, 102, 103, 104, 108, 109, 110, 114, 118, 119, 120, 121, 128, 129, 130, 131, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, and 172. In some embodiments, the single domain antibody comprises SEQ ID NOs: 92, 93, 94, 95, 96, 97, 101, 102, 103, 104, 108, 109, 110, 114, 118, 119, 120, 121, 128, 129, 130, 131, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, and 172. In some embodiments, the single domain antibody consists of SEQ ID NOs: 92, 93, 94, 95, 96, 97, 101, 102, 103, 104, 108, 109, 110, 114, 118, 119, 120, 121, 128, 129, 130, 131, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, and 172. [0014] In some embodiments, the CDR1 comprises SEQ ID NO: 88, the CDR2 comprises SEQ ID NO: 89, and the CDR3 comprises SEQ ID NO: 90. In some embodiments, the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to any one of SEQ ID NOs: 92, 93, 94, 95 and 96. [0015] In some embodiments, the CDR1 comprises SEQ ID NO: 88, the CDR2 comprises SEQ ID NO: 89, and the CDR3 comprises SEQ ID NO: 91. In some embodiments, the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 97. [0016] In some embodiments, the CDR1 comprises SEQ ID NO: 98, the CDR2 comprises SEQ ID NO: 99, and the CDR3 comprises SEQ ID NO: 100. In some embodiments, the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to any one of SEQ ID NOs: 101, 102, 103, and 104. [0017] In some embodiments, the CDR1 comprises SEQ ID NO: 105, the CDR2 comprises SEQ ID NO: 106, and the CDR3 comprises SEQ ID NO: 107. In some embodiments, the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to any one of SEQ ID NOs: 108, 109, and 110. [0018] In some embodiments, the CDR1 comprises SEQ ID NO: 111, the CDR2 comprises SEQ ID NO: 112, and the CDR3 comprises SEQ ID NO: 113. In some embodiments, the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 114. [0019] In some embodiments, the CDR1 comprises SEQ ID NO: 115, the CDR2 comprises SEQ ID NO: 116, and the CDR3 comprises SEQ ID NO: 117. In some embodiments, the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to any one of SEQ ID NOs: 118, 119, 120, and 121. 338699-2168 [0020] In some embodiments, the CDR1 comprises SEQ ID NO: 122, the CDR2 comprises SEQ ID NO: 124, and the CDR3 comprises SEQ ID NO: 126. In some embodiments, the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 128. [0021] In some embodiments, the CDR1 comprises SEQ ID NO: 123, the CDR2 comprises SEQ ID NO: 124, and the CDR3 comprises SEQ ID NO: 126. In some embodiments, the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 129. [0022] In some embodiments, the CDR1 comprises SEQ ID NO: 122, the CDR2 comprises SEQ ID NO: 125, and the CDR3 comprises SEQ ID NO: 126. In some embodiments, the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 130. [0023] In some embodiments, the CDR1 comprises SEQ ID NO: 122, the CDR2 comprises SEQ ID NO: 124, and the CDR3 comprises SEQ ID NO: 127. In some embodiments, the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 131. [0024] In some embodiments, the CDR1 comprises SEQ ID NO: 123, the CDR2 comprises SEQ ID NO: 125, and the CDR3 comprises SEQ ID NO: 127. In some embodiments, the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 132. [0025] In some embodiments, the CDR1 comprises SEQ ID NO: 133, the CDR2 comprises SEQ ID NO: 134, and the CDR3 comprises SEQ ID NO: 135. In some embodiments, the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 136. [0026] In some embodiments, the CDR1 comprises SEQ ID NO: 137, the CDR2 comprises SEQ ID NO: 138, and the CDR3 comprises SEQ ID NO: 139. In some embodiments, the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 140. [0027] In some embodiments, the CDR1 comprises SEQ ID NO: 141, the CDR2 comprises SEQ ID NO: 142, and the CDR3 comprises SEQ ID NO: 143. In some embodiments, the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 144. [0028] In some embodiments, the CDR1 comprises SEQ ID NO: 145, the CDR2 comprises SEQ ID NO: 146, and the CDR3 comprises SEQ ID NO: 147. In some embodiments, the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 148. 338699-2168 [0029] In some embodiments, the CDR1 comprises SEQ ID NO: 149, the CDR2 comprises SEQ ID NO: 150, and the CDR3 comprises SEQ ID NO: 151. In some embodiments, the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 152. [0030] In some embodiments, the CDR1 comprises SEQ ID NO: 153, the CDR2 comprises SEQ ID NO: 154, and the CDR3 comprises SEQ ID NO: 155. In some embodiments, the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 156. [0031] In some embodiments, the CDR1 comprises SEQ ID NO: 157, the CDR2 comprises SEQ ID NO: 158, and the CDR3 comprises SEQ ID NO: 159. In some embodiments, the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 160. [0032] In some embodiments, the CDR1 comprises SEQ ID NO: 161, the CDR2 comprises SEQ ID NO: 162, and the CDR3 comprises SEQ ID NO: 163. In some embodiments, the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 164. [0033] In some embodiments, the CDR1 comprises SEQ ID NO: 165, the CDR2 comprises SEQ ID NO: 166, and the CDR3 comprises SEQ ID NO: 167. In some embodiments, the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 168. [0034] In some embodiments, the CDR1 comprises SEQ ID NO: 169, the CDR2 comprises SEQ ID NO: 170, and the CDR3 comprises SEQ ID NO: 171. In some embodiments, the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 172. [0035] In some embodiments, the CDR1 comprises SEQ ID NO: 88, the CDR2 comprises SEQ ID NO: 89, and the CDR3 comprises SEQ ID NO: 90. In some embodiments, the single domain antibody comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 93. In some embodiments, the single domain antibody comprises or consists of the amino acid sequence of SEQ ID NO: 93. [0036] In some aspects, disclosed herein is a polynucleotide encoding the single domain of any of the above aspects or embodiments. [0037] In some aspects, disclosed herein is a vector comprising the polynucleotide of any of the above aspects or embodiments. [0038] In some aspects, disclosed herein is a host comprising the polynucleotide or vector of any of the above aspects or embodiments. 338699-2168 [0039] In some aspects, disclosed herein is a method of producing the single domain antibody of any one of the above aspects or embodiments, comprising: (a) introducing the vector of any of the above aspects or embodiments into a host cell under conditions permitting expression of the polynucleotide encoding the single domain antibody; and (b) isolating the single domain antibody from the culture supernatant. [0040] In some aspects, disclosed herein is a pharmaceutical composition comprising the single domain antibody of any one of the above aspects or embodiments and a pharmaceutically acceptable carrier. [0041] In some aspects, disclosed herein is a binding agent comprising the single domain antibody of any one of the above aspects or embodiments. [0042] In some aspects, disclosed herein is a multi-specific binding agent comprising: (a) a first antigen-binding domain that specifically binds to human 5T4 comprising the single domain antibody of any one of the above aspects or embodiments; and (b) a second antigen-binding domain that specifically binds to an antigen expressed on an immune cell. In some embodiments, the antigen expressed on the immune cell is selected from CD3, CD2, an αβ T cell receptor, a γδ T cell receptor (TCR). In some embodiments, the γδ T cell receptor is a Vδ2 TCR. In some embodiments, the Vδ2 TCR is a human Vδ2 T cell receptor. In some embodiments, the human Vδ2 T cell receptor is a Vγ9Vδ2 T cell receptor. In some embodiments, the first and second antigen-binding domains are present in the same polypeptide chain. In some embodiments, the first antigen-binding domain and second antigen-binding domain are covalently linked via a peptide linker. In some embodiments, the multi-specific binding agent comprises an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 362 or 363. In some embodiments, the multi-specific binding agent comprises or consists of the amino acid sequence of SEQ ID NOs: 362 or 363. In some embodiments, the first and second antigen-binding domains are present on different polypeptide chains. In some embodiments, the multi-specific binding agent comprises an Fc domain comprising a first and a second Fc domain monomer. In some embodiments, the multi- specific binding agent comprises (a) a first polypeptide comprising the first antigen-binding domain that binds to a human 5T4 protein and the first Fc domain monomer; and (b) a second polypeptide comprising the second antigen-binding domain that binds to a human Vδ2 T cell receptor and the second Fc domain monomer. In some embodiments, the human Vδ2 T cell receptor is a Vγ9Vδ2 T cell receptor. In some embodiments, the antigen-binding domain that binds to the Vγ9Vδ2 T cell receptor comprises SEQ ID NO: 22. In some embodiments, the antigen-binding domain that binds to the Vγ9Vδ2 T cell receptor consists of SEQ ID NO: 22. In some embodiments, the multi-specific binding agent induces proliferation of Vγ9Vδ2 T cells. In some embodiments, the first and second Fc domain monomers comprise a mutation at 338699-2168 position 234 and/or 235 according to the EU numbering system. In some embodiments, the first and second Fc domain monomers comprise an L234F and an L235E substitution. In some embodiments, (a) the first Fc domain monomer comprises a T366W substitution and the second Fc polypeptide comprises T366S, L368A and Y407V substitutions, or vice versa, according to the EU numbering system; and/or (b) the cysteine residue at position 220 according to the EU numbering system in the first or second Fc domain monomers have been deleted or substituted. [0043] In some aspects, disclosed herein is a multi-specific binding agent comprising: (a) a first antigen-binding domain that binds to human 5T4, where the first antigen-binding domain comprises a complementarity determining region (CDR)1 comprising SEQ ID NO: 88, a CDR2 comprising SEQ ID NO: 89, and a CDR3 comprising SEQ ID NO: 90; and (b) a second antigen- binding domain that binds to Vδ2, where the second antigen-binding domain comprises a CDR1 comprising SEQ ID NO: 18, a CDR2 comprising SEQ ID NO: 19, and a CDR3 comprising SEQ ID NO: 20. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 93 and the second antigen-binding domain comprises SEQ ID NO: 22. In some embodiments, the first and second antigen-binding domains are comprised in the same polypeptide chain. [0044] In some as aspects, disclosed herein is a multi-specific binding agent comprising (a) a first polypeptide comprising (i) a first antigen-binding domain that binds to a human 5T4 protein and comprises a complementarity determining region (CDR)1 comprising SEQ ID NO: 88, a CDR2 comprising SEQ ID NO: 89, and a CDR3 comprising SEQ ID NO: 90; and (ii) a first Fc domain monomer; (b) a second polypeptide comprising (i) a second antigen-binding domain that binds to a human Vδ2 T cell receptor and comprises a CDR1 comprising SEQ ID NO: 18, a CDR2 comprising SEQ ID NO: 19, and a CDR3 comprising SEQ ID NO: 20; and (ii) a second Fc domain monomer. In some embodiments, the first Fc domain monomer comprises SEQ ID NO: 358 and the second Fc domain monomer comprises SEQ ID NO: 359. In some embodiments, the first Fc domain monomer comprises SEQ ID NO: 359 and the second Fc domain monomer comprises SEQ ID NO: 358. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 369. In some embodiments, the second antigen-binding domain comprises SEQ ID NO: 381. In some embodiments, (a) the first polypeptide comprises SEQ ID NO: 369; and (b) the second polypeptide comprises SEQ ID NO: 381. [0045] In some aspects, disclosed herein is a polynucleotide encoding the multi-specific binding agent of any one of the above aspects or embodiments. [0046] In some aspects, disclosed herein is an expression vector comprising the polynucleotide of any of the above aspects or embodiments. 338699-2168 [0047] In some aspects, disclosed herein is a host cell comprising the polynucleotide or the expression vector of any one of the above aspects or embodiments. In some embodiments, the cell is a CHO cell. [0048] In some aspects, disclosed herein is a method of producing the multi-specific binding agent of any one of the above aspects or embodiments, comprising culturing the host cell of any one of the above aspects or embodiments, under conditions sufficient to express the multi- specific binding agent from the polynucleotide or expression vector therein and purifying the expressed multi-specific binding agent from the culture supernatant. [0049] In some aspects, disclosed herein is a pharmaceutical composition comprising the single domain antibody, the binding agent, or the multi-specific binding agent according to any one the above aspects or embodiments, and a pharmaceutically-acceptable excipient. [0050] In some aspects disclosed herein is a method of treatment a cancer in a subject in need thereof, comprising administration to the subject the single domain antibody, the binding agent, the multi-specific binding agent, or the pharmaceutical composition of any one of the above aspects or embodiments. In some embodiments, the subject is human. In some embodiments, the cancer expresses 5T4. In some embodiments, the subject is suffering from a cervical, colorectal, head and neck, glioblastoma, melanoma, urothelial, gastric, renal cell carcinoma, lung cancer, pancreatic cancer, mesothelioma, pre-B acute lymphoblastic leukemia, or hepatocellular cancer. [0051] In some aspects, disclosed herein is a method of expanding Vγ9Vδ2 T cells comprising contacting the Vγ9Vδ2 T cells with single domain, the binding agent, the multi-specific binding agent, or the pharmaceutical composition of any one of the above aspects or embodiments. BRIEF DESCRIPTION OF THE DRAWINGS [0052] FIG.1A-FIG.1B show the expression of 5T4 on tumor cells (FIG.1A) and percentage of Vγ9+Vδ2+ T cells out of the total CD3+ T cells in different patient cancer samples (FIG.1B) . (m)/(p)CRC = (metastatic)/(primary) colorectal cancer; ccRCC = clear cell renal cell cancer; GBM = glioblastoma multiforme; H&N = head & neck cancer; EC = esophagus carcinoma; UCC = urothelial cell carcinoma. [0053] FIG.2 shows the expression of 5T4 on non-malignant urothelial cells. [0054] FIG 3A-FIG.3C show the binding profile of 5T4 binding agents to recombinant human 5T4 as determined by ELISA. FIG.3A shows results for LV1138, LV1139, LV1140, LV1141, LV1142, LV1143, LV1144, and LV1145. FIG.3B shows results for LV1145, LV1146, LV1147, LV1148, LV1149, LV1150, LV1151, and LV1152. FIG.3C shows results for LV1292. [0055] FIG.4A-FIG.4C show the binding profile of multi-specific binding agents comprising LV1138, LV1139, LV1140, or LV1142 with a Vδ2-binding arm to CHO-K1 cells that were 338699-2168 transfected with human 5T4 (FIG. 4A), murine 5T4 (FIG. 4B), or cynomolgus monkey 5T4 (FIG.4C). [0056] FIG.5A-FIG.5B show the binding profile of multi-specific binding agents comprising LV1138, LV1139, LV1140, or LV1142 with a Vδ2-binding arm to U-251 (FIG.5A) or A-431 (FIG.5B) cancer cells as determined by flow cytometry. [0057] FIG.6 shows the binding profile of multi-specific binding agents comprising LV1138, LV1139, LV1140, or LV1142 with a Vδ2-binding arm to human Vγ9Vδ2 T cells as determined by flow cytometry. [0058] FIG.7A-FIG.7H show the Vγ9Vδ2 T cell degranulation (FIGs.7A, 7C, 7E, and 7G) and cytotoxicity (FIGs.7B, 7D, 7F, and 7H) against U-251 cells expressing 5T4 induced by increasing concentrations of multi-specific binding agents comprising LV1138, LV1139, LV1140, or LV1142 with a Vδ2-binding arm in the presence or absence of primary Vγ9Vδ2 T cells. [0059] FIG.8A-FIG.8H show the Vγ9Vδ2 T cell degranulation (FIGs.7A, 7C, 7E, and 7G) and cytotoxicity (FIGs.7B, 7D, 7F, and 7H) against A-431 cells expressing 5T4 induced by increasing concentrations of multi-specific binding agents comprising LV1138, LV1139, LV1140, or LV1142 with a Vδ2-binding arm in the presence or absence (control) of primary Vγ9Vδ2 T cells. [0060] FIG. 9A-FIG. 9D show Vγ9Vδ2 T cell degranulation as measured by CD107a expression induced by 5T4-multi-specific binding agents in patient-derived cervical squamous cell carcinoma (FIG. 9A), cervical adenocarcinoma (FIG. 9B), metastatic colorectal cancer (FIG.9C), and glioblastoma (FIG.9D). [0061] FIG.10A-FIG.10E show tumor cell lysis induced by 5T4-multi-specific binding agents in patient-derived squamous cell carcinoma at 18 hours (FIG.10A) and 7 days (FIG.10B), cervical adenocarcinoma at 18 hours (FIG.10C) and 7 days (FIG.10D), and in glioblastoma at 18 hours (FIG.10E). [0062] Fig.11A-FIG.11B show Vγ9Vδ2 T cell degranulation (FIG.11A) and percent tumor lysis (FIG.11B) in head and neck, melanoma, glioblastoma, cervical, colorectal, renal, and urothelial cell cancers incubated with Vγ9Vδ2 T cells and with or without LAVA-378. [0063] FIG.12A-FIG.12B show increased tumor cell lysis when LAVA-378 and Vγ9Vδ2 T cells are incubated with urothelial cancer cells (FIG.12A) and absence of an increase in cell lysis when LAVA-378 and Vγ9Vδ2 T cells are incubated with non-malignant urothelial cells (FIG.12B). [0064] FIG.13 shows the increase in tumor lysis when LAVA-378 is incubated with urothelial cancer cells in the absence of additional expanded (allogeneic) Vγ9Vδ2-T cells. 338699-2168 [0065] FIG.14A-FIG.14B show 5T4 expression on patient colorectal cancer samples (FIG. 14A) and Vγ9Vδ2 T cell degranulation as measured by CD107a expression after incubation with LAVA-378 and LAVA-380 in the presence of expanded Vγ9Vδ2-T cells (FIG.14B). [0066] FIG. 15A-FIG. 15B show chromatographs illustrating retention of pyroglutamate- containing (LAVA-1395) (FIG. 15A) and non-pyroglutamate containing (LAVA-1433) (FIG. 15B) multi-specific binding agents in reverse-phase HPLC. [0067] FIG.16 illustrates the different CDR numbering systems (Kabat, Chothia, IMGT, and Combined) for the 6H4 VHH. DETAILED DESCRIPTION Definitions [0068] The terms used in this specification generally have their ordinary meaning in the art, within the context of this disclosure and in the specific context where each term is used. Certain terms are discussed below or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the disclosure and how to make and use them. The scope and meaning of any use of a term will be apparent from the specific context in which the term is used. As such, the definitions set forth herein are intended to provide illustrative guidance in ascertaining particular embodiments of the disclosure, without limitation to particular compositions or biological systems. All publications, patent applications, patents, GenBank or other accession numbers and other references mentioned herein are incorporated by reference in their entirety for all purposes. [0069] The terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims, unless clearly indicated otherwise. [0070] The term “about” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or in some instances ±10%, or in some instances ±5%, or in some instances ±1%, or in some instances ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. [0071] The term “5T4”, when used herein, refers to the human 5T4 protein, also termed trophoblast glycoprotein, abbreviated as TPBG (UniProt Q13641-TPBG_HUMAN). The sequence of human 5T4 is set forth in SEQ ID NO:382. [0072] The term “Vδ2”, when used herein, refers to the rearranged δ2 chain of the human Vγ9Vδ2-T cell receptor (TCR). UniProtKB - A0JD36 (A0JD36_HUMAN) gives an example of a variable TRDV2 sequence. The sequence of human Vδ2 is set forth in SEQ ID NO:383. [0073] The term “human Vγ9”, when used herein, refers to the rearranged γ9 chain of the Vγ9Vδ2-T cell receptor (TCR). UniProtKB – Q99603_HUMAN gives an example of a variable TRGV9 sequence. 338699-2168 [0074] The terms “antibody” and “binding agent” are used interchangeably and refer to a polypeptide or complex of polypeptides comprising at least one antigen-binding domain that specifically binds to a target antigen and includes intact immunoglobulin molecules, antigen- binding fragments thereof, and derivatives of either. Binding agents, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies and humanized antibodies, and antibody fragments provided by any known technique, such as enzymatic cleavage, peptide synthesis and recombinant techniques. In some embodiments a binding agent has the ability to specifically bind to an antigen under typical physiological conditions with a half-life of significant periods of time, such as at least about 30 minutes, at least about one hour, at least about two hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functionally-defined period (such as a time sufficient to induce, promote, enhance, and/or modulate a physiological response associated with antibody binding to the antigen and/or time sufficient for the antibody to recruit an effector activity). [0075] As used herein, the terms “antigen-binding domain” or “antigen-binding region” refer to a portion of a binding agent that specifically binds a target antigen or target epitope. Antigen- binding domains may comprise variable regions of both the heavy and light chains of an antibody or antigen-binding fragments thereof, including single domain antibodies. [0076] The terms “single domain antibody,” “Nanobody®,” “VHH,” “VHH antibody,” “single domain binding agent,” or “VHH domain” as used herein refer to an antigen-binding fragment comprising a single monomeric variable domain. Single-domain antibodies are well known to the skilled person, see e.g., Hamers-Casterman et al. (1993) Nature 363:446, Roovers et al. (2007) Curr Opin Mol Ther 9:327, and Krah et al. (2016) Immunopharmacol Immunotoxicol 38:21. Single-domain antibodies may contain only the variable domain of an immunoglobulin chain, i.e., CDR1, CDR2, and CDR3 and framework regions. Examples of single-domain antibodies are variable fragments of heavy-chain-only antibodies, antibodies that naturally do not comprise light chains, single-domain antibodies derived from conventional antibodies and engineered antibodies. Single-domain antibodies may be derived from any species. For example, naturally occurring single-domain antibodies can be derived from antibodies raised in Camelidae species, for example in camel, dromedary, llama, alpaca, and guanaco. Like an intact immunoglobulin, a single-domain antibody is able to bind selectively to a single specific antigen. [0077] The term “hinge region” as used herein is intended to refer to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216-230 according to the EU numbering. 338699-2168 [0078] The term “CH2 region” or “CH2 domain” as used herein is intended to refer to the CH2 region of an immunoglobulin heavy chain. Thus, for example the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to the EU numbering. However, the CH2 region may also be any of the other subtypes as described herein. [0079] The term “CH3 region” or “CH3 domain” as used herein is intended to refer to the CH3 region of an immunoglobulin heavy chain. Thus, for example the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the EU numbering. However, the CH3 region may also be any of the other subtypes as described herein. [0080] The terms “complementarity determining region” or “CDR,” as used herein, refer to the amino acid sequences within antibody variable regions which confer antigen specificity and binding affinity. For example, in general, there are three CDRs in each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3). The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme), Al- Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme), IMGT (Lefranc et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev Comp Immunol. 2003 Jan;27(1):55-77), or a combination thereof. Various methods for CDR definition and amino acid numbering can be compared on www.abysis.org (UCL). [0081] The constant regions of an antibody, if present, may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells and T cells) and components of the complement system such as C1q, the first component in the classical pathway of complement activation. [0082] The term “antigen” refers to a molecule or a portion of a molecule capable of being bound by an antibody or an antigen-binding fragment thereof and additionally capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen. [0083] The term “antigen-binding fragment,” as used herein refers to a polypeptide comprising at least one complementarity determining region (CDR) that binds to at least one epitope of an antigen of interest (e.g., 5T4). In this regard, an antigen-binding fragment may comprise 1, 2, 3, 4, 5, or all 6 CDRs of a variable heavy chain (VH) and/or variable light chain (VL) sequence. Antigen-binding fragments include proteins that comprise a portion of a full length immunoglobulin, generally the antigen-binding or variable region thereof, such as Fab, F(ab’)2, Fab’, Fd, Fv fragments, minibodies, diabodies, single domain antibodies (dAb, also known as VHH, camelid antibodies, or nanobodies), single-chain variable fragments (scFv), rIgG, 338699-2168 antibody mimetics, and any other modified configuration of the immunoglobulin molecule that comprises an antigen-binding fragment of the required specificity. [0084] As used herein, the terms “antigen-binding domain” or “antigen-binding region” refer to a portion of a binding agent that specifically binds a target antigen or target epitope. Antigen- binding domains may comprise variable regions of both the heavy and light chains of an antibody or antigen-binding fragments thereof, including single domain antibodies. [0085] The term “immunoglobulin” as used herein refers to a class of structurally related glycoproteins typically consisting of two pairs of polypeptide chains, one pair of light (L) chains and one pair of heavy (H) chains, all four potentially inter-connected by disulfide bonds, although some mammalian species also produce heavy-chain only antibodies. [0086] The terms “immunoglobulin heavy chain”, “heavy chain of an immunoglobulin,” or “heavy chain” as used herein refer to one of the chains of an immunoglobulin. A heavy chain typically comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH) which defines the isotype of the immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, or IgE). The heavy chain constant region typically comprises three domains, CH1, CH2, and CH3. The heavy chain constant region further comprises a hinge region. Within the structure of the immunoglobulin (e.g., IgG), the two heavy chains are inter-connected via disulfide bonds in the hinge region. Equally to the heavy chains, each light chain is typically comprised of several regions: a light chain variable region (VL) and a light chain constant region (CL). Furthermore, the VH and VL regions may be subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. [0087] The term “isotype” as used herein, refers to the immunoglobulin (sub)class (for instance IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) or any allotype thereof, such as IgG1m(za) and IgG1m(f) that is encoded by heavy chain constant region genes. Each heavy chain isotype can be combined with either a kappa (κ) or lambda (λ) light chain. An immunoglobulin disclosed herein can possess any isotype. [0088] The terms “parent antibody” or “parent binding agent,” is to be understood as a binding agent which is identical to a binding agent disclosed herein, but wherein the parent binding agent does not have one or more of the specified mutations. [0089] A “variant binding agent,” “variant antibody,” “binding agent variant,” “antibody variant,” or a “variant of a parent antibody” is a binding agent which comprises one or more mutations 338699-2168 (substitutions, deletions, or insertions) as compared to a binding agent or parent binding agent described here. Amino acid substitutions may exchange a native amino acid for another naturally-occurring amino acid, or for a non-naturally-occurring amino acid derivative. The amino acid substitution may be conservative or non-conservative. [0090] In some embodiments, one or more amino acid substitutions are introduced into a 5T4 binding agent described herein. In some embodiments, the one or more amino acid substitutions is a conservative amino acid substitution. The phrases "conservative amino acid substitution" or "conservative mutation" refer to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and Schirmer, R. H., Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz, G. E. and Schirmer, R. H., supra). Examples of conservative mutations include amino acid substitutions of amino acids within the sub-groups above, for example, lysine for arginine and vice versa such that a positive charge may be maintained; glutamic acid for aspartic acid and vice versa such that a negative charge may be maintained; serine for threonine such that a free -OH can be maintained; and glutamine for asparagine such that a free -NH2 can be maintained. Examples of amino acid classes are provided in Table 1, Table 2, and Table 3 below. Table 1: Amino Acid Residue Classes for Conservative Substitutions Acidic Residues Asp (D) and Glu (E) Basic Residues Lys (K), Arg (R), and His (H) Hydrophilic Uncharged Residues Ser (S), Thr (T), Asn (N), and Gln (Q) Aliphatic Uncharged Residues Gly (G), Ala (A), Val (V), Leu (L), and Ile (I) Non-polar Uncharged Residues Cys (C), Met (M), and Pro (P) Aromatic Residues Phe (F), Tyr (Y), and Trp (W) 338699-2168 Table 2: Alternative Conservative Amino Acid Residue Substitution Classes 1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y W Table 3: Alternative Physical and Functional Classifications of Amino Acid Residues Alcohol group-containing residues S and T Aliphatic residues I, L, V, and M Cycloalkenyl-associated residues F, H, W, and Y Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and Y Negatively charged residues D and E Polar residues C, D, E, H, K, N, Q, R, S, and T Positively charged residues H, K, and R Small residues A, C, D, G, N, P, S, T, and V Very small residues A, G, and S Residues involved in turn formation A, C, D, E, G, H, K, N, Q, R, S, P, and T Flexible residues Q, T, K, S, G, N, D, E, and R [0091] In some embodiments, the one or more amino acid substitutions is a non-conservative amino acid substitution. "Non-conservative substitutions" involve amino acid substitutions between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc. In this case, it is preferable for the non-conservative amino acid substitution to not interfere with or inhibit the biological activity of the binding agent (i.e., does not interfere with binding to the target antigen). The non-conservative amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent binding agent. [0092] In the context of the present disclosure, a substitution in a variant is indicated as: Original amino acid – position – substituted amino acid; The three-letter code, or one letter code, are used, including the codes Xaa and X to indicate amino acid residue. Accordingly, the notation “T366W” means that the variant comprises a substitution of threonine with tryptophan in the variant amino acid position corresponding to the amino acid in position 366 in the parent antibody. [0093] Furthermore, the term “a substitution” includes a substitution into any one of the other nineteen natural amino acids, or into other amino acids, such as non-natural amino acids. For example, a substitution of amino acid T in position 366 includes each of the following substitutions: 366A, 366C, 366D, 366G, 366H, 366F, 366I, 366K, 366L, 366M, 366N, 366P, 366Q, 366R, 366S, 366E, 366V, 366W, and 366Y. 338699-2168 [0094] The term “full-length immunoglobulin” when used herein, refers to an immunoglobulin which contains all heavy and light chain constant and variable domains corresponding to those that are normally found in a wild-type immunoglobulin of that isotype. [0095] The terms “chimeric antibody” or “chimeric binding agent” refer to a binding agent wherein the variable region is derived from a non-human species (e.g., derived from rodents) and the constant region is derived from a different species, such as human. Chimeric antibodies may be generated by genetic engineering. Chimeric monoclonal antibodies for therapeutic applications are developed to reduce antibody immunogenicity. [0096] In some embodiments, the binding agents described herein are humanized immunoglobulins or antigen-binding fragments thereof. A “humanized” immunoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a non- human (such as a mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is termed a “donor,” and the human immunoglobulin providing the framework is termed an “acceptor.” Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. Humanized immunoglobulins can be constructed by means of genetic engineering (for example, see U.S. Patent No.5,585,089). [0097] The terms “humanized antibody” or “humanized binding agent,” refers to a genetically engineered non-human binding agent, which contains a humanized light chain and/or a humanized heavy chain. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized antibody may have a limited number of substitutions by amino acids taken from the donor framework. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen-binding or other immunoglobulin functions. [0098] As used herein, the term “multi-specific” refers to the capability of a binding agent described herein to specifically bind two or more different target antigens or to specifically bind two or more different epitopes on a target antigen. [0099] The terms “multi-specific antibody” and “multi-specific binding agent” are used interchangeably and refer to a binding agent comprising two or more antigen-binding domains (e.g., two, three, four, or more), each binding to a different target antigen or to a different epitope on the same target antigen. [0100] The terms “bispecific antibody” and “bispecific binding agent” are used interchangeably and refer to a binding agent comprising not more than two antigen-binding domains, each binding to a different target antigen or to a different epitope on the same target antigen. 338699-2168 [0101] Examples of different classes of bispecific and multi-specific binding agents include but are not limited to (i) IgG-like molecules with complementary CH3 domains to force heterodimerization; (ii) recombinant IgG-like dual targeting molecules, wherein the two sides of the molecule each contain the Fab fragment or part of the Fab fragment of at least two different binding agents; (iii) IgG fusion molecules, wherein full length IgG antibodies are fused to extra Fab fragment or parts of Fab fragment; (iv) Fc fusion molecules, wherein single chain Fv molecules or stabilized diabodies are fused to heavy-chain constant- domains, Fc-regions or parts thereof; (v) Fab fusion molecules, wherein different Fab fragments are fused together, fused to heavy-chain constant-domains, Fc-regions or parts thereof; and (vi) scFv-and diabody-based and heavy chain-based antibodies (e.g., domain antibodies, Nanobodies®) wherein different single chain Fv molecules or different diabodies or different heavy-chain antibodies (e.g. domain antibodies, Nanobodies®) are fused to each other or to another protein or carrier molecule fused to heavy-chain constant-domains, Fc-regions or parts thereof. [0102] Examples of IgG-like molecules with complementary CH3 domains molecules include but are not limited to the Triomab® (Trion Pharma/Fresenius Biotech), the Knobs-into-Holes (Genentech), CrossMAbs (Roche) and the electrostatically-matched (Amgen, Chugai, Oncomed), the LUZ-Y (Genentech, Wranik et al. J. Biol. Chem.2012, 287(52): 43331-9, doi: 10.1074/jbc.M112.397869. Epub 2012 Nov 1), DIG-body and PIG-body (Pharmabcine, WO2010134666, WO2014081202), the Strand Exchange Engineered Domain body (SEEDbody)(EMD Serono), the Biclonics (Merus, WO2013157953), FcΔAdp (Regeneron), bispecific IgG1 and IgG2 (Pfizer/Rinat), Azymetric scaffold (Zymeworks/Merck,), mAb-Fv (Xencor), bivalent bispecific antibodies (Roche, WO2009080254) and DuoBody® molecules (Genmab). [0103] Examples of recombinant IgG-like dual targeting molecules include, but are not limited to, Dual Targeting (DT)-Ig (GSK/Domantis, WO2009058383), Two-in-one Antibody (Genentech, Bostrom, et al 2009. Science 323, 1610–1614), Cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star), ZybodiesTM (Zyngenia, LaFleur et al. MAbs. 2013 Mar- Apr;5(2):208-18), approaches with common light chain, κλBodies (NovImmune, WO2012023053), US2020319181 (Merus) and CovX-body® (CovX/Pfizer, Doppalapudi, V.R., et al 2007. Bioorg. Med. Chem. Lett.17,501–506). [0104] Examples of IgG fusion molecules include but are not limited to Dual Variable Domain (DVD)-Ig (Abbott), Dual domain double head antibodies (Unilever; Sanofi Aventis), IgG-like Bispecific (ImClone/Eli Lilly, Lewis et al. Nat Biotechnol. 2014 Feb;32(2):191-8), Ts2Ab (MedImmune/AZ, Dimasi et al. J Mol Biol. 2009 Oct 30;393(3):672-92) and BsAb 338699-2168 (Zymogenetics, WO2010111625), HERCULES (Biogen Idec), scFv fusion (Novartis), scFv fusion (Changzhou Adam Biotech Inc) and TvAb (Roche). [0105] Examples of Fc fusion molecules include but are not limited to scFv/Fc Fusions (Academic Institution, Pearce et al Biochem Mol Biol Int.1997 Sep;42(6):1179), SCORPION (Emergent BioSolutions/Trubion, Blankenship JW, et al. AACR 100th Annual meeting 2009 (Abstract #5465); Zymogenetics/BMS, WO2010111625), Dual Affinity Retargeting Technology (Fc-DARTTM) (MacroGenics) and Dual(ScFv)2-Fab (National Research Center for Antibody Medicine – China). [0106] Examples of Fab fusion bispecific antibodies include but are not limited to F(ab)2 (Medarex/AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock® (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol) and Fab-Fv (UCB-Celltech). [0107] Examples of scFv-, diabody-based and domain antibodies include but are not limited to Bispecific T Cell Engager (BiTE®) (Micromet, Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DARTTM) (MacroGenics), Single-chain Diabody (Academic, Lawrence FEBS Lett.1998 Apr 3;425(3):479-84), TCR-like Antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack, WO2010059315) and COMBODY molecules (Epigen Biotech, Zhu et al. Immunol Cell Biol. 2010 Aug;88(6):667-75), dual targeting nanobodies® (Ablynx, Hmila et al., FASEB J.2010), dual targeting heavy chain only domain antibodies. [0108] Antibody or antigen-binding fragment binding to a target antigen is typically demonstrated by a dissociation constant (KD). The term “KD” is intended to refer to the dissociation equilibrium constant of a particular antibody-antigen interaction. The ratio of dissociation rate (koff) to association rate (kon) of a binding agent to a monovalent antigen (koff/kon) is the dissociation constant KD, which is inversely related to affinity. The lower the KD value, the higher the affinity of the antibody. The value of KD varies for different complexes of antibody and antigen and depends on both kon and koff. The dissociation constant KD for a binding agent provided herein can be determined using any method provided herein or any other method well known to those skilled in the art. [0109] In the context of antibody binding to an antigen, the terms “binds” or “specifically binds” refer to the binding of a binding agent to a predetermined antigen or target (e.g. human 5T4 or Vδ2) to which binding typically is with an affinity corresponding to a KD of about 10-6 M or less, e.g.10-7 M or less, such as about 10-8 M or less, such as about 10-9 M or less, about 10- 10 M or less, or about 10-11 M or even less, e.g. when determined as described in the Examples herein. KD values can be determined using surface plasmon resonance (SPR) technology in a BIAcore T200 or bio-layer interferometry (BLI) in an Octet RED96 instrument using the antigen as the ligand and the binding domain or binding agent as the analyte. Specific binding 338699-2168 means that the binding agent binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100-fold lower, for instance at least 1,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The degree with which the affinity is lower is dependent on the KD of the binding moiety or binding molecule, so that when the KD of the binding moiety or binding molecule is very low (that is, the binding moiety or binding molecule is highly specific), then the degree with which the affinity for the antigen is lower than the affinity for a non-specific antigen may be at least 10,000-fold. [0110] “Polynucleotide” is used interchangeably with “oligonucleotide,” and “nucleic acid” herein, and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide. In some embodiments, the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions. However, it may be suitable in some instances, as discussed herein, for the nucleic acid to comprise one or more insertions, deletions, inversions, and/or substitutions. [0111] As used herein, the term “host cell” refers to any type of cell that comprises a vector or polynucleotide described herein and is typically used in the manufacturing of proteins (e.g., in the manufacturing of a binding agent). [0112] The term “cancer” refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells. [0113] The term “subject” as used herein refers to a vertebrate or an invertebrate, and includes mammals, birds, fish, reptiles, and amphibians. Subjects include humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species. Subjects include farm animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats, and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like; and aquatic animals such as fish, shrimp, and crustaceans. [0114] The terms “first” and “second” antigen-binding domains when used herein do not refer to their orientation/position in the antibody, i.e., they have no meaning with regard to the N- or C-terminus. The terms “first” and “second” only serve to correctly and consistently refer to the two different antigen-binding domains in the claims and the description. 338699-2168 [0115] “% sequence identity”, when used herein, refers to the number of identical nucleotide or amino acid positions shared by different sequences (i.e., % identity = # of identical positions/total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment. The percent identity between two nucleotide or amino acid sequences may e.g., be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. 5T4 Binding agents [0116] 5T4 is an antagonist of the Wnt/β-catenin pathway. Human 5T4 is a 420 amino acid protein. The amino acid sequence of wild type human 5T4 is provided as SEQ ID NO: 382. Representative 5T4 polynucleotide sequences (NCBI Gene ID: 7162) include NM_001166392.2 among others, each of which are available at the 5T4 gene page on the NCBI website. [0117] In some embodiments, the present disclosure provides binding agents comprising at least one antigen-binding domain that specifically binds to human 5T4 – referred to herein as 5T4 binding agents. In some embodiments, the 5T4 binding agent is an immunoglobulin or an antigen-binding fragment thereof. In some embodiments, the 5T4 binding agent is a single domain antibody. [0118] In some embodiments, the 5T4 binding agent comprises a first antigen-binding domain comprising a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 88, 98, 105, 111, 115, 122, 123, 133, 137, 141, 145, 149, 153, 157, 161, 165, and 169; a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 89, 99, 106, 112, 116, 124, 125, 134, 138, 142, 146, 150, 154, 158, 162, 166 and 170; and a CDR3 comprising an amino acid sequence selected from SEQ ID NOs 90, 100, 107, 113, 117, 126, 127, 135, 139, 143, 147, 151, 155, 159, 163, 137 and 171. In some embodiments, the 5T4 binding agent comprises first antigen-binding domain comprising a CDR1, CDR2, and CDR3 combination selected from Table 4A, 4B, 4C, or 4D. In some embodiments, 5T4 binding agent comprises first antigen-binding domain comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 92, 93, 94, 95, 96, 97, 101, 102, 103, 104, 108, 109, 110, 114, 118, 119, 120, 121, 128, 129, 130, 131, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, and 172. In some embodiments, the 5T4 binding agent comprises first antigen-binding domain comprising an amino acid sequence selected from SEQ ID NOs: 92, 93, 94, 95, 96, 97, 101, 102, 103, 104, 108, 109, 110, 114, 118, 119, 120, 121, 128, 129, 130, 131, 132, 136, 140, 144, 148, 338699-2168 152, 156, 160, 164, 168, and 172. In some embodiments, the 5T4 binding agent comprises a first antigen-binding domain consisting of an amino acid sequence selected from SEQ ID NOs: 92, 93, 94, 95, 96, 97, 101, 102, 103, 104, 108, 109, 110, 114, 118, 119, 120, 121, 128, 129, 130, 131, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, and 172. [0119] Exemplary CDRs of 5T4 binding agents are shown in Table 4A – Table 4D. Table 4A: Exemplary 5T4-specific CDR (Kabat) and VHH Sequences Reference Component Sequence SEQ ID LV1139 CDR1 NYRMK 88 CDR2 TISHRVGRTYYADSVKG 89 CDR3 VAMGGSDYAPHDYDY 90 CDR3_var VDMGGSDYAPHDYDY 91 VHH EVQLVESGGGLVQPGGSLRLSCAANGFAFSNYRMKWVRQ 92 APGKGLEWVSTISHRVGRTYYADSVKGRFTISRDNAKNT VYLQMNSLKPEDTARYFCTRVAMGGSDYAPHDYDYTGQG TQVTVSS LV1139 (D) VHH DVQLVESGGGLVQPGGSLRLSCAANGFAFSNYRMKWVRQ 93 APGKGLEWVSTISHRVGRTYYADSVKGRFTISRDNAKNT VYLQMNSLKPEDTARYFCTRVAMGGSDYAPHDYDYTGQG TQVTVSS LV1139 H1 VHH DVQLVESGGGLVQPGGSLRLSCAANGFAFSNYRMKWVRQ 94 APGKGLEWVSTISHRVGRTYYADSVKGRFTISRDNAKNT VYLQMNSLKPEDTARYFCTRVAMGGSDYAPHDYDYTGQG TQVTVSS LV1139 H2 VHH EVQLVESGGGLVQPGGSLRLSCAANGFAFSNYRMKWVRQ 95 APGKGLEWVSTISHRVGRTYYADSVKGRFTISRDNSKNT LYLQMNSLRAEDTARYFCTRVAMGGSDYAPHDYDYTGQG TQVTVSS LV1139 H3 VHH EVQLVESGGGLVQPGGSLRLSCAANGFAFSNYRMKWVRQ 96 APGKGLEWVSTISHRVGRTYYADSVKGRFTISRDNAKNT LYLQMNSLRAEDTARYFCTRVAMGGSDYAPHDYDYTGQG TQVTVSS LV1139_var VHH EVQLVESGGGLVQPGGSLRLSCAANGFAFSNYRMKWVRQ 97 APGKGLEWVSTISHRVGRTYYADSVKGRFTISRDNAKNT VYLQMNSLKPEDTARYFCTRVDMGGSDYAPHDYDYTGQG TQVTVSS LV1138 CDR1 YYAIG 98 CDR2 YISRSGGSVYYADSVKG 99 CDR3 RSSAYSRRYDIYSGWTEYGY 100 VHH EVQLVESGGGLVQAGGSLRLSCAASGDTLDYYAIGWFRQ 101 APGKEREGVSYISRSGGSVYYADSVKGRFTISRDSAKNT VYLQMVSLKPEDTAVYYCAARSSAYSRRYDIYSGWTEYG YWGQGTQVTVSS LV1138 H1 VHH EVQLVESGGGLVQPGGSLRLSCAASGDTLDYYAIGWFRQ 102 APGKEREGVSYISRSGGSVYYADSVKGRFTISRDNSKNT LYLQMVSLRAEDTAVYYCAARSSAYSRRYDIYSGWTEYG YWGQGTQVTVSS LV1138 H2 VHH EVQLVESGGGLVQPGGSLRLSCAASGDTLDYYAIGWFRQ 103 APGKEREGVSYISRSGGSVYYADSVKGRFTISRDSAKNT LYLQMVSLRAEDTAVYYCAARSSAYSRRYDIYSGWTEYG YWGQGTQVTVSS LV1138 H3 VHH EVQLVESGGGLVQPGGSLRLSCAASGDTLDYYAIGWFRQ 104 APGKEREGVSYISRSGGSVYYADSVKGRFTISRDSAKNT 338699-2168 Reference Component Sequence SEQ ID VYLQMVSLRPEDTAVYYCAARSSAYSRRYDIYSGWTEYG YWGQGTQVTVSS LV1140 CDR1 AYGMT 105 CDR2 GIESSGDIRKYSDSVKG 106 CDR3 SRDRWVIGDTRKYDS 107 VHH EVQLVESGGGLVQPGGSLRLSCAASGFTFSAYGMTWVRQ 108 APGKGLEWVSGIESSGDIRKYSDSVKGRFTISRDNAKNT LYLQMNSLKPEDTAMYYCARSRDRWVIGDTRKYDSRGQG TQVTVSS LV1140 H1 VHH EVQLVESGGGLVQPGGSLRLSCAASGFTFSAYGMTWVRQ 109 APGKGLEWVSGIESSGDIRKYSDSVKGRFTISRDNAKNT LYLQMNSLRAEDTAMYYCARSRDRWVIGDTRKYDSRGQG TQVTVSS LV1140 H2 VHH EVQLVESGGGLVQPGGSLRLSCAASGFTFSAYGMTWVRQ 110 APGKGLEWVSGIESSGDIRKYSDSVKGRFTISRDNAKNT LYLQMNSLRPEDTAMYYCARSRDRWVIGDTRKYDSRGQG TQVTVSS LV1141 CDR1 RYTYKTMG 111 CDR2 AIRWSGGATYYTDSVKG 112 CDR3 GRDWVVGGRGAFDY 113 VHH EVQLVESGGGVVQAGGSLKLSCAASGHTFSRYTYKTMGW 114 IRQAPGKEREFVAAIRWSGGATYYTDSVKGRFTISRDNA KNTVYLQMNSLKPEDTAIYYCAAGRDWVVGGRGAFDYWG QGTQVTVSS LV1142 CDR1 MGVID 115 CDR2 GIASGGATNVIDSVKG 116 CDR3 RQFGPDA 117 VHH EVQLVESGGGLVQAGGSLRLSCAASGSIFSMGVIDWHRQ 118 APGKQREWVGGIASGGATNVIDSVKGRFTISRDGAKNTV YLQMSSLKPEDTAVYYCHARQFGPDAWGQGTQVTVSS LV1142 H1 VHH EVQLVESGGGLVQPGGSLRLSCAASGSIFSMGVIDWHRQ 119 APGKQREWVGGIASGGATNVIDSVKGRFTISRDNSKNTL YLQMSSLRAEDTAVYYCHARQFGPDAWGQGTQVTVSS LV1142 H2 VHH EVQLVESGGGLVQPGGSLRLSCAASGSIFSMGVIDWHRQ 120 APGKQREWVGGIASGGATNVIDSVKGRFTISRDNAKNTV YLQMSSLRAEDTAVYYCHARQFGPDAWGQGTQVTVSS LV1142 H3 VHH EVQLVESGGGLVQPGGSLRLSCAASGSIFSMGVIDWHRQ 121 APGKQREWVGGIASGGATNVIDSVKGRFTISRDGAKNTV YLQMSSLRPEDTAVYYCHARQFGPDAWGQGTQVTVSS LV1143 CDR1 ANVMD 122 CDR1_var SNVMD 123 CDR2 GIAPDGSTGYADSVKG 124 CDR2_var GITSGGDTGYTASVKG 125 CDR3 RGNDY 126 CDR3_var RGDDY 127 VHH EVQLVESGGGLVQAGGSLRLSCVASGSGFSANVMDWYRQ 128 APGKQRQWVGGIAPDGSTGYADSVKGRFTISKDSAKNTV YLQMNSLKPEDTAVYYCKWRGNDYWGQGTQVTVSS LV1143_CDR1_v VHH EVQLVESGGGLVQAGGSLRLSCVASGSGFSSNVMDWYRQ 129 ar APGKQRQWVGGIAPDGSTGYADSVKGRFTISKDSAKNTV YLQMNSLKPEDTAVYYCKWRGNDYWGQGTQVTVSS 338699-2168 Reference Component Sequence SEQ ID LV1143_CDR2_v VHH EVQLVESGGGLVQAGGSLRLSCVASGSGFSANVMDWYRQ 130 ar APGKQRQWVGGITSGGDTGYTASVKGRFTISKDSAKNTV YLQMNSLKPEDTAVYYCKWRGNDYWGQGTQVTVSS LV1143_CDR3_v VHH EVQLVESGGGLVQAGGSLRLSCVASGSGFSANVMDWYRQ 131 ar APGKQRQWVGGIAPDGSTGYADSVKGRFTISKDSAKNTV YLQMNSLKPEDTAVYYCKWRGDDYWGQGTQVTVSS LV1143_allCDR_ VHH EVQLVESGGGLVQAGGSLRLSCVASGSGFSSNVMDWYRQ 132 var APGKQRQWVGGITSGGDTGYTASVKGRFTISKDSAKNTV YLQMNSLKPEDTAVYYCKWRGDDYWGQGTQVTVSS LV1144 CDR1 SNVMD 133 CDR2 GITSGGDTGYTASVKG 134 CDR3 RGDDY 135 VHH EVQLVESGGGLVQTGGSLSLSCAASRSTFTSNVMDWYRQ 136 APGKQREWVGGITSGGDTGYTASVKGRFTISRDNAKNTV YLQMNSLTPEDTAVYYCNVRGDDYWGQGTQVTVSS LV1145 CDR1 IEDMG 137 CDR2 DISRRGGTAYADAVKG 138 CDR3 WNSHHDAGGY 139 VHH EVQLVESGGGLVQPGGSLRLSCAASGTIFRIEDMGWSRQ 140 GPGKERESVADISRRGGTAYADAVKGRFTISRDNAKNTV FLEMNNLKPEDTAVYYCNAWNSHHDAGGYWGQGTQVTVS S LV1146 CDR1 KYTLNNMG 141 CDR2 TITRGGTTNYAGSVKG 142 CDR3 DDASVRNY 143 VHH EVQLVESGGGLVQAGGSLSLSCTASGMRYTKYTLNNMGW 144 YRQAPGKQRELVATITRGGTTNYAGSVKGRFTISRDNAK STVYLQMNSLKPEDTAVYYCNADDASVRNYWGQGTQVTV SS LV1147 CDR1 TYALA 145 CDR2 AIWGSGSITTYSTSVKG 146 CDR3 SLSGGIRASAYNY 147 VHH EVQLVESGGGLVQAGGSLRLSCAASGRTFSTYALAWFRQ 148 APRKEREFVAAIWGSGSITTYSTSVKGRFTISRDNAKNT VYLQMNSLKPEDTAVYYCAASLSGGIRASAYNYWGQGTQ VTVSS LV1148 CDR1 FSTMG 149 CDR2 AFTSGGTTNYADFVKG 150 CDR3 TATVLFGTTSDSDY 151 VHH EVQLVESGGGLVQAGGSLRLSCVASRNIFWFSTMGWYRQ 152 TPGKQRELVAAFTSGGTTNYADFVKGRFTISRDNAKNTV YLQMNSLNPEDTAVYYCKATATVLFGTTSDSDYWGQGTQ VTVSS LV1149 CDR1 SYAMG 153 CDR2 AITWSGHNTYYADSVKG 154 CDR3 RLDSGSYYYTEEYDY 155 VHH EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQ 156 APGKEREFVAAITWSGHNTYYADSVKGRFTISRDNAKNT VYLQMNSLKPEDTAVYYCAARLDSGSYYYTEEYDYWGQG TQVTVSS LV1150 CDR1 NYRMK 157 338699-2168 Reference Component Sequence SEQ ID CDR2 TISHRVGRTYYADSVKG 158 CDR3 VDMGGSDYAPHDYDY 159 VHH EVQLVESGGGLVQPGGSLRLSCAANGFTFSNYRMKWVRQ 160 APGKGLEWVSTISHRVGRTYYADSVKGRFTISRDNAKNT VYLQMTSLKPEDTARYFCTRVDMGGSDYAPHDYDYTGQG TQVTVSS LV1151 CDR1 SYTMG 161 CDR2 SLSWSGGGTRYADSVKG 162 CDR3 HTEIPSDAMGWAFQGY 163 VHH EVQLVESGGGLVQAGDSLRLSCTGSEDTFGSYTMGWFRQ 164 APGKEREFVASLSWSGGGTRYADSVKGRFTISRDNAKNA VYPQMNDLKADDTAVYYCNKHTEIPSDAMGWAFQGYWGR GTQVTVSS LV1152 CDR1 IWSMG 165 CDR2 TVTSEGRIDYGGPVKG 166 CDR3 TGYTDYDDDRLRHDDY 167 VHH EVQLVESGGGSVQPGGSLRLICVASGTSFSIWSMGWYRQ 168 APGKQRELVATVTSEGRIDYGGPVKGRFTISRDSLKNTV YLQMNNLKPEDTAVYLCTYTGYTDYDDDRLRHDDYWGQG TQVTVSS LV1292 CDR1 IDAMD 169 CDR2 SITRGGITSYTNYADSVKG 170 CDR3 REIMVREGGMWSVVPVDY 171 VHH EVQLVESGGGLVQPGGSLRLSCAASGSIFSIDAMDWYRQ 172 APGRQRELVASITRGGITSYTNYADSVKGRFTIPRDNAK NTVYLQMNSLLPEDTAVYYCNVREIMVREGGMWSVVPVD YWGQGTQVTVSS Table 4B: Exemplary 5T4-specific CDR Sequences (IMGT) Reference Component Sequence SEQ ID LV1139 CDR1 GFAFSNYR 173 LV1139 (D) CDR2 ISHRVGRT 174 LV1139 H1 CDR TRVAMGGSDYAPHDYDY LV1139 H2 3 175 LV1139 H3 LV1138 CDR1 GDTLDYYA 176 LV1138 H1 CDR ISRSGGSV LV1138 H2 2 177 LV1138 H3 CDR3 AARSSAYSRRYDIYSGWTEYGY 178 LV1140 CDR1 GFTFSAYG 179 LV1140 H1 CDR2 IESSGDIR LV1140 H2 180 CDR3 ARSRDRWVIGDTRKYDS 181 LV1141 CDR1 GHTFSRYTYKT 182 CDR2 IRWSGGAT 183 CDR3 AAGRDWVVGGRGAFDY 184 LV1142 CDR1 GSIFSMGV 185 LV1142 H1 IASGG V1142 H2 CDR AT L 2 186 LV1142 H3 CDR3 HARQFGPDA 187 338699-2168 Reference Component Sequence SEQ ID LV1143 CDR1 GSGFSANV 188 CDR2 IAPDGST 189 CDR3 KWRGNDY 190 LV1144 CDR1 RSTFTSNV 191 CDR2 ITSGGDT 192 CDR3 NVRGDDY 193 LV1145 CDR1 GTIFRIED 194 CDR2 ISRRGGT 195 CDR3 NAWNSHHDAGGY 196 LV1146 CDR1 GMRYTKYTLNN 197 CDR2 ITRGGTT 198 CDR3 NADDASVRNY 199 LV1147 CDR1 GRTFSTYA 200 CDR2 IWGSGSIT 201 CDR3 AASLSGGIRASAYNY 202 LV1148 CDR1 RNIFWFST 203 CDR2 FTSGGTT 204 CDR3 KATATVLFGTTSDSDY 205 LV1149 CDR1 GRTFSSYA 206 CDR2 ITWSGHNT 207 CDR3 AARLDSGSYYYTEEYDY 208 LV1150 CDR1 GFTFSNYR 209 CDR2 ISHRVGRT 210 CDR3 TRVDMGGSDYAPHDYDY 211 LV1151 CDR1 EDTFGSYT 212 CDR2 LSWSGGGT 213 CDR3 NKHTEIPSDAMGWAFQGY 214 LV1152 CDR1 GTSFSIWS 215 CDR2 VTSEGRI 216 CDR3 TYTGYTDYDDDRLRHDDY 217 LV1292 CDR1 GSIFSIDA 218 CDR2 ITRGGITSYT 219 CDR3 NVREIMVREGGMWSVVPVDY 220 Table 4C: Exemplary 5T4-specific CDR Sequences (Chothia) Reference Component Sequence SEQ ID LV1139 CDR1 GFAFSNY 221 LV1139 (D) LV1139 H1 CDR2 SHRVGR 222 LV1139 H2 CDR3 VAMGGSDYAPHDYDY 223 LV1139 H3 LV1138 CDR1 GDTLDYY 224 338699-2168 Reference Component Sequence SEQ ID LV1138 H1 CDR2 SRSGGS 225 LV1138 H2 38 H3 CDR3 RSSAYSRRYDIYSGWTEYGY LV11 226 LV1140 CDR1 GFTFSAY 227 LV1140 H1 ESSGDI LV1140 H2 CDR2 228 CDR3 SRDRWVIGDTRKYDS 229 LV1141 CDR1 GHTFSRYTYK 230 CDR2 RWSGGA 231 CDR3 GRDWVVGGRGAFDY 232 LV1142 CDR1 GSIFSMG 233 LV1142 H1 ASGGA LV1142 H2 CDR2 234 LV1142 H3 CDR3 RQFGPDA 235 LV1143 CDR1 GSGFSAN 236 CDR2 APDGS 237 CDR3 RGNDY 238 LV1144 CDR1 RSTFTSN 239 CDR2 TSGGD 240 CDR3 RGDDY 241 LV1145 CDR1 GTIFRIE 242 CDR2 SRRGG 243 CDR3 WNSHHDAGGY 244 LV1146 CDR1 GMRYTKYTLN 245 CDR2 TRGGT 246 CDR3 DDASVRNY 247 LV1147 CDR1 GRTFSTY 248 CDR2 WGSGSI 249 CDR3 SLSGGIRASAYNY 250 LV1148 CDR1 RNIFWFS 251 CDR2 TSGGT 252 CDR3 TATVLFGTTSDSDY 253 LV1149 CDR1 GRTFSSY 254 CDR2 TWSGHN 255 CDR3 RLDSGSYYYTEEYDY 256 LV1150 CDR1 GFTFSNY 257 CDR2 SHRVGR 258 CDR3 VDMGGSDYAPHDYDY 259 LV1151 CDR1 EDTFGSY 260 CDR2 SWSGGG 261 CDR3 HTEIPSDAMGWAFQGY 262 LV1152 CDR1 GTSFSIW 263 CDR2 TSEGR 264 CDR3 TGYTDYDDDRLRHDDY 265 338699-2168 Reference Component Sequence SEQ ID LV1292 CDR1 GSIFSID 266 CDR2 TRGGITSY 267 CDR3 REIMVREGGMWSVVPVDY 268 Table 4D: Exemplary 5T4-specific CDR Sequences (Combined) Reference Component Sequence SEQ ID LV1139 CDR1 GFAFSNYRMK 269 LV1139 (D) TISHRVG LV1139 H1 CDR2 RTYYADSVKG 270 LV1139 H2 CDR3 TRVAMGGSDYAPHDYDY 271 LV1139 H3 LV1138 CDR1 GDTLDYYAIG 272 LV1138 H1 YISRSGGSVYYADSVK LV1138 H2 CDR2 G 273 LV1138 H3 CDR3 AARSSAYSRRYDIYSGWTEYGY 274 LV1140 CDR1 GFTFSAYGMT 275 LV1140 H1 40 H2 CDR GIESSGDIRKYSDSVKG LV11 2 276 CDR3 ARSRDRWVIGDTRKYDS 277 LV1141 CDR1 GHTFSRYTYKTMG 278 CDR2 AIRWSGGATYYTDSVKG 279 CDR3 AAGRDWVVGGRGAFDY 280 LV1142 CDR1 GSIFSMGVID 281 LV1142 H1 2 H2 CDR2 GIASGGATNVIDSVKG LV114 282 LV1142 H3 CDR3 HARQFGPDA 283 LV1143 CDR1 GSGFSANVMD 284 CDR2 GIAPDGSTGYADSVKG 285 CDR3 KWRGNDY 286 LV1144 CDR1 RSTFTSNVMD 287 CDR2 GITSGGDTGYTASVKG 288 CDR3 NVRGDDY 289 LV1145 CDR1 GTIFRIEDMG 290 CDR2 DISRRGGTAYADAVKG 291 CDR3 NAWNSHHDAGGY 292 LV1146 CDR1 GMRYTKYTLNNMG 293 CDR2 TITRGGTTNYAGSVKG 294 CDR3 NADDASVRNY 295 LV1147 CDR1 GRTFSTYALA 296 CDR2 AIWGSGSITTYSTSVKG 297 CDR3 AASLSGGIRASAYNY 298 LV1148 CDR1 RNIFWFSTMG 299 CDR2 AFTSGGTTNYADFVKG 300 CDR3 KATATVLFGTTSDSDY 301 338699-2168 Reference Component Sequence SEQ ID LV1149 CDR1 GRTFSSYAMG 302 CDR2 AITWSGHNTYYADSVKG 303 CDR3 AARLDSGSYYYTEEYDY 304 LV1150 CDR1 GFTFSNYRMK 305 CDR2 TISHRVGRTYYADSVKG 306 CDR3 TRVDMGGSDYAPHDYDY 307 LV1151 CDR1 EDTFGSYTMG 308 CDR2 SLSWSGGGTRYADSVKG 309 CDR3 NKHTEIPSDAMGWAFQGY 310 LV1152 CDR1 GTSFSIWSMG 311 CDR2 TVTSEGRIDYGGPVKG 312 CDR3 TYTGYTDYDDDRLRHDDY 313 LV1292 CDR1 GSIFSIDAMD 314 CDR2 SITRGGITSYTNYADSVKG 315 CDR3 NVREIMVREGGMWSVVPVDY 316 [0120] One of skill in the art will recognize that there are many numbering systems known in the art to define the CDR sequences of a binding agent, including AbM, Kabat, Chothia, and IMGT systems. An overview of these systems is provided in Dondelinger et al., Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface/Residue Definition. Front Immunol.2018 Oct 16;9:2278. CDR sequences of the 5T4- specific antigen-binding domains as defined by Kabat, IMGT, and Chothia are provided in Tables 4A, 4B, and 4C above. The description of the binding agents herein refers to the CDRs as defined by the Kabat numbering system. However, one of skill in the art is able to correlate a CDR sequence defined by one numbering system to the same CDR sequence as defined by another system. [0121] In some embodiments, a combination of CDR numbering systems may be used. In such embodiments, CDR sequences of a given binding agent as determined by multiple numbering systems (e.g., a CDR1 sequence as determined by the Kabat, IMGT, and Chothia numbering systems) are compiled into a single sequence that encompasses the entirety of each of the CDR amino acid ranges in the variable region. As an illustrative example, CDRs for the 6H4 VHH described in Tables 5A, 5B, and 5C are shown in Fig.16. The N- and C- terminal ranges for each CDR region are indicated by dashed lines. The combined CDR sequences covering the entirety of these ranges are shown in the last row. Illustrative combined CDRs for 5T4 binding agents are shown in Table 4D. [0122] As an illustrative example, a 6H4 VHH comprising a CDR1 amino acid sequence comprising SEQ ID NO: 27, a CDR2 amino acid sequence comprising SEQ ID NO: 28, and a CDR3 amino acid sequence comprising SEQ ID NO: 29 as defined by the Kabat numbering 338699-2168 system will be understood by one of skill in the art to be the equivalent of (1) a 6H4 VHH comprising a CDR1 amino acid sequence comprising SEQ ID NO: 46, a CDR2 amino acid sequence comprising SEQ ID NO: 47, and a CDR3 amino acid sequence comprising SEQ ID NO: 48 as defined by the IMGT numbering system; (2) a 6H4 VHH comprising a CDR1 amino acid sequence comprising SEQ ID NO: 64, a CDR2 amino acid sequence comprising SEQ ID NO: 65, and a CDR3 amino acid sequence comprising SEQ ID NO: 66 as defined by the Chothia numbering system; and (3) a 6H4 VHH comprising a CDR1 amino acid sequence comprising SEQ ID NO: 85, a CDR2 amino acid sequence comprising SEQ ID NO: 86, and a CDR3 amino acid sequence comprising SEQ ID NO: 87 as defined by combining all the numbering systems. [0123] In some embodiments, the first antigen-binding domain comprise one or more framework regions (FR). In some embodiments, the binding agent disclosed herein are of the format FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Exemplary framework regions are shown in Table 5 below. In some embodiments, the first antigen-binding domain comprises an FR1 amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 317, 321, 325, 329, and 333. In some embodiments, the first antigen-binding domain comprises an FR1 amino acid sequence that comprises or consists of SEQ ID NOs: 317, 321, 325, 329, and 333. In some embodiments, the first antigen-binding domain comprises an FR2 amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 318, 322, 326, 330, and 334. In some embodiments, the first antigen-binding domain comprises an FR2 amino acid sequence that comprises or consists of SEQ ID NOs: 318, 322, 326, 330, and 334. In some embodiments, the first antigen-binding domain comprises an FR3 amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 319, 323, 327, 331, and 335. In some embodiments, the first antigen-binding domain comprises an FR3 amino acid sequence that comprises or consists of SEQ ID NOs: 319, 323, 327, 331, and 335. In some embodiments, the first antigen-binding domain comprises an FR4 amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 320, 324, 328, 332, and 336. In some embodiments, the first antigen-binding domain comprises an FR4 amino acid sequence that comprises or consists of SEQ ID NOs: 320, 324, 328, 332, and 336. In some embodiments, the first antigen-binding domain comprises or consists of, from N’ to C’ terminus, SEQ ID NOs: 317, 88, 318, 89, 319, 90, and 320. In some embodiments, the first antigen-binding domain comprises or consists of, from N’ to C’ terminus, SEQ ID NOs: 321, 98, 322, 99, 323, 100, and 324. In some embodiments, the first 338699-2168 antigen-binding domain comprises or consists of, from N’ to C’ terminus, SEQ ID NOs: 325, 105, 326, 106, 327, 107, and 328. In some embodiments, the first antigen-binding domain comprises or consists of, from N’ to C’ terminus, SEQ ID NOs: 329, 111, 330, 112, 331, 113, and 332. In some embodiments, the first antigen-binding domain comprises or consists of, from N’ to C’ terminus, SEQ ID NOs: 333, 115, 334, 116, 335, 117, and 336. Table 5: Exemplary Framework Regions Ref Domain Sequence SEQ ID LV1139 FR1 EVQLVESGGGLVQPGGSLRLSCAANGFAFS 317 FR2 WVRQAPGKGLEWVS 318 FR3 RFTISRDNAKNTVYLQMNSLKPEDTARYFCTR 319 FR4 TGQGTQVTVSS 320 LV1138 FR1 EVQLVESGGGLVQAGGSLRLSCAASGDTLD 321 FR2 WFRQAPGKEREGVS 322 FR3 RFTISRDSAKNTVYLQMVSLKPEDTAVYYCAA 323 FR4 WGQGTQVTVSS 324 LV1140 FR1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS 325 FR2 WVRQAPGKGLEWVS 326 FR3 RFTISRDNAKNTLYLQMNSLKPEDTAMYYCAR 327 FR4 RGQGTQVTVSS 328 LV1141 FR1 EVQLVESGGGVVQAGGSLKLSCAASGHTFS 329 FR2 WIRQAPGKEREFVA 330 FR3 RFTISRDNAKNTVYLQMNSLKPEDTAIYYCAA 331 FR4 WGQGTQVTVSS 332 LV1142 FR1 EVQLVESGGGLVQAGGSLRLSCAASGSIFS 333 FR2 WHRQAPGKQREWVG 334 FR3 RFTISRDGAKNTVYLQMSSLKPEDTAVYYCHA 335 FR4 WGQGTQVTVSS 336 [0124] In some embodiments, the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 88, a CDR2 amino acid sequence comprising SEQ ID NO: 89, and a CDR3 amino acid sequence comprising SEQ ID NO: 90. In some embodiments, one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 88, wherein one or two amino acids are modified. In some embodiments, CDR2 comprises the amino acid sequence of SEQ ID NO: 89, wherein one or two amino acids are modified. In some embodiments, CDR3 comprises the amino acid sequence of SEQ ID NO: 90, wherein one or two amino acids are modified. In some embodiments, the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 88, a CDR2 amino acid 338699-2168 sequence consisting of SEQ ID NO: 89, and a CDR3 amino acid sequence consisting of SEQ ID NO: 90. In some embodiments, the CDR3 sequence differs from the CDR3 sequence set forth in SEQ ID NO: 90 in one position, preferably position 2, wherein the substitution preferably is to D. In some embodiments, the CDR3 sequence comprises or consists of SEQ ID NO: 91. [0125] In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 92. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 92. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 92. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 93. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 93. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 93. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 94. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 94. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 94. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 95. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 95. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 95. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 96. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 96. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 96. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 97. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 97. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 97. [0126] In some embodiments, the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 98, a CDR2 amino acid sequence comprising SEQ ID NO: 99, and a CDR3 amino acid sequence comprising SEQ ID NO: 100. In some embodiments, one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 98, wherein one or two amino acids are modified. In some embodiments, CDR2 comprises the amino acid 338699-2168 sequence of SEQ ID NO: 99, wherein one or two amino acids are modified. In some embodiments, CDR3 comprises the amino acid sequence of SEQ ID NO: 100, wherein one or two amino acids are modified. In some embodiments, the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 98, a CDR2 amino acid sequence consisting of SEQ ID NO: 99, and a CDR3 amino acid sequence consisting of SEQ ID NO: 100. [0127] In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 101. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 101. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 101. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 102. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 102. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 102. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 103. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 103. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 103. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 104. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 104. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 104. [0128] In some embodiments, the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 105, a CDR2 amino acid sequence comprising SEQ ID NO: 106, and a CDR3 amino acid sequence comprising SEQ ID NO: 107. In some embodiments, one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 105, wherein one or two amino acids are modified. In some embodiments, CDR2 comprises the amino acid sequence of SEQ ID NO: 106, wherein one or two amino acids are modified. In some embodiments, CDR3 comprises the amino acid sequence of SEQ ID NO: 107, wherein one or two amino acids are modified. In some embodiments, the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 105, a CDR2 amino acid sequence consisting of SEQ ID NO: 106, and a CDR3 amino acid sequence consisting of SEQ ID NO: 107. 338699-2168 [0129] In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 108. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 108. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 108. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 109. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 109. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 109. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 110. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 110. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 110. [0130] In some embodiments, the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 111, a CDR2 amino acid sequence comprising SEQ ID NO: 112, and a CDR3 amino acid sequence comprising SEQ ID NO: 113. In some embodiments, one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 111, wherein one or two amino acids are modified. In some embodiments, CDR2 comprises the amino acid sequence of SEQ ID NO: 112, wherein one or two amino acids are modified. In some embodiments, CDR3 comprises the amino acid sequence of SEQ ID NO: 113, wherein one or two amino acids are modified. In some embodiments, the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 111, a CDR2 amino acid sequence consisting of SEQ ID NO: 112, and a CDR3 amino acid sequence consisting of SEQ ID NO: 113. [0131] In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 114. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 114. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 114. [0132] In some embodiments, the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 115, a CDR2 amino acid sequence comprising SEQ ID NO: 116, and a CDR3 amino acid sequence comprising SEQ ID NO: 117. In some embodiments, one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion). In 338699-2168 some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 115, wherein one or two amino acids are modified. In some embodiments, CDR2 comprises the amino acid sequence of SEQ ID NO: 116, wherein one or two amino acids are modified. In some embodiments, CDR3 comprises the amino acid sequence of SEQ ID NO: 117, wherein one or two amino acids are modified. In some embodiments, the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 115, a CDR2 amino acid sequence consisting of SEQ ID NO: 116, and a CDR3 amino acid sequence consisting of SEQ ID NO: 117. [0133] In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 118. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 118. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 118. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 119 In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 119. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 119. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 120 In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 120. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 120. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 121 In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 121. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 121. [0134] In some embodiments, the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 122, a CDR2 amino acid sequence comprising SEQ ID NO: 124, and a CDR3 amino acid sequence comprising SEQ ID NO: 125. In some embodiments, one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 122, wherein one or two amino acids are modified. In some embodiments, CDR2 comprises the amino acid sequence of SEQ ID NO: 124, wherein one or two amino acids are modified. In some embodiments, CDR3 comprises the amino acid sequence of SEQ ID NO: 126, wherein one or two amino acids are modified. In some embodiments, the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 122, a CDR2 amino acid 338699-2168 sequence consisting of SEQ ID NO: 124, and a CDR3 amino acid sequence consisting of SEQ ID NO: 126. In some embodiments, the CDR1 sequence differs from the CDR1 sequence set forth in SEQ ID NO: 122 in one position, preferably position 1, wherein the substitution preferably is to S. In some embodiments, the CDR1 sequence comprises or consists of SEQ ID NO: 123. In some embodiments, the CDR2 sequence differs from the CDR2 sequence set forth in SEQ ID NO: 124 in one, two, three, four, five or six positions, preferably positions 3, 4, 5, 7, 11 and/or 12 wherein the substitutions preferably are to T, S, G, D, T and A, respectively. In some embodiments, the CDR2 sequence comprises or consists of SEQ ID NO: 125. In some embodiments, the CDR3 sequence differs from the CDR3 sequence set forth in SEQ ID NO: 126 in one position, preferably position 3, wherein the substitution preferably is to D. In some embodiments, the CDR3 sequence comprises or consists of SEQ ID NO: 127. [0135] In some embodiments, the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 123, a CDR2 amino acid sequence comprising SEQ ID NO: 125, and a CDR3 amino acid sequence comprising SEQ ID NO: 127. In some embodiments, one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 123, wherein one or two amino acids are modified. In some embodiments, CDR2 comprises the amino acid sequence of SEQ ID NO: 125, wherein one or two amino acids are modified. In some embodiments, CDR3 comprises the amino acid sequence of SEQ ID NO: 127, wherein one or two amino acids are modified. In some embodiments, the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 123, a CDR2 amino acid sequence consisting of SEQ ID NO: 125, and a CDR3 amino acid sequence consisting of SEQ ID NO: 127. [0136] In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 128. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 128. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 128. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 129. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 129. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 129. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 130. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 130. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 338699-2168 130. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 131. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 131. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 131. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 132. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 132. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 132. [0137] In some embodiments, the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 133, a CDR2 amino acid sequence comprising SEQ ID NO: 134, and a CDR3 amino acid sequence comprising SEQ ID NO: 135. In some embodiments, one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 133, wherein one or two amino acids are modified. In some embodiments, CDR2 comprises the amino acid sequence of SEQ ID NO: 134, wherein one or two amino acids are modified. In some embodiments, CDR3 comprises the amino acid sequence of SEQ ID NO: 135, wherein one or two amino acids are modified. In some embodiments, the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 133, a CDR2 amino acid sequence consisting of SEQ ID NO: 134, and a CDR3 amino acid sequence consisting of SEQ ID NO: 135. [0138] In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 136. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 136. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 136. [0139] In some embodiments, the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 137, a CDR2 amino acid sequence comprising SEQ ID NO: 138, and a CDR3 amino acid sequence comprising SEQ ID NO: 139. In some embodiments, one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 137, wherein one or two amino acids are modified. In some embodiments, CDR2 comprises the amino acid sequence of SEQ ID NO: 338699-2168 138, wherein one or two amino acids are modified. In some embodiments, CDR3 comprises the amino acid sequence of SEQ ID NO: 139, wherein one or two amino acids are modified. In some embodiments, the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 137, a CDR2 amino acid sequence consisting of SEQ ID NO: 138, and a CDR3 amino acid sequence consisting of SEQ ID NO: 139. [0140] In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 140. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 140. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 140. [0141] In some embodiments, the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 141, a CDR2 amino acid sequence comprising SEQ ID NO: 142, and a CDR3 amino acid sequence comprising SEQ ID NO: 143. In some embodiments, one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 141, wherein one or two amino acids are modified. In some embodiments, CDR2 comprises the amino acid sequence of SEQ ID NO: 142, wherein one or two amino acids are modified. In some embodiments, CDR3 comprises the amino acid sequence of SEQ ID NO: 143, wherein one or two amino acids are modified. In some embodiments, the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 141, a CDR2 amino acid sequence consisting of SEQ ID NO: 142, and a CDR3 amino acid sequence consisting of SEQ ID NO: 143. [0142] In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 144. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 144. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 144. [0143] In some embodiments, the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 145, a CDR2 amino acid sequence comprising SEQ ID NO: 146, and a CDR3 amino acid sequence comprising SEQ ID NO: 147. In some embodiments, one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 145, wherein one or two amino acids are modified. In some embodiments, CDR2 comprises the amino acid sequence of SEQ ID NO: 146, wherein one or two amino acids are modified. In some 338699-2168 embodiments, CDR3 comprises the amino acid sequence of SEQ ID NO: 147, wherein one or two amino acids are modified. In some embodiments, the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 145, a CDR2 amino acid sequence consisting of SEQ ID NO: 146, and a CDR3 amino acid sequence consisting of SEQ ID NO: 147. [0144] In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 148. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 148. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 148. [0145] In some embodiments, the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 149, a CDR2 amino acid sequence comprising SEQ ID NO: 150, and a CDR3 amino acid sequence comprising SEQ ID NO: 151. In some embodiments, one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 149, wherein one or two amino acids are modified. In some embodiments, CDR2 comprises the amino acid sequence of SEQ ID NO: 150, wherein one or two amino acids are modified. In some embodiments, CDR3 comprises the amino acid sequence of SEQ ID NO: 151, wherein one or two amino acids are modified. In some embodiments, the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 149, a CDR2 amino acid sequence consisting of SEQ ID NO: 150, and a CDR3 amino acid sequence consisting of SEQ ID NO: 151. [0146] In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 152. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 152. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 152. [0147] In some embodiments, the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 153, a CDR2 amino acid sequence comprising SEQ ID NO: 154, and a CDR3 amino acid sequence comprising SEQ ID NO: 155. In some embodiments, one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 153, wherein one or two amino acids are modified. In some embodiments, CDR2 comprises the amino acid sequence of SEQ ID NO: 154, wherein one or two amino acids are modified. In some 338699-2168 embodiments, CDR3 comprises the amino acid sequence of SEQ ID NO: 155, wherein one or two amino acids are modified. In some embodiments, the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 153, a CDR2 amino acid sequence consisting of SEQ ID NO: 154, and a CDR3 amino acid sequence consisting of SEQ ID NO: 155. [0148] In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 156. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 156. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 156. [0149] In some embodiments, the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 157, a CDR2 amino acid sequence comprising SEQ ID NO: 158, and a CDR3 amino acid sequence comprising SEQ ID NO: 159. In some embodiments, one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 157, wherein one or two amino acids are modified. In some embodiments, CDR2 comprises the amino acid sequence of SEQ ID NO: 158, wherein one or two amino acids are modified. In some embodiments, CDR3 comprises the amino acid sequence of SEQ ID NO: 159, wherein one or two amino acids are modified. In some embodiments, the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 157, a CDR2 amino acid sequence consisting of SEQ ID NO: 158, and a CDR3 amino acid sequence consisting of SEQ ID NO: 159. [0150] In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 160. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 160. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 160. [0151] In some embodiments, the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 161, a CDR2 amino acid sequence comprising SEQ ID NO: 162, and a CDR3 amino acid sequence comprising SEQ ID NO: 163. In some embodiments, one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 161, wherein one or two amino acids are modified. In some embodiments, CDR2 comprises the amino acid sequence of SEQ ID NO: 162, wherein one or two amino acids are modified. In some 338699-2168 embodiments, CDR3 comprises the amino acid sequence of SEQ ID NO: 163, wherein one or two amino acids are modified. In some embodiments, the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 161, a CDR2 amino acid sequence consisting of SEQ ID NO: 162, and a CDR3 amino acid sequence consisting of SEQ ID NO: 163. [0152] In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 164. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 164. In some embodiments, the first antigen-binding domain consists of SEQ ID NO:164. [0153] In some embodiments, the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 165, a CDR2 amino acid sequence comprising SEQ ID NO: 166, and a CDR3 amino acid sequence comprising SEQ ID NO: 167. In some embodiments, one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 165, wherein one or two amino acids are modified. In some embodiments, CDR2 comprises the amino acid sequence of SEQ ID NO: 166, wherein one or two amino acids are modified. In some embodiments, CDR3 comprises the amino acid sequence of SEQ ID NO: 167, wherein one or two amino acids are modified. In some embodiments, the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 165, a CDR2 amino acid sequence consisting of SEQ ID NO: 166, and a CDR3 amino acid sequence consisting of SEQ ID NO: 167. [0154] In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 168. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 168. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 168. [0155] In some embodiments, the binding agent described herein comprises a first antigen- binding domain comprising a CDR1 amino acid sequence comprising SEQ ID NO: 169, a CDR2 amino acid sequence comprising SEQ ID NO: 170, and a CDR3 amino acid sequence comprising SEQ ID NO: 171. In some embodiments, one or more of CDR1, CDR2, or CDR3 comprise one or two amino acid modification (e.g., a substitution, insertion, or deletion). In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 169, wherein one or two amino acids are modified. In some embodiments, CDR2 comprises the amino acid sequence of SEQ ID NO: 170, wherein one or two amino acids are modified. In some 338699-2168 embodiments, CDR3 comprises the amino acid sequence of SEQ ID NO: 171, wherein one or two amino acids are modified. In some embodiments, the first antigen-binding domain comprises a CDR1 amino acid sequence consisting of SEQ ID NO: 169, a CDR2 amino acid sequence consisting of SEQ ID NO: 170, and a CDR3 amino acid sequence consisting of SEQ ID NO: 171. [0156] In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 172. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 172. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 172. [0157] In some embodiments, the 5T4 binding agents described herein have a binding affinity (KD) for human 5T4 of less than 10−7 M, such as approximately less than 10−8 M, less than 10−9 M, or less than 10−10 M when determined by surface plasmon resonance (SPR) using recombinant human 5T4 as the analyte and the 5T4 binding agent as the ligand. In some embodiments, the binding agent binds to human 5T4 with an affinity that is at least 1.1-, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2.0-, 2.5-, 3.0-, 3.5-, 4.0-, 4.5-, 5.0-, 6.0-, 7.0-, 8.0-, 9.0-, or 10.0-fold or greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein). [0158] Methods of testing binding agents for the ability to bind a target antigen are known in the art and include any antibody-antigen binding assay, such as, for example, radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assays (see, e.g., Janeway et al., infra, U.S. Patent Application Publication No. 2002/0197266 Al, and U.S. Patent No.7,338,929). [0159] In some embodiments, the 5T4 binding agents and multi-specific binding agents comprising the same described herein are capable of binding to recombinant 5T4 protein with a KD of less than 10 nM, for example less than 5 nM, less than 2 nM, less than 1 nM or less than 0.5 nM, as determined by Bio-Layer Interferometry as used in Example 2 herein. [0160] In some embodiments, the 5T4 binding agents or multi-specific binding agents described herein bind to CHO-K1 cells transfected with human 5T4 with an EC50 of less than 10 nM, less than 8 nM, less than 6 nM, less than 4 nM or less than 2 nM, when tested as described in Example 5 herein. [0161] In some embodiments, the 5T4 binding agents or multi-specific binding agents described herein bind to 5T4-expressing U-251 cells with an EC50 of less than 5 nM, less than 4 nM, or less than 2 nM, when tested as described in Example 6 herein. [0162] In some embodiments, the 5T4 binding agents or multi-specific binding agents described herein bind to 5T4-expressing A-431 cells with an EC50 of less than 5 nM, less than 4 nM, or less than 2 nM when tested as described in Example 6 herein. 338699-2168 [0163] In some embodiments, the 5T4 binding agents or multi-specific binding agents described herein bind to Vγ9Vδ2 T cells with an EC50 of less than 5 nM, such as less than 2 nM, when tested as described in Example 8 herein. [0164] In some embodiments, the 5T4 binding agents or multi-specific binding agents described herein are not capable of binding cynomolgus 5T4 or murine 5T4. [0165] In some embodiments, the 5T4 binding agents or multi-specific binding agents described herein are capable of mediating killing of 5T4-expressing tumor cells, such as U- 251 or A-431, by Vγ9Vδ2 T cells. [0166] Preferably, the 5T4 binding agents or multi-specific binding agents described herein are capable of inducing killing of U-251 cells through activation of Vγ9Vδ2 T cells with an EC50 value of 100 pM or less, such as 20 pM or less, e.g., 10 pM or less, or even 7 pM or less, or 5 pM or less when tested as described in Example 9 herein. [0167] Preferably, the 5T4 binding agents or multi-specific binding agents described herein are capable of inducing killing of A-431 cells through activation of Vγ9Vδ2 T cells with an EC50 value of 100 pM or less, such as 20 pM or less, e.g., 10 pM or less, or even 7 pM or less, or 5 pM or less when tested as described in Example 9 herein. [0168] In some embodiments, the 5T4 binding agents or multi-specific binding agents described herein are able to induce tumor lysis in cervical squamous cell carcinoma, cervical adenocarcinoma, metastatic colorectal cancer, and/or glioblastoma when tested as described in Example 10 herein. [0169] In some embodiments, the 5T4 binding agents or multi-specific binding agents described herein are not capable of enhancing killing of 5T4-negative cells, such as 5T4 negative human cells, by Vγ9Vδ2 T cells. [0170] In some embodiments, the 5T4 binding agents or multi-specific binding agents described herein are capable of enhancing killing of 5T4-positive cells, such as 5T4 positive tumor cells, by Vγ9Vδ2 T cells. [0171] In some embodiments, the 5T4 binding agents and multi-specific binding agents comprising the same described herein are: (a) capable of binding to recombinant human 5T4 with a KD of less than 10 nM, for example less than 5 nM, less than 2 nM, less than 1 nM, or less than 0.5 nM, as determined by Bio-Layer Interferometry according to the methods described in Example 2 herein; and (b) capable of inducing killing of A-431 cells through activation of Vγ9Vδ2 T cells with an EC50 value of 10 pM or less, or even 7 pM or less, or 5 pM or less when tested as described in Example 9 herein. [0172] In some embodiments, the 5T4 binding agents and multi-specific binding agents comprising the same described herein are: 338699-2168 (a) capable of binding to recombinant human 5T4 with a KD of less than 10 nM, for example less than 5 nM, less than 2 nM, less than 1 nM, or less than 0.5 nM, as determined by Bio-Layer Interferometry according to the methods described in Example 2 herein; and (b) capable of inducing killing of U-251 cells through activation of Vγ9Vδ2 T cells with an EC50 value of 10 pM or less, or even 7 pM or less, or 5 pM or less when tested as described in Example 9 herein. [0173] In some embodiments, the 5T4 binding agents and multi-specific binding agents comprising the same described herein have all of the following properties: (a) bind recombinant human 5T4 with a KD of less than 50 nM, for example less than 15 nM, less than 5 nM, less than 4 nM, less than 3 nM, or less than 2 nM, as determined by Bio-Layer Interferometry according to the method described in Example 2 herein; (b) bind 5T4-expressing U-251 cells with an EC50 of less than 5 nM, less than 4 nM, or less than 2 nM, when tested for example as described in Example 6 herein; (c) binds 5T4-expressing A-431 cells with an EC50 of less than 5 nM, less than 4 nM, or less than 2 nM, when tested for example as described in Example 6 herein; (d) bind Vγ9Vδ2 T cells with an EC50 of less than 5 nM, such as less than 2 nM, when tested as described in Example 8 herein; (e) are capable of inducing killing of U-251 cells through activation of Vγ9Vδ2 T cells with an EC50 value of 100 pM or less, such as 20 pM or less, e.g., 10 pM or less, or even 7 pM or less, or 5 pM or less when tested as described in Example 9 herein; (f) are capable of inducing killing of A-431 cells through activation of Vγ9Vδ2 T cells with an EC50 value of 100 pM or less, such as 20 pM or less, e.g., 10 pM or less, or even 7 pM or less, or 5 pM or less when tested as described in Example 9 herein; and (g) are able to induce tumor lysis in cervical squamous cell carcinoma, cervical adenocarcinoma, metastatic colorectal cancer, and/or glioblastoma when tested as described in Example 10 herein. [0174] In the context of the present disclosure, “capable of competing” or “able to compete” or “competes” refers to any detectably significant reduction in the propensity for a particular binding molecule (e.g., a 5T4 antibody) to bind a particular binding partner (e.g., 5T4) in the presence of another molecule (e.g., a different 5T4 antibody) that binds the binding partner. Typically, competition means that saturating binding of the first binding agent to the antigen (5T4) blocks or prevents binding of the second binding agent, as determined by, e.g., biolayer interferometry (BLI) as described in Example 3 herein using sufficient amounts of the two (or more) competing binding agents. Additional methods for determining binding specificity by competitive inhibition may be found in for instance Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988), Colligan et 338699-2168 al., eds., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley InterScience N. Y., (1992, 1993), and Muller, Meth. Enzymol.92, 589-601 (1983). [0175] In some embodiments, disclosed herein are binding agents comprising an antigen- binding domain capable of binding human 5T4 wherein the binding agent is capable of competing for binding to human 5T4 with a binding agent having the sequence set forth in SEQ ID NOs: 92, 93, 94, 95, 96, 97, 101, 102, 103, 104, 108, 109, 110, 114, 118, 119, 120, 121, 128, 129, 130, 131, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, and 172, e.g. when tested as described in Example 3 herein. [0176] In some embodiments, the binding agent is capable of competing for binding to human 5T4 with a binding agent having the sequence set forth in SEQ ID NO:91 and with a binding agent having the sequence set forth in SEQ ID NO:96. [0177] In some embodiments, the binding agent is capable of competing for binding to human 5T4 with a binding agent having the sequence set forth in SEQ ID NO:91 and with a binding agent having the sequence set forth in SEQ ID NO:140. [0178] In some embodiments, the binding agent is capable of competing for binding to human 5T4 with a binding agent having the sequence set forth in SEQ ID NO:96 and with a binding agent having the sequence set forth in SEQ ID NO:140. [0179] In some embodiments, the binding agent is capable of competing for binding to human 5T4 with a binding agent having the sequence set forth in SEQ ID NO:91, with a binding agent having the sequence set forth in SEQ ID NO:96, and with a binding agent having the sequence set forth in SEQ ID NO:140. [0180] In some embodiments, the binding agent is capable of competing for binding to human 5T4 with a binding agent having the sequence set forth in SEQ ID NO:108 and with a binding agent having the sequence set forth in SEQ ID NO:114. [0181] In some embodiments, the binding agent is capable of competing for binding to human 5T4 with a binding agent having the sequence set forth in SEQ ID NO:118 and with a binding agent having the sequence set forth in SEQ ID NO:128. [0182] In some embodiments, the binding agent is capable of competing for binding to human 5T4 with a binding agent having the sequence set forth in SEQ ID NO:118 and with a binding agent having the sequence set forth in SEQ ID NO:136. [0183] In some embodiments, the binding agent is capable of competing for binding to human 5T4 with a binding agent having the sequence set forth in SEQ ID NO:128 and with a binding agent having the sequence set forth in SEQ ID NO:136. [0184] In some embodiments, the binding agent is capable of competing for binding to human 5T4 with a binding agent having the sequence set forth in SEQ ID NO:118, with a binding 338699-2168 agent having the sequence set forth in SEQ ID NO:128, and with a binding agent having the sequence set forth in SEQ ID NO:136. [0185] In some embodiments, the binding agent of the present disclosure binds to the same epitope on 5T4 as binding agent LV1138, LV1139, LV1140, LV1141, LV1142, LV1143, LV1144, LV1145, LV1146, LV1147, LV1148, LV1149, LV1150, LV1151, LVA1152, LV1292 or any of the variants of these binding agents set forth in SEQ ID NOs: 93, 94, 95, 96, 97, 102, 103, 104, 109, 110, 119, 120, 121, 129, 130, 131, or 132 disclosed herein and/or to the same epitope on Vδ2 as binding agent 5C8 or 6H4 (WO2015156673) or any of the variants of 5C8 set forth in SEQ ID NOs: 8, 9, 13, 17, 21, 22, or 26. There are several methods available for mapping binding agent binding epitopes on target antigens known in the art, including but not limited to: crosslinking coupled mass spectrometry, allowing identification of peptides that are part of the epitope, and X-ray crystallography identifying individual residues on the antigen that form the epitope. Epitope residues can be determined as being all amino acid residues with at least one atom less than or equal to 5 Å from the binding agent.5 Å was chosen as the epitope cutoff distance to allow for atoms within a van der Waals radius plus a possible water-mediated hydrogen bond. Next, epitope residues can be determined as being all amino acid residues with at least one atom less than or equal to 8 Å. Less than or equal to 8 Å is chosen as the epitope cutoff distance to allow for the length of an extended arginine amino acid. Crosslinking coupled mass spectrometry begins by binding the binding agent and the antigen with a mass labeled chemical crosslinker. Next the presence of the complex is confirmed using high mass MALDI detection. Because after crosslinking chemistry the Ab/Ag complex is extremely stable, many various enzymes and digestion conditions can be applied to the complex to provide many different overlapping peptides. Identification of these peptides is performed using high resolution mass spectrometry and MS/MS techniques. Identification of the crosslinked peptides is determined using mass tag linked to the cross-linking reagents. After MS/MS fragmentation and data analysis, peptides that are crosslinked and are derived from the antigen are part of the epitope, while peptides derived from the antibody are part of the paratope. All residues between the most N- and C-terminal crosslinked residue from the individual crosslinked peptides found are considered to be part of the epitope or paratope. [0186] In some embodiments, the binding agent binds to the same epitope on 5T4 as a binding agent having the sequence set forth in any one of SEQ ID NOs: 92, 93, 94, 95, 96, 97, 101, 102, 103, 104, 108, 109, 110, 114, 118, 119, 120, 121, 128, 129, 130, 131, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, and 172. [0187] In some embodiments, the binding agent binds to the same epitope on 5T4 as a binding agent having the sequence set forth in SEQ ID NO:91 and with a binding agent having the sequence set forth in SEQ ID NO:96. 338699-2168 [0188] In some embodiments, the binding agent binds to the same epitope on 5T4 as a binding agent having the sequence set forth in SEQ ID NO:91 and with a binding agent having the sequence set forth in SEQ ID NO:140. [0189] In some embodiments, the binding agent binds to the same epitope on 5T4 as a binding agent having the sequence set forth in SEQ ID NO:96 and with a binding agent having the sequence set forth in SEQ ID NO:140. [0190] In some embodiments, the binding agent binds to the same epitope on 5T4 as a binding agent having the sequence set forth in SEQ ID NO:91, with a binding agent having the sequence set forth in SEQ ID NO:96, and with a binding agent having the sequence set forth in SEQ ID NO:140. [0191] In some embodiments, the binding agent binds to the same epitope on 5T4 as a binding agent having the sequence set forth in SEQ ID NO:108 and with a binding agent having the sequence set forth in SEQ ID NO:114. [0192] In some embodiments, the binding agent binds to the same epitope on 5T4 as a binding agent having the sequence set forth in SEQ ID NO:118 and with a binding agent having the sequence set forth in SEQ ID NO:128. [0193] In some embodiments, the binding agent binds to the same epitope on 5T4 as a binding agent having the sequence set forth in SEQ ID NO:118 and with a binding agent having the sequence set forth in SEQ ID NO:136. [0194] In some embodiments, the binding agent binds to the same epitope on 5T4 as a binding agent having the sequence set forth in SEQ ID NO:128 and with a binding agent having the sequence set forth in SEQ ID NO:136. [0195] In some embodiments, the binding agent binds to the same epitope on 5T4 as a binding agent having the sequence set forth in SEQ ID NO:118, with a binding agent having the sequence set forth in SEQ ID NO:128, and with a binding agent having the sequence set forth in SEQ ID NO:136. Multi-specific Binding Agents [0196] In some embodiments, the 5T4 binding agent is a multi-specific binding agent. In some embodiments, the multi-specific binding agent comprises a first antigen-binding domain that specifically binds to human 5T4 described above and a second antigen-binding domain that specifically binds to a second target antigen or epitope (e.g., a bispecific binding agent). In some embodiments, the multi-specific binding agent comprises a first antigen-binding domain that specifically binds to human 5T4 described above, a second antigen-binding domain that 338699-2168 specifically binds to a second target antigen or epitope, and a third antigen-binding domain that specifically binds to a third target antigen or epitope. [0197] In some embodiments, the first, second, and/or third antigen-binding domain is a single-domain antibody. In some embodiments, the first, second, and/or third antigen-binding region is humanized. In some embodiments, the multi-specific binding agent is a bispecific binding agent, wherein the first antigen-binding domain is a VHH and the second antigen- binding region is a VHH. [0198] In some embodiments, the multi-specific binding agent is in a VHH-Fc format, i.e., the binding agent comprises two or more single-domain antigen-binding domains that are linked to each other via a human Fc domain dimer. In this format, each single-domain antigen-binding domain is fused to an Fc region polypeptide and the two fusion polypeptides form a dimeric bispecific binding agent via disulfide bridges in the hinge region. Such constructs typically do not contain full, or any, CH1 or light chain sequences. Figure 12B of WO 2006/064136 provides an illustration of an example of this embodiment. [0199] In some embodiments, the multi-specific binding agents provided herein comprise a first antigen-binding domain that specifically binds to human 5T4 and a second antigen-binding domain that specifically binds to an antigen expressed on an immune cell. The term “immune cell” refers to cells involved in mounting innate and adaptive immune responses, including but not limited to lymphocytes (such as T-cells and B-cells), natural killer (NK) cells, NKT cells, macrophages, monocytes, eosinophils, basophils, neutrophils, dendritic cells, and mast cells. In some embodiments, the immune cell is a T cell, such as a CD4+ T cell, a CD8+ T cell (also referred to as a cytotoxic T cell or CTL), or a γδ T cell. In some embodiments, the immune cell is a γδ T cell. [0200] In some embodiments, the second antigen-binding domain specifically binds to a target antigen selected from CD3, CD2, αβ T cell receptors,
Figure imgf000049_0001
T cell receptors (e.g., Vδ2 TCR, Vδ1 TCR, Vγ9, or Vδ3 TCR), CD56, CD16, CD19, and CD20. In some embodiments, the Vδ2 TCR is a human Vδ2 T cell receptor. In some embodiments, the human Vδ2 T cell receptor is a Vγ9Vδ2 T cell receptor. [0201] In some embodiments, the second antigen-binding domain specifically binds to a γδ T cell receptor. Antigen-binding domains that specifically bind to Vδ1+ T cells are known in the art. See e.g., WO 2021/032963. Antigen-binding domains that specifically bind to Vδ2+ T cell receptors are known in the art. See e.g., WO 2015/156673, WO 2022/008646, WO 2022/122973, and WO 2023/242319, each discloses antigen-binding domains that specifically bind to Vδ2, and each is incorporated herein by reference in its entirety. [0202] In some embodiments, the second antigen-binding domain specifically binds to human Vδ2. In some embodiments, the multi-specific binding agents provided herein are capable of 338699-2168 binding the Vδ2 chain of a Vγ9Vδ2-TCR. “Capable of binding the Vδ2 chain of a Vγ9Vδ2-TCR” means that the binding agent disclosed herein or multi-specific binding agent comprising the same can bind the Vδ2 chain as a separate molecule and/or as part of a Vγ9Vδ2-TCR. This can be referred to as a Vδ2 binding agent. However, the binding agent or multi-specific binding agent comprising the same will not bind to the Vγ9 chain as a separate molecule. Exemplary Vγ9Vδ2 TCR antigen-binding domain sequences are shown in Table 6A, Table 6B, Table 6C, and Table 6D. Table 6A: Exemplary Vδ2 TCR-specific CDR (Kabat) and VHH Sequences Reference Component Sequence SEQ ID 5C8 CDR1 NYAMG 1 CDR2 AISWSGGSTSYADSVKG 2 CDR3 QFSGADYGFGRLGIRGYEYDY 3 VHH EVQLVESGGGLVQAGGSLRLSCAASGRPFSNYAMGWF 4 RQAPGKEREFVAAISWSGGSTSYADSVKGRFTISRDN AKNTVYLQMNSPKPEDTAIYYCAAQFSGADYGFGRLG IRGYEYDYWGQGTQVTVSS 5C8 var1 CDR1 NYAMS 5 5C8 var2 CDR2 AISWSGGSTSYADSVKG 6 CDR3 QFSGADYGFGRLGIRGYEYDY 7 VHH (var1) EVQLLESGGGSVQPGGSLRLSCAASGRPFSNYAMSWF 8 RQAPGKEREFVSAISWSGGSTSYADSVKGRFTISRDN SKNTLYLQMNSLRAEDTAVYYCAAQFSGADYGFGRLG IRGYEYDYWGQGTQVTVSS VHH (var2) EVQLLESGGGLVQPGGSLRLSCAASGRPFSNYAMSWF 9 RQAPGKEREFVSAISWSGGSTSYADSVKGRFTISRDN SKNTLYLQMNSLRAEDTAVYYCAAQFSGADYGFGRLG IRGYEYDYWGQGTLVTVSS 5C8 var1 (Y105F) CDR1 NYAMS 10 CDR2 AISWSGGSTSYADSVKG 11 CDR3 QFSGADFGFGRLGIRGYEYDY 12 VHH EVQLLESGGGSVQPGGSLRLSCAASGRPFSNYAMSWF 13 RQAPGKEREFVSAISWSGGSTSYADSVKGRFTISRDN SKNTLYLQMNSLRAEDTAVYYCAAQFSGADFGFGRLG IRGYEYDYWGQGTQVTVSS 5C8 var1 (Y105S) CDR1 NYAMS 14 CDR2 AISWSGGSTSYADSVKG 15 CDR3 QFSGADSGFGRLGIRGYEY 16 VHH EVQLLESGGGSVQPGGSLRLSCAASGRPFSNYAMSWF 17 RQAPGKEREFVSAISWSGGSTSYADSVKGRFTISRDN SKNTLYLQMNSLRAEDTAVYYCAAQFSGADSGFGRLG IRGYEYDYWGQGTQVTVSS 5C8 var1 (Y105F- CDR1 NYAMS 18 R109A) CDR2 AISWSGGSTSYADSVKG 19 CDR3 QFSGADFGFGALGIRGYEYDY 20 VHH EVQLLESGGGSVQPGGSLRLSCAASGRPFSNYAMSWF 21 RQAPGKEREFVSAISWSGGSTSYADSVKGRFTISRDN SKNTLYLQMNSLRAEDTAVYYCAAQFSGADFGFGALG IRGYEYDYWGQGTQVTVSS 5C8 var1 (E1D- VHH DVQLLESGGGSVQPGGSLRLSCAASGRPFSNYAMSWF 22 Y105F-R109A) RQAPGKEREFVSAISWSGGSTSYADSVKGRFTISRDN SKNTLYLQMNSLRAEDTAVYYCAAQFSGADFGFGALG IRGYEYDYWGQGTQVTVSS 338699-2168 Reference Component Sequence SEQ ID 5C8 var1 (Y105F- CDR1 NYAMS 23 G108A) CDR2 AISWSGGSTSYADSVKG 24 CDR3 QFSGADFGFARLGIRGYEYDY 25 VHH EVQLLESGGGSVQPGGSLRLSCAASGRPFSNYAMSWF 26 RQAPGKEREFVSAISWSGGSTSYADSVKGRFTISRDN SKNTLYLQMNSLRAEDTAVYYCAAQFSGADFGFARLG IRGYEYDYWGQGTQVTVSS 6H4 CDR1 NYGMG 27 CDR2 GISWSGGSTDYADSVKG 28 CDR3 VFSGAETAYYPSDDYDY 29 VHH EVQLVESGGGLVQAGGSLRLSCAASGRPFSNYGMGWF 30 RQAPGKKREFVAGISWSGGSTDYADSVKGRFTISRDN AKNTVYLQMNSLKPEDTAVYYCAAVFSGAETAYYPSD DYDYWGQGTQVTVSS Table 6B: Exemplary Vδ2 TCR-specific CDR Sequences (IMGT) Reference Component Sequence SEQ ID 5C8 CDR1 GRPFSNYA 31 5C8 var1 CDR2 ISWSGGST 32 5C8 var2 CDR3 AAQFSGADYGFGRLGIRGYEYDY 33 5C8 var1 (Y105F) CDR1 GRPFSNYA 34 CDR2 ISWSGGST 35 CDR3 AAQFSGADFGFGRLGIRGYEYDY 36 5C8 var1 (Y105S) CDR1 GRPFSNYA 37 CDR2 ISWSGGST 38 CDR3 AAQFSGADSGFGRLGIRGYEYDY 39 5C8 var1 (Y105F- CDR1 GRPFSNYA 40 R109A) CDR2 ISWSGGST 41 5C8 var1 (E1D- CDR3 AAQFSGADFGFGALGIRGYEYDY 42 105F-R109A) 5C8 var1 (Y105F- CDR1 GRPFSNYA 43 G108A) CDR2 ISWSGGST 44 CDR3 AAQFSGADFGFARLGIRGYEYDY 45 6H4 CDR1 GRPFSNYG 46 CDR2 ISWSGGST 47 CDR3 AAVFSGAETAYYPSDDYDY 48 Table 6C: Exemplary Vδ2 TCR-specific CDR Sequences (Chothia) Reference Component Sequence SEQ ID 5C8 CDR1 GRPFSNY 49 5C8 var1 CDR2 SWSGGS 50 5C8 var2 CDR3 QFSGADYGFGRLGIRGYEYDY 51 5C8 var1 (Y105F) CDR1 GRPFSNY 52 CDR2 SWSGGS 53 CDR3 QFSGADFGFGRLGIRGYEYDY 54 5C8 var1 (Y105S) CDR1 GRPFSNY 55 CDR2 SWSGGS 56 CDR3 QFSGADSGFGRLGIRGYEYDY 57 5C8 var1 (Y105F- CDR1 GRPFSNY 58 R109A) CDR2 SWSGGS 59 5C8 var1 (E1D- CDR3 QFSGADFGFGALGIRGYEYDY 60 105F-R109A) 5C8 var1 (Y105F- CDR1 GRPFSNY 61 G108A) CDR2 SWSGGS 62 CDR3 QFSGADFGFARLGIRGYEYDY 63 338699-2168 Reference Component Sequence SEQ ID 6H4 CDR1 GRPFSNY 64 CDR2 SWSGGS 65 CDR3 VFSGAETAYYPSDDYDY 66 Table 6D: Exemplary Vδ2 TCR-specific CDR Sequences (Combined) Reference Component Sequence SEQ ID 5C8 CDR1 GRPFSNYAMG 67 CDR2 AISWSGGSTSYADSVKG 68 CDR3 AAQFSGADYGFGRLGIRGYEYDY 69 5C8 var1 CDR1 GRPFSNYAMS 70 5C8 var2 CDR2 AISWSGGSTSYADSVKG 71 CDR3 AAQFSGADYGFGRLGIRGYEYDY 72 5C8 var1 (Y105F) CDR1 GRPFSNYAMS 73 CDR2 AISWSGGSTSYADSVKG 74 CDR3 AAQFSGADFGFGRLGIRGYEYDY 75 5C8 var1 (Y105S) CDR1 GRPFSNYAMS 76 CDR2 AISWSGGSTSYADSVKG 77 CDR3 AAQFSGADSGFGRLGIRGYEYDY 78 5C8 var1 (Y105F- CDR1 GRPFSNYAMS 79 R109A) CDR2 AISWSGGSTSYADSVKG 80 5C8 var1 (E1D- CDR3 AAQFSGADFGFGALGIRGYEYDY 81 Y105F-R109A) 5C8 var1 (Y105F- CDR1 GRPFSNYAMS 82 G108A) CDR2 AISWSGGSTSYADSVKG 83 CDR3 AAQFSGADFGFARLGIRGYEYDY 84 6H4 CDR1 GRPFSNYGMG 85 CDR2 GISWSGGSTDYADSVKG 86 CDR3 AAVFSGAETAYYPSDDYDY 87 [0203] In some embodiments, the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 88, a CDR2 of SEQ ID NO: 89, and a CDR3 of SEQ ID NO: 90 or 91; and (ii) a second antigen-binding domain that binds to Vδ2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs). In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 92, 93, 94, 95, 96, and 97, and the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain comprises a sequence selected from SEQ ID NOs: 92, 93, 94, 95, 96, and 97, and the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain consists of a sequence 338699-2168 selected from SEQ ID NOs: 92, 93, 94, 95, 96, and 97, and the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. [0204] In some embodiments, the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 98, a CDR2 of SEQ ID NO: 99, and a CDR3 of SEQ ID NO: 100; and (ii) a second antigen-binding domain that binds to Vδ2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs). In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 101, 102, 103, and 104, and the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain comprises any one of SEQ ID NOs: 101, 102, 103, and 104, and the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain consists of any one of SEQ ID NOs: 101, 102, 103, and 104, and the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. [0205] In some embodiments, the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 105, a CDR2 of SEQ ID NO: 106, and a CDR3 of SEQ ID NO: 107; and (ii) a second antigen-binding domain that binds to Vδ2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs). In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 108, 109, and 110, and the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain comprises any one of SEQ ID NOs: 108, 109, and 110, and the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain consists of any one of SEQ ID NOs: 108, 109, and 110, and the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. 338699-2168 [0206] In some embodiments, the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 111, a CDR2 of SEQ ID NO: 112, and a CDR3 of SEQ ID NO: 113; and (ii) a second antigen-binding domain that binds to Vδ2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs). In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 114, and the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 114, and the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 114, and the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. [0207] In some embodiments, the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 115, a CDR2 of SEQ ID NO: 116, and a CDR3 of SEQ ID NO: 117; and (ii) a second antigen-binding domain that binds to Vδ2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs). In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 118, 119, 120, and 121, and the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain comprises any one of SEQ ID NOs: 118, 119, 120, and 121, and the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain consists of any one of SEQ ID NOs: 118, 119, 120, and 121, and the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. [0208] In some embodiments, the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 122 or 123, a CDR2 of SEQ ID NO: 124 or 125, and a CDR3 of SEQ ID NO: 126 or 127; and (ii) a second antigen- 338699-2168 binding domain that binds to Vδ2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs). In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 128, 129, 130, 131, and 132, and the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen- binding domain comprises any one of SEQ ID NOs: 128, 129, 130, 131, and 132, and the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain consists of any one of SEQ ID NOs: 128, 129, 130, 131, and 132, and the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. [0209] In some embodiments, the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 133, a CDR2 of SEQ ID NO: 134, and a CDR3 of SEQ ID NO: 135; and (ii) a second antigen-binding domain that binds to Vδ2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs). In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 136, and the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 136, and the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 136, and the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. [0210] In some embodiments, the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 137, a CDR2 of SEQ ID NO: 138, and a CDR3 of SEQ ID NO: 139; and (ii) a second antigen-binding domain that binds to Vδ2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs). In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, 338699-2168 at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 140, and the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 140, and the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 140, and the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. [0211] In some embodiments, the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 141, a CDR2 of SEQ ID NO: 142, and a CDR3 of SEQ ID NO: 143; and (ii) a second antigen-binding domain that binds to Vδ2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs). In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 144, and the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 144, and the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 144, and the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. [0212] In some embodiments, the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 145, a CDR2 of SEQ ID NO: 146, and a CDR3 of SEQ ID NO: 147; and (ii) a second antigen-binding domain that binds to Vδ2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs). In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 148, and the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% 338699-2168 identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 148, and the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 148, and the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. [0213] In some embodiments, the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 149, a CDR2 of SEQ ID NO: 150, and a CDR3 of SEQ ID NO: 151; and (ii) a second antigen-binding domain that binds to Vδ2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs). In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 152, and the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 152, and the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 152, and the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. [0214] In some embodiments, the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 153, a CDR2 of SEQ ID NO: 154, and a CDR3 of SEQ ID NO: 155; and (ii) a second antigen-binding domain that binds to Vδ2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs). In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 156, and the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 156, and the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain consists of SEQ ID 338699-2168 NO: 156, and the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. [0215] In some embodiments, the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 157, a CDR2 of SEQ ID NO: 158, and a CDR3 of SEQ ID NO: 159; and (ii) a second antigen-binding domain that binds to Vδ2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs). In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 160, and the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 160, and the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 160, and the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. [0216] In some embodiments, the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 161, a CDR2 of SEQ ID NO: 162, and a CDR3 of SEQ ID NO: 163; and (ii) a second antigen-binding domain that binds to Vδ2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs). In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 164, and the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 164, and the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 164, and the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. [0217] In some embodiments, the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 165, a CDR2 of SEQ 338699-2168 ID NO: 166, and a CDR3 of SEQ ID NO: 167; and (ii) a second antigen-binding domain that binds to Vδ2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs). In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 168, and the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 168, and the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 168, and the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. [0218] In some embodiments, the multi-specific binding agent comprises (i) a first antigen- binding domain that binds to 5T4 and comprises a CDR1 of SEQ ID NO: 169, a CDR2 of SEQ ID NO: 170, and a CDR3 of SEQ ID NO: 171; and (ii) a second antigen-binding domain that binds to Vδ2 and comprises a combination of CDR sequences selected from Table 6A (e.g., the CDR combinations for any of the described VHHs). In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 172, and the second antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 172, and the second antigen-binding domain comprises a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 172, and the second antigen-binding domain consists of a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 22, 26, and 30. [0219] In some embodiments, any of the multi-specific binding agents disclosed herein are modified to reduce or eliminate the formation of pyroglutamic acid at the N-terminus of the modified multi-specific binding agents as compared to the unmodified multi-specific binding agents of this disclosure. In some embodiments, the glutamate residue (E) or glutamine (Q) residue at the first position is modified to an aspartic acid residue (D). In some embodiments, 338699-2168 any of the multi-specific binding agents of the present disclosure may comprise an aspartic acid (D) residue in the first position. [0220] In some embodiments, the 5T4 binding agent, or multi-specific binding agent comprising the same, comprises an aspartic acid or a glutamic acid residue at the N-terminal amino acid position and elutes earlier in reversed-phase HPLC than a 5T4 binding agent or multi-specific binding agent comprising the same that comprises a pyroglutamate residue at the N-terminal amino acid position. [0221] In some embodiments, the Vδ2 binding agent, or multi-specific binding agent comprising the same, comprises an aspartic acid or a glutamic acid residue at the N-terminal amino acid position and elutes earlier in reversed-phase HPLC than a Vδ2 binding agent or multi-specific binding agent comprising the same that comprises a pyroglutamate residue at the N-terminal amino acid position. [0222] In some embodiments, the second antigen-binding domain is capable of binding the Vγ9 chain of a human Vγ9Vδ2 T cell receptor. In some embodiments, the multi-specific binding agents provided herein are capable of binding the Vγ9 chain of a Vγ9Vδ2-TCR. “Capable of binding the Vγ9 chain of a Vγ9Vδ2-TCR” means that the binding agent disclosed herein or multi-specific binding agent comprising the same can bind the Vγ9 chain as a separate molecule and/or as part of a Vγ9Vδ2-TCR. This can be referred to as a Vγ9 binding agent. However, the binding agent or multi-specific binding agent comprising the same will not bind to the Vδ2 chain as a separate molecule. Exemplary Vγ9Vδ2 TCR antigen-binding domain sequences are known in the art, see e.g., WO 2015/156673. [0223] In some embodiments, the multi-specific binding agents provided herein are able to activate Vγ9Vδ2-T cells. “Able to activate Vγ9Vδ2-T cells” in the context of the present disclosure refers to activation of Vγ9Vδ2-T cells in the presence of a multi-specific binding agent, such as a bispecific binding agent, disclosed herein in the presence of a target cell expressing 5T4. Preferably the activation of the Vγ9Vδ2-T cells is measurable through gene- expression and/or (surface) marker expression (e.g., activation markers, such as CD25, CD69, or CD107a) and/or secretory protein (e.g., cytokines or chemokines) profiles. In some embodiments, the binding agent is able to induce activation (e.g., upregulation of CD69 and/or CD25 expression) resulting in degranulation (marked by an increase in CD107a expression; see Examples 9, 10, and 13 herein) and cytokine production (e.g., TNF, IFN-γ) by Vγ9Vδ2-T cells. Preferably, the multi-specific binding agent that is used is able to increase CD107a expression on Vγ9Vδ2 T cells to at least 10%, more preferably at least 20%, more preferably at least 40%, most preferably at least 90%, when used in an assay as described in Example 9 herein, wherein e.g., 10% means that 10% of the total number of Vγ9Vδ2 T cells is positive for CD107a. In another embodiment, the number of Vγ9Vδ2 T cells positive for CD107a is 338699-2168 increased 1.5-fold, such as 2-fold, e.g., 5-fold, in the presence of an multi-specific binding agent of the disclosure. In another embodiment, the multi-specific binding agents of the disclosure have an EC50 value for increasing the percentage of CD107a positive Vγ9Vδ2 T cells of 100 pM or less, such as 25 pM or less, e.g., 12 pM or less, when tested using Vγ9Vδ2 T cells and 5T4 expressing U-251 target cells as described herein in Example 9. In another embodiment, the multi-specific binding agent is not capable of inducing degranulation of Vγ9Vδ2 T cells marked by an increase in CD107a in the absence of 5T4-expressing cells. [0224] In some embodiments, the binding agents disclosed herein are able to induce cytotoxicity in the presence of Vγ9Vδ2-T cells. "Able to induce cytotoxicity" in the context of the present disclosure is meant that Vγ9Vδ2-T cells are activated in the presence of a multi- specific binding agent, such as a bispecific binding agent, of the disclosure in the presence of a target cell expressing 5T4, and induce the death of the target cell through any means of cellular cytotoxicity, such as but not limited to apoptosis, necroptosis and pyroptosis. Linkers [0225] In some embodiments, the multi-specific binding agents disclosed herein comprise one or more linkers. As used herein, the term “linker” refers to a short stretch of amino acids used to connect two functional domains (e.g., antigen-binding domains or Fc domains) together in a polypeptide chain. For example, in some embodiments of the multi-specific binding agents of the disclosure, the antigen-binding domains are linked together on a polypeptide chain via one or more peptide linkers. Peptide linkers can also be used to attach other domains (such as half-life extension domains) to the multi-specific binding agents of the disclosure. [0226] The first and second antigen-binding regions in the multi-specific binding agents may be arranged in various ways. In one embodiment, antigen-binding domains are connected to each other via a linker, such as a covalent linker. In one embodiment, the first antigen-binding domain and the second antigen-binding domain are covalently linked to each other via a peptide linker, e.g. a linker having a length of from 1 to 20 amino acids, e.g. from 1 to 10 amino acids, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. [0227] Suitable linkers are known in the art and include, for example, peptide linkers containing flexible amino acid residues such as glycine, alanine, and serine. In some embodiments, a linker can contain motifs, e.g., multiple or repeating motifs, of GA, GS, GG, GGA, GGS, GGG, GGGA (SEQ ID NO: 337), GGGS (SEQ ID NO: 338), GGGG (SEQ ID NO: 339), GGGGA (SEQ ID NO: 340), GGGGS (SEQ ID NO: 341), GGGGG (SEQ ID NO: 342), GGAG (SEQ ID NO: 343), GGSG (SEQ ID NO: 344), AGGG (SEQ ID NO: 345), or SGGG (SEQ ID NO: 346). In one embodiment, the peptide linker comprises or consists of GGGGS 338699-2168 (SEQ ID NO: 341). In some embodiments, the linker comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 repeating motifs. Fc domains [0228] Multi-specific binding agents disclosed herein, such as bispecific binding agent, may contain additional molecules, domains, or polypeptide sequences beyond the first and second antigen-binding domains. [0229] In some embodiments, the present disclosure provides a 5T4 binding agent or multi- specific binding agent comprising the same, further comprising an Fc domain. In some embodiments, the Fc domain comprises a first and a second Fc domain monomer. [0230] As used herein, “Fc domain” or “Fc region” describes the minimum region (in the context of a larger polypeptide) or smallest protein folded structure (in the context of an isolated protein) that can bind to or be bound by an Fc receptor (FcR). The Fc domain is the fragment of an antibody which would be typically generated after digestion with papain, and generally includes the two CH2-CH3 regions of an immunoglobulin and a hinge region. As used herein, “Fc domain monomer” describes the single chain protein that, when associated with another Fc domain monomer, forms a functional Fc domain. The association of two Fc domain monomers creates one Fc domain. When two Fc domain monomers associate, the resulting Fc domain has Fc receptor binding activity. Thus, an Fc domain is a dimeric structure that can bind an Fc receptor. Unless otherwise noted, all references herein to an “Fc domain” are to be understood as referring to a dimeric Fc domain, in which each Fc domain monomer comprises the referenced mutation. The Fc domain mediates the effector functions of antibodies with cell surface receptors called Fc receptors and proteins of the complement system. [0231] It will be understood that Fc domain as used herein includes the polypeptides comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus, Fc domain refers to the last two constant region immunoglobulin domains (CH2, CH3) and optionally the flexible hinge N-terminal to these domains. Unless otherwise noted, all references to amino acid positions in Fc domains and Fc domain monomers are according to the EU index as set forth in Kabat (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). It is noted that polymorphisms have been observed at a number of Fc domain positions, including but not limited to Kabat 270, 272, 312, 315, 356, and 358, and thus slight differences between the sequences provided herein and sequences in the art may exist. 338699-2168 [0232] The Fc domain may be derived from any of a variety of different antibody isotypes, including but not limited to, a wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA, IgE, or IgM. In some embodiments, the Fc domain is derived from a human IgG1. [0233] There are many known polymorphs for the IgG1 Fc domain, including the “DEL” polymorph and the “EEM” polymorph. The DEL polymorph comprises the amino acids D-E-L at positions 356, 357, and 358, respectively (e.g., SEQ ID NO: 348). The EEM polymorph comprises the amino acids E-E-M at positions 356, 357, and 358, respectively (e.g., SEQ ID NO: 347). Two binding agents that are otherwise identical except for the presence of a DEL Fc domain or an EEM Fc domain are expected to demonstrate similar properties in terms of ligand binding and therapeutic efficacy. In some embodiments, the Fc domain is a DEL Fc domain. In some embodiments of the technology, the Fc domain is an EEM Fc domain. [0234] In some embodiments, the Fc domain is a variant Fc domain that forms a variant Fc domain with a desirable property, such as increased half-life, compared to naturally occurring (wild-type) Fc sequences. As used herein, a “variant Fc domain” refers to a non-naturally occurring Fc domain, for example an Fc domain comprising one or more non-naturally occurring amino acid residues, one or more amino acid substitutions relative to a wild-type human constant domain, or one or more amino acid deletion, addition and/or modification. [0235] In one embodiment, the multi-specific binding agent further comprises a half-life extension domain, i.e., a domain which prolongs the half-life of the molecules in the circulation of a human patient. In some embodiments, the multi-specific binding agent has a terminal half- life that is longer than about 168 hours when administered to a human subject. Most preferably the terminal half-life is 336 hours or longer. The “terminal half-life” of a binding agent, when used herein refers to the time taken for the serum concentration of the polypeptide to be reduced by 50%, in vivo in the final phase of elimination. [0236] The serum half-life of binding agents comprising Fc domains may be increased by increasing the binding affinity of the Fc domain for FcRn. In some embodiments, the Fc domain variant has enhanced serum half-life relative to a comparable molecule. In a particular embodiment, the Fc domain variant comprises at least one amino acid substitution at one or more positions selected from the group consisting of M252Y, S254T and T256E (referred to herein as “YTE”; e.g., SEQ ID NO: 349 and 350). In another embodiment, the Fc domain variant comprises a Y at position 252 (referred to herein as “Y”, e.g., SEQ ID NO: 351 and 352). Optionally, the Fc domain variant may comprise a non-naturally occurring amino acid residue at additional and/or alternative positions known to one skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; PCT Patent Publications WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO 04/035752; WO 04/074455; WO 04/099249; WO 04/063351; WO 05/070963; WO 05/040217; WO 05/092925; and WO 06/020114). 338699-2168 [0237] Examples for means to extend serum half-life of the binding agents of the disclosure include peptides, proteins, or domains of proteins, which are fused or otherwise attached to the binding agents. The group of peptides, proteins or protein domains includes peptides binding to other proteins with preferred pharmacokinetic profile in the human body such as serum albumin (see WO 2009/127691). As used herein, the term "human serum albumin" refers to the albumin protein present in human blood plasma. Human serum albumin is the most abundant protein in the blood. It constitutes about half of the blood serum protein. In some embodiments, a human serum albumin has the sequence of UniProt ID NO: P02768. [0238] An alternative concept of such half-life extending peptides includes peptides binding to the neonatal Fc receptor (FcRn, see WO 2007/098420), which can also be used in the polypeptides and binding agents of the present disclosure. The concept of attaching larger domains of proteins or complete proteins includes the fusion of human serum albumin, variants, or mutants of human serum albumin (see WO 2011/051489, WO 2012/059486, WO 2012/150319, WO 2013/135896, WO 2014/072481, WO 2013/075066) or domains thereof as well as the fusion of constant region of immunoglobulins (Fc domains) and variants thereof, as described herein. Such variants of Fc domains may be optimized/modified in order to allow the desired pairing of dimers or multimers, to abolish Fc receptor binding (e.g., the Fcγ receptor), to enhance binding to FcRn, or for other reasons. A further concept known in the art to extend the half-life of small protein compounds in the human body is the pegylation of those compounds such as the polypeptide or binding agent of the present disclosure. [0239] In an exemplary embodiment, the Fc domain monomer allows assembly of two or more polypeptide chains in a covalent manner, for example by disulfide linking between cysteine residues. In this way the Fc domain monomer acts as a dimerization domain, allowing assembly of two polypeptide chains to form a dimer. In some embodiments, such dimers comprise two polypeptides, each polypeptide including an antigen-binding domain described herein linked to an Fc domain monomer, thereby forming a bivalent binding agent. In some embodiments, such dimers comprise two polypeptides, one polypeptide comprising an antigen-binding domain described herein linked to an Fc domain monomer, and one polypeptide comprising an Fc domain monomer, thereby forming a monovalent binding agent. [0240] In some embodiments, the Fc domain may be a heterodimer comprising two Fc monomers, wherein the first antigen-binding domain is fused to the first Fc monomer and the second antigen-binding domain is fused to the second Fc monomer and wherein the first and second Fc monomers comprise asymmetric amino acid mutations that favor the formation of heterodimers over the formation of homodimers (see e.g. Ridgway et al. (1996) 'Knobs-into- holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng 9:617). The CH3 regions of the Fc monomers may comprise said asymmetric amino acid 338699-2168 mutations, for example the first Fc monomer may comprise a T366W substitution and the second Fc monomer may comprise T366S, L368A and Y407V substitutions, or vice versa, wherein the amino acid positions correspond to human IgG1 according to the EU numbering system. [0241] Furthermore, the cysteine residues at position 220 in the first and second Fc monomers may have been deleted or substituted, wherein the amino acid position corresponds to human IgG1 according to the EU numbering system. [0242] Furthermore, the first and/or second Fc monomers may contain mutations that render the binding agent inert, i.e., unable to, or having reduced ability to, mediate Fc effector functions (e.g., Fc-mediated cross-linking of FcRs). The inert Fc domain may in addition not be able to bind complement component (e.g., C1q) and/or one or more Fc receptors. In some embodiments, the first and second Fc monomers may comprise a mutation at position 234 and/or 235, for example the first and second Fc monomers may comprise an L234F and an L235E substitution, wherein the amino acid positions correspond to human IgG1 according to the EU numbering system. In some embodiments, the first and second Fc monomers may comprise an L234A and L235A substitution. In some embodiments, the first and second Fc domain monomers may further comprise a P329G substitution and/or a D265A substitution. Exemplary Fc domain monomer sequences are shown in TABLE 7. Hinge sequences are shown in bold and italicized text; DEL and EEM polymorphisms are shown in boxed text; mutations relative to WT IgG1 are shown in bold and underlined text. Table 7: Fc domain sequences Fc domain AA Sequence SEQ ID: WT IgG1 (EEM) EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC 347 VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G WT IgG1 (DEL) EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC 348 VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G IgG1 – YTE (EEM) EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTC 349 VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G IgG1 – YTE (DEL) EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTC 350 VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT 338699-2168 Fc domain AA Sequence SEQ ID: VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G IgG1 – Y (EEM) EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTC 351 VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G IgG1 – Y (DEL) EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTC 352 VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G IgG1 L234F L235E EPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTC 353 (Effector) VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G IgG1 T366W (Knob) EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC 354 VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G IgG1 T366S L368A EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC 355 Y407V (Hole) VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G IgG1 Effector+Knob AAASDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCV 356 VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG IgG1 Effector+Hole AAASDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCV 357 VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG IgG1 (Effector+Knob) + AAASDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCV 358 K VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 338699-2168 Fc domain AA Sequence SEQ ID: IgG1 (Effector+Hole) + AAASDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCV 359 K VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K [0243] In some embodiments, the Fc domains described in Table 7 comprise a wild type IgG1 hinge (e.g., SEQ ID NO: 360 – EPKSCDKTHTCPPCP). In some embodiments, the Fc domains described in Table 7 comprise a modified IgG1 hinge (e.g., SEQ ID NO: 361 – AAASDKTHTCPPCP). In some embodiments, the Fc domain sequences may further comprise a lysine (K) residue at the C terminus. Exemplary Multi-specific Binding Agents [0244] In some embodiments, the present disclosure provides a multi-specific binding agent comprising a first antigen-binding domain that binds to 5T4 and a second antigen-binding domain that binds to Vδ2. In some embodiments, the first and second antigen-binding domains are comprised in the same polypeptide chain. In some embodiments, the multi-specific binding agent comprises the following format, from N’ to C’ terminus: first antigen-binding domain – linker – second antigen-binding domain. In some embodiments, the multi-specific binding agent comprises the following format, from N’ to C’ terminus: second antigen-binding domain – linker – first antigen-binding domain. In some embodiments, the linker is a glycine-serine linker. In some embodiments, the linker is G4S and comprises SEQ ID NO: 341. [0245] In some embodiments, the multi-specific binding agent comprises (i) a first antigen- binding domain comprising a CDR1 of SEQ ID NO: 88, a CDR2 of SEQ ID NO: 89 and a CDR3 of SEQ ID NO: 90 and (ii) a second antigen-binding domain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2 and a CDR3 of SEQ ID NO: 3. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 92 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 92 and the second antigen-binding domain comprises SEQ ID NO: 4. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 92 and the second antigen-binding domain consists of SEQ ID NO: 4. In some embodiments, the multi-specific binding agent comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at 338699-2168 least 99% identical to SEQ ID NO: 362. In some embodiments, the multi-specific binding agent comprises SEQ ID NO: 362. In some embodiments, the multi-specific binding agent consists of SEQ ID NO: 362. Such a multi-specific binding agent is referred to herein as LAVA-378. [0246] In some embodiments, the multi-specific binding agent comprises (i) a first antigen- binding domain comprising a CDR1 of SEQ ID NO: 115, a CDR2 of SEQ ID NO: 116 and a CDR3 of SEQ ID NO: 117 and (ii) a second antigen-binding domain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2 and a CDR3 of SEQ ID NO: 3. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 118 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 118 and the second antigen-binding domain comprises SEQ ID NO: 4. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 118 and the second antigen-binding domain consists of SEQ ID NO: 4. In some embodiments, the multi-specific binding agent comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 363. In some embodiments, the multi- specific binding agent comprises SEQ ID NO: 363. In some embodiments, the multi-specific binding agent consists of SEQ ID NO: 363. Such a multi-specific binding agent is referred to herein as LAVA-380. [0247] In some embodiments, the multi-specific binding agent comprises (i) a first antigen- binding domain comprising a CDR1 of SEQ ID NO: 98, a CDR2 of SEQ ID NO: 99 and a CDR3 of SEQ ID NO: 100 and (ii) a second antigen-binding domain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2 and a CDR3 of SEQ ID NO: 3. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 101 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 101 and the second antigen-binding domain comprises SEQ ID NO: 4. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 101 and the second antigen-binding domain consists of SEQ ID NO: 4. In some embodiments, the multi-specific binding agent comprises an amino acid sequence that is at least 90%, at least 91%, at least 338699-2168 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 364. In some embodiments, the multi-specific binding agent comprises SEQ ID NO: 364. In some embodiments, the multi-specific binding agent consists of SEQ ID NO: 364. [0248] In some embodiments, the multi-specific binding agent comprises (i) a first antigen- binding domain comprising a CDR1 of SEQ ID NO: 105, a CDR2 of SEQ ID NO: 106 and a CDR3 of SEQ ID NO: 107 and (ii) a second antigen-binding domain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2 and a CDR3 of SEQ ID NO: 3. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 108 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 108 and the second antigen-binding domain comprises SEQ ID NO: 4. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 108 and the second antigen-binding domain consists of SEQ ID NO: 4. In some embodiments, the multi-specific binding agent comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 365. In some embodiments, the multi- specific binding agent comprises SEQ ID NO: 365. In some embodiments, the multi-specific binding agent consists of SEQ ID NO: 365. [0249] Exemplary embodiments of such single chain multi-specific binding agents are provided in TABLE 8. Linkers are shown in bold. Table 8: Exemplary single chain multi-specific binding agents. Bispecific Format Amino acid sequence SEQ agent ID LV1139 x Vδ2 LV1139- EVQLVESGGGLVQPGGSLRLSCAANGFAFSNYRMKWVRQAPGK 362 (LAVA-378) G4S-5C8 GLEWVSTISHRVGRTYYADSVKGRFTISRDNAKNTVYLQMNSL KPEDTARYFCTRVAMGGSDYAPHDYDYTGQGTQVTVSSGGGGS EVQLVESGGGLVQAGGSLRLSCAASGRPFSNYAMGWFRQAPGK EREFVAAISWSGGSTSYADSVKGRFTISRDNAKNTVYLQMNSP KPEDTAIYYCAAQFSGADYGFGRLGIRGYEYDYWGQGTQVTVS S LV1142 x Vδ2 LV1142- EVQLVESGGGLVQAGGSLRLSCAASGSIFSMGVIDWHRQAPGK 363 (LAVA-380) G4S-5C8 QREWVGGIASGGATNVIDSVKGRFTISRDGAKNTVYLQMSSLK PEDTAVYYCHARQFGPDAWGQGTQVTVSSGGGGSEVQLVESGG GLVQAGGSLRLSCAASGRPFSNYAMGWFRQAPGKEREFVAAIS WSGGSTSYADSVKGRFTISRDNAKNTVYLQMNSPKPEDTAIYY CAAQFSGADYGFGRLGIRGYEYDYWGQGTQVTVSS 338699-2168 Bispecific Format Amino acid sequence SEQ agent ID LV1138xVδ2 LV1138- EVQLVESGGGLVQAGGSLRLSCAASGDTLDYYAIGWFRQAPGK 364 G4S-5C8 EREGVSYISRSGGSVYYADSVKGRFTISRDSAKNTVYLQMVSL KPEDTAVYYCAARSSAYSRRYDIYSGWTEYGYWGQGTQVTVSS GGGGSEVQLVESGGGLVQAGGSLRLSCAASGRPFSNYAMGWFR QAPGKEREFVAAISWSGGSTSYADSVKGRFTISRDNAKNTVYL QMNSPKPEDTAIYYCAAQFSGADYGFGRLGIRGYEYDYWGQGT QVTVSS LV1140xVδ2 LV1140- EVQLVESGGGLVQPGGSLRLSCAASGFTFSAYGMTWVRQAPGK 365 G4S-5C8 GLEWVSGIESSGDIRKYSDSVKGRFTISRDNAKNTLYLQMNSL KPEDTAMYYCARSRDRWVIGDTRKYDSRGQGTQVTVSSGGGGS EVQLVESGGGLVQAGGSLRLSCAASGRPFSNYAMGWFRQAPGK EREFVAAISWSGGSTSYADSVKGRFTISRDNAKNTVYLQMNSP KPEDTAIYYCAAQFSGADYGFGRLGIRGYEYDYWGQGTQVTVS S [0250] In some embodiments the multi-specific binding agent further comprises one or more Fc domains. In such embodiments, the multi-specific binding agent comprises a first and a second polypeptide, wherein the first polypeptide comprises a first antigen-binding domain that binds to 5T4 and a first Fc domain monomer, and wherein the second polypeptide comprises a second antigen-binding domain and a second Fc domain monomer. The Fc domain monomers associate to form a dimeric multi-specific binding agent. In some embodiments, the Fc domain monomers comprise one or more mutations to facilitate dimerization and/or reduce effector function. [0251] Exemplary embodiments of such first and second polypeptides are provided below in TABLE 9. Antigen-binding domains are shown in underlined text. Hinges are shown in italicized and bolded text. Fc domains are shown in regular text. For all of the 5T4 VHH – Fc fusion proteins, the Fc domain corresponds to SEQ ID NO: 357 or 359 (i.e., effector mutations + hole mutations, w/ or w/o a C-terminal lysine). For all of the Vδ2 VHH – Fc fusions proteins, the Fc domain corresponds to SEQ ID NO: 356 or 358 (i.e., effector mutations + knob mutations, w/ or w/o a C-terminal lysine). Table 9: Exemplary VHH-Fc Binding Agents Reference AA Sequence SEQ ID: LV1139 EVQLVESGGGLVQPGGSLRLSCAANGFAFSNYRMKWVRQAPGKGLEWVSTISHR 366 IgG1 VGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTARYFCTRVAMGGSDYAP (Effector+ HDYDYTGQGTQVTVSSAAASDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMIS Hole) RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG LV1139 EVQLVESGGGLVQPGGSLRLSCAANGFAFSNYRMKWVRQAPGKGLEWVSTISHR 367 IgG1 VGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTARYFCTRVAMGGSDYAP (Effector+ HDYDYTGQGTQVTVSSAAASDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMIS Hole) +K RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ 338699-2168 Reference AA Sequence SEQ ID: (LAVA- VSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSR 395) WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK LV1139 DVQLVESGGGLVQPGGSLRLSCAANGFAFSNYRMKWVRQAPGKGLEWVSTISHR 368 (D) IgG1 VGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTARYFCTRVAMGGSDYAP (Effector+ HDYDYTGQGTQVTVSSAAASDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV Hole) LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG LV1139 369 (D) IgG1 DVQLVESGGGLVQPGGSLRLSCAANGFAFSNYRMKWVRQAPGKGLEWVSTISHR VGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTARYFCTRVAMGGSDYAP (Effector+ HDYDYTGQGTQVTVSSAAASDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMIS Hole) +K RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ (LAVA- VSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSR 433) WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK LV1138 EVQLVESGGGLVQAGGSLRLSCAASGDTLDYYAIGWFRQAPGKEREGVSYISRS 370 IgG1 GGSVYYADSVKGRFTISRDSAKNTVYLQMVSLKPEDTAVYYCAARSSAYSRRYD IYSGWTEYGYWGQGTQVTVSSAAASDKTHTCPPCPAPEFEGGPSVFLFPPKPKD (Effector+ TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV Hole) SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG LV1138 EVQLVESGGGLVQAGGSLRLSCAASGDTLDYYAIGWFRQAPGKEREGVSYISRS 371 IgG1 GGSVYYADSVKGRFTISRDSAKNTVYLQMVSLKPEDTAVYYCAARSSAYSRRYD IYSGWTEYGYWGQGTQVTVSSAAASDKTHTCPPCPAPEFEGGPSVFLFPPKPKD (Effector+ TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV Hole) +K SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK LV1140 EVQLVESGGGLVQPGGSLRLSCAASGFTFSAYGMTWVRQAPGKGLEWVSGIESS 372 IgG1 GDIRKYSDSVKGRFTISRDNAKNTLYLQMNSLKPEDTAMYYCARSRDRWVIGDT RKYDSRGQGTQVTVSSAAASDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMIS (Effector+ RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV Hole) LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG LV1140 EVQLVESGGGLVQPGGSLRLSCAASGFTFSAYGMTWVRQAPGKGLEWVSGIESS 373 IgG1 GDIRKYSDSVKGRFTISRDNAKNTLYLQMNSLKPEDTAMYYCARSRDRWVIGDT RKYDSRGQGTQVTVSSAAASDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMIS (Effector+ RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV Hole) +K LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK LV1141 EVQLVESGGGVVQAGGSLKLSCAASGHTFSRYTYKTMGWIRQAPGKEREFVAAI 374 IgG1 RWSGGATYYTDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAAGRDWVVG GRGAFDYWGQGTQVTVSSAAASDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLM (Effector+ ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL Hole) TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK NQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG LV1141 EVQLVESGGGVVQAGGSLKLSCAASGHTFSRYTYKTMGWIRQAPGKEREFVAAI 375 IgG1 RWSGGATYYTDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAAGRDWVVG GRGAFDYWGQGTQVTVSSAAASDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL 338699-2168 Reference AA Sequence SEQ ID: (Effector+ TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK Hole) +K NQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK LV1142 EVQLVESGGGLVQAGGSLRLSCAASGSIFSMGVIDWHRQAPGKQREWVGGIASG 376 IgG1 GATNVIDSVKGRFTISRDGAKNTVYLQMSSLKPEDTAVYYCHARQFGPDAWGQG TQVTVSSAAASDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVV (Effector+ VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK Hole) EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPG LV1142 EVQLVESGGGLVQAGGSLRLSCAASGSIFSMGVIDWHRQAPGKQREWVGGIASG 377 IgG1 GATNVIDSVKGRFTISRDGAKNTVYLQMSSLKPEDTAVYYCHARQFGPDAWGQG TQVTVSSAAASDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVV (Effector+ VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK Hole) +K EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK 5C8var1- EVQLLESGGGSVQPGGSLRLSCAASGRPFSNYAMSWFRQAPGKEREFVSAISWS 378 (Y105F- GGSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAQFSGADFGFG R109A) ALGIRGYEYDYWGQGTQVTVSSAAASDKTHTCPPCPAPEFEGGPSVFLFPPKPK IgG1 DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV (Effector+ VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD Knob) ELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 5C8var1- 379 (Y105F- EVQLLESGGGSVQPGGSLRLSCAASGRPFSNYAMSWFRQAPGKEREFVSAISWS R109A) GGSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAQFSGADFGFG IgG1 ALGIRGYEYDYWGQGTQVTVSSAAASDKTHTCPPCPAPEFEGGPSVFLFPPKPK (Effector+ DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV Knob) + K VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL (LAVA- TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 328) 5C8 var1 DVQLLESGGGSVQPGGSLRLSCAASGRPFSNYAMSWFRQAPGKEREFVSAISWS 380 (E1D- GGSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAQFSGADFGFG Y105F- ALGIRGYEYDYWGQGTQVTVSSAAASDKTHTCPPCPAPEFEGGPSVFLFPPKPK R109A)- DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD IgG1 ELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL (Effector+ TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Knob) 5C8 var1 DVQLLESGGGSVQPGGSLRLSCAASGRPFSNYAMSWFRQAPGKEREFVSAISWS 381 (E1D- GGSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAQFSGADFGFG Y105F- ALGIRGYEYDYWGQGTQVTVSSAAASDKTHTCPPCPAPEFEGGPSVFLFPPKPK R109A)- DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD IgG1 ELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL (Effector+ TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Knob) +K (LAVA- 424) 338699-2168 [0252] In some embodiments, the multi-specific binding agent comprises (i) a first polypeptide comprising a first antigen-binding domain that binds 5T4 comprising a CDR1 of SEQ ID NO: 88, a CDR2 of SEQ ID NO: 89 and a CDR3 of SEQ ID NO: 90 and a first Fc domain monomer; and (ii) a second polypeptide comprising a second antigen-binding domain that binds to Vδ2 and comprises a CDR1 of SEQ ID NO: 18, a CDR2 of SEQ ID NO: 19 and a CDR3 of SEQ ID NO: 20 and a second Fc domain monomer. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 92 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 21. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 92 and the second antigen-binding domain comprises SEQ ID NO: 21. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 92 and the second antigen-binding domain consists of SEQ ID NO: 21. In some embodiments, the first Fc domain monomer comprises SEQ ID NO: 356 or 358 and the second Fc domain monomer comprises SEQ ID NO: 357 or 359. In some embodiments, the first Fc domain monomer comprises SEQ ID NO: 357 or 359 and the second Fc domain monomer comprises SEQ ID NO: 356 or 358. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 367 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 379. In some embodiments, the first polypeptide comprises SEQ ID NO: 367 and the second polypeptide comprises SEQ ID NO: 379. In some embodiments, the first polypeptide consists of SEQ ID NO: 367, referred to herein as LAVA-395, and the second polypeptide consists of SEQ ID NO: 379, referred to herein as LAVA-328. Such a multi-specific binding agent is referred to herein as LAVA-1395. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 366 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 378. In some embodiments, the first polypeptide comprises SEQ ID NO: 366 and the second polypeptide comprises SEQ ID NO: 378. In some embodiments, the first polypeptide consists of SEQ ID NO: 366 and the second polypeptide consists of SEQ ID NO: 378. 338699-2168 [0253] In some embodiments, the multi-specific binding agent comprises (i) a first polypeptide comprising a first antigen-binding domain that binds 5T4 comprising a CDR1 of SEQ ID NO: 88, a CDR2 of SEQ ID NO: 89 and a CDR3 of SEQ ID NO: 90 and a first Fc domain monomer; and (ii) a second polypeptide comprising a second antigen-binding domain that binds to Vδ2 and comprises a CDR1 of SEQ ID NO: 18, a CDR2 of SEQ ID NO: 19 and a CDR3 of SEQ ID NO: 20 and a second Fc domain monomer. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 93 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 22. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 93 and the second antigen-binding domain comprises SEQ ID NO: 22. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 93 and the second antigen-binding domain consists of SEQ ID NO: 22. In some embodiments, the first Fc domain monomer comprises SEQ ID NO: 356 or 358 and the second Fc domain monomer comprises SEQ ID NO: 357 or 359. In some embodiments, the first Fc domain monomer comprises SEQ ID NO: 357 or 359 and the second Fc domain monomer comprises SEQ ID NO: 356 or 358. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 369 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 381. In some embodiments, the first polypeptide comprises SEQ ID NO: 369 and the second polypeptide comprises SEQ ID NO: 381. In some embodiments, the first polypeptide consists of SEQ ID NO: 369, referred to herein as LAVA-433, and the second polypeptide consists of SEQ ID NO: 381, referred to herein as LAVA-424. Such a multi-specific binding agent is referred to herein as LAVA-1433. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 368 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 380. In some embodiments, the first polypeptide comprises SEQ ID NO: 368 and the second polypeptide comprises SEQ ID NO: 380. In some embodiments, the first polypeptide consists of SEQ ID NO: 368 and the second polypeptide consists of SEQ ID NO: 380. 338699-2168 [0254] In some embodiments, the multi-specific binding agent comprises (i) a first polypeptide comprising a first antigen-binding domain that binds 5T4 comprising a CDR1 of SEQ ID NO: 98, a CDR2 of SEQ ID NO: 99, and a CDR3 of SEQ ID NO: 100 and a first Fc domain monomer; and (ii) a second polypeptide comprising a second antigen-binding domain that binds to Vδ2 and comprises a CDR1 of SEQ ID NO: 18, a CDR2 of SEQ ID NO: 19 and a CDR3 of SEQ ID NO: 20 and a second Fc domain monomer. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 101 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 21. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 101 and the second antigen-binding domain comprises SEQ ID NO: 21. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 101 and the second antigen-binding domain consists of SEQ ID NO: 21. In some embodiments, the first Fc domain monomer comprises SEQ ID NO: 356 or 358 and the second Fc domain monomer comprises SEQ ID NO: 357 or 359. In some embodiments, the first Fc domain monomer comprises SEQ ID NO: 357 or 359 and the second Fc domain monomer comprises SEQ ID NO: 356 or 358. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 371 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 379. In some embodiments, the first polypeptide comprises SEQ ID NO: 371 and the second polypeptide comprises SEQ ID NO: 379. In some embodiments, the first polypeptide consists of SEQ ID NO: 371, and the second polypeptide consists of SEQ ID NO: 379. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 370 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 378. In some embodiments, the first polypeptide comprises SEQ ID NO: 370 and the second polypeptide comprises SEQ ID NO: 378. In some embodiments, the first polypeptide consists of SEQ ID NO: 370 and the second polypeptide consists of SEQ ID NO: 378. 338699-2168 [0255] In some embodiments, the multi-specific binding agent comprises (i) a first polypeptide comprising a first antigen-binding domain that binds 5T4 comprising a CDR1 of SEQ ID NO: 98, a CDR2 of SEQ ID NO: 99 and a CDR3 of SEQ ID NO: 100 and a first Fc domain monomer; and (ii) a second polypeptide comprising a second antigen-binding domain that binds to Vδ2 and comprises a CDR1 of SEQ ID NO: 18, a CDR2 of SEQ ID NO: 19 and a CDR3 of SEQ ID NO: 20 and a second Fc domain monomer. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 101 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 22. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 101 and the second antigen-binding domain comprises SEQ ID NO: 22. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 101 and the second antigen-binding domain consists of SEQ ID NO: 22. In some embodiments, the first Fc domain monomer comprises SEQ ID NO: 356 or 358 and the second Fc domain monomer comprises SEQ ID NO: 357 or 359. In some embodiments, the first Fc domain monomer comprises SEQ ID NO: 357 or 359 and the second Fc domain monomer comprises SEQ ID NO: 356 or 358. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 371 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 381. In some embodiments, the first polypeptide comprises SEQ ID NO: 371 and the second polypeptide comprises SEQ ID NO: 381. In some embodiments, the first polypeptide consists of SEQ ID NO: 371, and the second polypeptide consists of SEQ ID NO: 381. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 370 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 380. In some embodiments, the first polypeptide comprises SEQ ID NO: 370 and the second polypeptide comprises SEQ ID NO: 380. In some embodiments, the first polypeptide consists of SEQ ID NO: 370 and the second polypeptide consists of SEQ ID NO: 380. 338699-2168 [0256] In some embodiments, the multi-specific binding agent comprises (i) a first polypeptide comprising a first antigen-binding domain that binds 5T4 comprising a CDR1 of SEQ ID NO: 105, a CDR2 of SEQ ID NO: 106, and a CDR3 of SEQ ID NO: 107 and a first Fc domain monomer; and (ii) a second polypeptide comprising a second antigen-binding domain that binds to Vδ2 and comprises a CDR1 of SEQ ID NO: 18, a CDR2 of SEQ ID NO: 19 and a CDR3 of SEQ ID NO: 20 and a second Fc domain monomer. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 108 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 21. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 108 and the second antigen-binding domain comprises SEQ ID NO: 21. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 108 and the second antigen-binding domain consists of SEQ ID NO: 21. In some embodiments, the first Fc domain monomer comprises SEQ ID NO: 356 or 358 and the second Fc domain monomer comprises SEQ ID NO: 357 or 359. In some embodiments, the first Fc domain monomer comprises SEQ ID NO: 357 or 359 and the second Fc domain monomer comprises SEQ ID NO: 356 or 358. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 373 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 379. In some embodiments, the first polypeptide comprises SEQ ID NO: 373 and the second polypeptide comprises SEQ ID NO: 379. In some embodiments, the first polypeptide consists of SEQ ID NO: 373, and the second polypeptide consists of SEQ ID NO: 379. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 372 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 378. In some embodiments, the first polypeptide comprises SEQ ID NO: 372 and the second polypeptide comprises SEQ ID NO: 378. In some embodiments, the first polypeptide consists of SEQ ID NO: 372 and the second polypeptide consists of SEQ ID NO: 378. 338699-2168 [0257] In some embodiments, the multi-specific binding agent comprises (i) a first polypeptide comprising a first antigen-binding domain that binds 5T4 comprising a CDR1 of SEQ ID NO: 105, a CDR2 of SEQ ID NO: 106, and a CDR3 of SEQ ID NO: 107 and a first Fc domain monomer; and (ii) a second polypeptide comprising a second antigen-binding domain that binds to Vδ2 and comprises a CDR1 of SEQ ID NO: 18, a CDR2 of SEQ ID NO: 19 and a CDR3 of SEQ ID NO: 20 and a second Fc domain monomer. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 108 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 22. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 108 and the second antigen-binding domain comprises SEQ ID NO: 22. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 108 and the second antigen-binding domain consists of SEQ ID NO: 22. In some embodiments, the first Fc domain monomer comprises SEQ ID NO: 356 or 358 and the second Fc domain monomer comprises SEQ ID NO: 357 or 359. In some embodiments, the first Fc domain monomer comprises SEQ ID NO: 357 or 359 and the second Fc domain monomer comprises SEQ ID NO: 356 or 358. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 373 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 381. In some embodiments, the first polypeptide comprises SEQ ID NO: 373 and the second polypeptide comprises SEQ ID NO: 381. In some embodiments, the first polypeptide consists of SEQ ID NO: 373, and the second polypeptide consists of SEQ ID NO: 381. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 372 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 380. In some embodiments, the first polypeptide comprises SEQ ID NO: 372 and the second polypeptide comprises SEQ ID NO: 380. In some embodiments, the first polypeptide consists of SEQ ID NO: 372 and the second polypeptide consists of SEQ ID NO: 380. 338699-2168 [0258] In some embodiments, the multi-specific binding agent comprises (i) a first polypeptide comprising a first antigen-binding domain that binds 5T4 comprising a CDR1 of SEQ ID NO: 111, a CDR2 of SEQ ID NO: 112, and a CDR3 of SEQ ID NO: 113 and a first Fc domain monomer; and (ii) a second polypeptide comprising a second antigen-binding domain that binds to Vδ2 and comprises a CDR1 of SEQ ID NO: 18, a CDR2 of SEQ ID NO: 19 and a CDR3 of SEQ ID NO: 20 and a second Fc domain monomer. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 114 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 21. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 114 and the second antigen-binding domain comprises SEQ ID NO: 21. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 114 and the second antigen-binding domain consists of SEQ ID NO: 21. In some embodiments, the first Fc domain monomer comprises SEQ ID NO: 356 or 358 and the second Fc domain monomer comprises SEQ ID NO: 357 or 359. In some embodiments, the first Fc domain monomer comprises SEQ ID NO: 357 or 359 and the second Fc domain monomer comprises SEQ ID NO: 356 or 358. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 375 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 379. In some embodiments, the first polypeptide comprises SEQ ID NO: 375 and the second polypeptide comprises SEQ ID NO: 379. In some embodiments, the first polypeptide consists of SEQ ID NO: 375, and the second polypeptide consists of SEQ ID NO: 379. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 374 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 378. In some embodiments, the first polypeptide comprises SEQ ID NO: 374 and the second polypeptide comprises SEQ ID NO: 378. In some embodiments, the first polypeptide consists of SEQ ID NO: 374 and the second polypeptide consists of SEQ ID NO: 378. 338699-2168 [0259] In some embodiments, the multi-specific binding agent comprises (i) a first polypeptide comprising a first antigen-binding domain that binds 5T4 comprising a CDR1 of SEQ ID NO: 111, a CDR2 of SEQ ID NO: 112, and a CDR3 of SEQ ID NO: 113 and a first Fc domain monomer; and (ii) a second polypeptide comprising a second antigen-binding domain that binds to Vδ2 and comprises a CDR1 of SEQ ID NO: 18, a CDR2 of SEQ ID NO: 19 and a CDR3 of SEQ ID NO: 20 and a second Fc domain monomer. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 114 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 22. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 114 and the second antigen-binding domain comprises SEQ ID NO: 22. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 114 and the second antigen-binding domain consists of SEQ ID NO: 22. In some embodiments, the first Fc domain monomer comprises SEQ ID NO: 356 or 358 and the second Fc domain monomer comprises SEQ ID NO: 357 or 359. In some embodiments, the first Fc domain monomer comprises SEQ ID NO: 357 or 359 and the second Fc domain monomer comprises SEQ ID NO: 356 or 358. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 375 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 381. In some embodiments, the first polypeptide comprises SEQ ID NO: 375 and the second polypeptide comprises SEQ ID NO: 381. In some embodiments, the first polypeptide consists of SEQ ID NO: 375, and the second polypeptide consists of SEQ ID NO: 381. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 374 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 380. In some embodiments, the first polypeptide comprises SEQ ID NO: 374 and the second polypeptide comprises SEQ ID NO: 380. In some embodiments, the first polypeptide consists of SEQ ID NO: 374 and the second polypeptide consists of SEQ ID NO: 380. 338699-2168 [0260] In some embodiments, the multi-specific binding agent comprises (i) a first polypeptide comprising a first antigen-binding domain that binds 5T4 comprising a CDR1 of SEQ ID NO: 115, a CDR2 of SEQ ID NO: 116, and a CDR3 of SEQ ID NO: 117 and a first Fc domain monomer; and (ii) a second polypeptide comprising a second antigen-binding domain that binds to Vδ2 and comprises a CDR1 of SEQ ID NO: 18, a CDR2 of SEQ ID NO: 19 and a CDR3 of SEQ ID NO: 20 and a second Fc domain monomer. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 118 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 21. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 118 and the second antigen-binding domain comprises SEQ ID NO: 21. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 118 and the second antigen-binding domain consists of SEQ ID NO: 21. In some embodiments, the first Fc domain monomer comprises SEQ ID NO: 356 or 358 and the second Fc domain monomer comprises SEQ ID NO: 357 or 359. In some embodiments, the first Fc domain monomer comprises SEQ ID NO: 357 or 359 and the second Fc domain monomer comprises SEQ ID NO: 356 or 358. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 377 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 379. In some embodiments, the first polypeptide comprises SEQ ID NO: 377 and the second polypeptide comprises SEQ ID NO: 379. In some embodiments, the first polypeptide consists of SEQ ID NO: 377, and the second polypeptide consists of SEQ ID NO: 379. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 376 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 378. In some embodiments, the first polypeptide comprises SEQ ID NO: 376 and the second polypeptide comprises SEQ ID NO: 378. In some embodiments, the first polypeptide consists of SEQ ID NO: 376 and the second polypeptide consists of SEQ ID NO: 378. 338699-2168 [0261] In some embodiments, the multi-specific binding agent comprises (i) a first polypeptide comprising a first antigen-binding domain that binds 5T4 comprising a CDR1 of SEQ ID NO: 115, a CDR2 of SEQ ID NO: 116, and a CDR3 of SEQ ID NO: 117 and a first Fc domain monomer; and (ii) a second polypeptide comprising a second antigen-binding domain that binds to Vδ2 and comprises a CDR1 of SEQ ID NO: 18, a CDR2 of SEQ ID NO: 19 and a CDR3 of SEQ ID NO: 20 and a second Fc domain monomer. In some embodiments, the first antigen-binding domain comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 118 and the second antigen-binding domain comprises an amino acid sequence that this at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 22. In some embodiments, the first antigen-binding domain comprises SEQ ID NO: 118 and the second antigen-binding domain comprises SEQ ID NO: 22. In some embodiments, the first antigen-binding domain consists of SEQ ID NO: 118 and the second antigen-binding domain consists of SEQ ID NO: 22. In some embodiments, the first Fc domain monomer comprises SEQ ID NO: 356 or 358 and the second Fc domain monomer comprises SEQ ID NO: 357 or 359. In some embodiments, the first Fc domain monomer comprises SEQ ID NO: 357 or 359 and the second Fc domain monomer comprises SEQ ID NO: 356 or 358. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 377 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 381. In some embodiments, the first polypeptide comprises SEQ ID NO: 377 and the second polypeptide comprises SEQ ID NO: 381. In some embodiments, the first polypeptide consists of SEQ ID NO: 377, and the second polypeptide consists of SEQ ID NO: 381. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 376 and the second polypeptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 380. In some embodiments, the first polypeptide comprises SEQ ID NO: 376 and the second polypeptide comprises SEQ ID NO: 380. In some embodiments, the first polypeptide consists of SEQ ID NO: 376 and the second polypeptide consists of SEQ ID NO: 380. 338699-2168 Polynucleotides [0262] In some embodiments, the present disclosure provides a polynucleotide encoding any one of the 5T4 binding agents described herein. In some embodiments, the polynucleotide encodes a polypeptide sequence having at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOs: 92, 93, 94, 95, 96, 97, 101, 102, 103, 104, 108, 109, 110, 114, 118, 119, 120, 121, 128, 129, 130, 131, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, and 172. [0263] Methods of determining sequence similarity or identity between two or more nucleic acid sequences or amino acid sequences are known in the art. For example, sequence similarity or identity may be determined using the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math.2, 482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch J Mol. Biol. 48, 443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85, 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), the Best Fit sequence program described by Devereux et al. Nucl. Acid Res.12, 387-395 (1984), or by inspection. [0264] Another suitable algorithm is the BLAST algorithm, described in Altschul et al. J. Mol. Biol.215, 403-410, (1990) and Karlin et al. Proc. Natl. Acad. Sci. USA 90, 5873-5787 (1993). A particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al. Methods in Enzymology, 266, 460-480 (1996); blast.wustl/edu/blast/README.html. WU-BLAST-2 uses several search parameters, which are optionally set to the default values. The parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity. Further, an additional useful algorithm is gapped BLAST as reported by Altschul et al, (1997) Nucleic Acids Res.25, 3389-3402. Unless otherwise indicated, percent identity is determined herein using the algorithm available at the internet address: blast.ncbi.nlm.nih.gov/Blast.cgi. [0265] In some embodiments, polynucleotide sequences described herein have been codon- optimized for improved expression. Without wishing to be bound by theory, it is believed that codon optimization of the nucleotide sequence increases the translation efficiency of the mRNA transcripts. Codon optimization of the nucleotide sequence may involve substituting a native codon for another codon that encodes the same amino acid, but can be translated by tRNA that is more readily available within a cell, thus increasing translation efficiency. 338699-2168 Optimization of the nucleotide sequence may also reduce secondary mRNA structures that would interfere with translation, thus increasing translation efficiency. [0266] The polynucleotides provided by the present disclosure may be not naturally occurring or may have a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques, such as those described in Sambrook et al., supra. The nucleic acids can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. See, for example, Sambrook et al., supra, and Ausubel et al. supra. For example, a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides). Examples of modified nucleotides that can be used to generate the nucleic acids include, but are not limited to, 5-flurouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopenenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5’-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio- N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids provided by the present disclosure can be purchased from companies, such as integrated DNA Technologies (Coralville, IA, USA). [0267] In some embodiments, the polynucleotides described herein comprise a nucleic acid sequence encoding a signal peptide at the 5’ end of the nucleic acid sequence encoding the binding agents described herein. In some embodiments, the signal peptide facilitates secretion of the encoded protein from a host cell (e.g., secretion of the 5T4 binding agents or multi- specific binding agents comprising the same). A person of skill in the art will recognize that signal peptides are cleaved during intracellular transport such that the mature proteins do not express the signal peptide. Signal peptides suitable for use according to the present disclosure are known in the art, for example signal peptides derived from GMCSF, the Igk -chain, CD8α, CD33, or TPA. 338699-2168 Vectors [0268] In some embodiments, the present disclosure provides a vector comprising a polynucleotide encoding a 5T4 binding agent or multi-specific binding agent comprising the same. [0269] In some embodiments, the vector comprising a polynucleotide encoding a 5T4 binding agent or multi-specific binding agent comprising the same is a viral vector. Illustrative viral vectors include, but are not limited to, adeno- associated virus (AAV), retrovirus (e.g., lentivirus), herpes simplex virus, adenovirus, and vaccinia virus. AAV (rAAV) vectors are typically composed of, at a minimum, a transgene and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). In particular embodiments, the rAAV comprises ITRs and capsid sequences isolated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In some embodiments, the ITR sequences of one AAV serotype and the capsid sequences of a different AAV serotype may be used to create a chimeric rAAV. In some embodiments, engineering and selection methods can be applied to AAV capsids to make them more likely to transduce cells of interest. Construction of rAAV vectors, production, and purification thereof have been disclosed, e.g., in U.S. Patent Nos. 9,169,494; 9,169,492; 9,012,224; 8,889,641; 8,809,058; and 8,784,799, each of which is incorporated by reference herein in its entirety. [0270] Illustrative retroviruses suitable for use in particular embodiments, include, but are not limited to: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV) and lentivirus. Illustrative lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2); visna-maedi virus (VMV) virus; the caprine arthritis- encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV). In various embodiments, a lentiviral vector contemplated herein comprises one or more LTRs, and one or more of the following accessory elements: a cPPT/FLAP, a Psi packaging signal, an export element, polyA sequences, and may optionally comprise a WPRE or HPRE, an insulator element, a selectable marker, and a cell suicide gene. In particular embodiments, lentiviral vectors contemplated herein may be integrative or non-integrating or integration defective lentivirus (e.g., having an integrase that lacks the capacity to integrate the viral genome into the genome of the host cells. Integration-incompetent viral vectors have been described in patent application WO 2006/010834, which is herein incorporated by reference in its entirety. 338699-2168 [0271] In particular embodiments, expression of heterologous sequences in viral vectors is increased by incorporating post-transcriptional regulatory elements, efficient polyadenylation sites, and optionally, transcription termination signals into the vectors. A variety of posttranscriptional regulatory elements can increase expression of a heterologous polynucleotide at the protein, e.g., woodchuck hepatitis vims posttranscriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); the posttranscriptional regulatory element present in hepatitis B vims (HPRE) (Huang et al., Mol. Cell. Biol., 5:3864); and the like (Liu et al., 1995, Genes Dev., 9: 1766). [0272] In some embodiments, the vector comprising a polynucleotide encoding a 5T4 binding agent or multi-specific binding agent comprising the same described herein is a plasmid. Numerous suitable plasmid expression vectors are known to those of skill in the art, and many are commercially available. The following vectors are provided by way of example; for eukaryotic host cells: pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). However, any other plasmid vector may be used so long as it is compatible with the host cell. Depending on the cell type utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544). [0273] In some embodiments, a polynucleotide sequence encoding a 5T4 binding agent or multi-specific binding agent comprising the same is operably linked to a control element, e.g., a transcriptional control element, such as a promoter. The transcriptional control element may be functional in either a eukaryotic cell (e.g., a mammalian cell) or a prokaryotic cell (e.g., bacterial or archaeal cell). In some embodiments, a polynucleotide sequence encoding a 5T4 binding agent or multi-specific binding agent comprising the same is operably linked to multiple control elements that allow expression of the polynucleotide in both prokaryotic and eukaryotic cells. Depending on the cell type utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544). [0274] Non-limiting examples of suitable eukaryotic promoters (promoters functional in a eukaryotic cell) include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, and mouse metallothionein-l. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. The expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector may also include appropriate sequences for amplifying expression. The expression 338699-2168 vector may also include nucleotide sequences encoding protein tags (e.g., 6xHis tag, hemagglutinin tag, green fluorescent protein, etc.) that are fused to the 5T4 binding agent or multi-specific binding agent comprising the same, thus resulting in a chimeric polypeptide. [0275] In some embodiments, a polynucleotide sequence encoding a 5T4 binding agent or multi-specific binding agent comprising the same is operably linked to an inducible or a constitutive promoter. [0276] A number of transfection techniques are generally known in the art (see, e.g., Graham et al., Virology, 52: 456-467 (1973); Sambrook et al., supra; Davis et al., Basic Methods in Molecular Biology, Elsevier (1986); and Chu et al., Gene, 13: 97 (1981). Transfection methods include calcium phosphate co-precipitation (see, e.g., Graham et al., supra), direct micro injection into cultured cells (see, e.g., Capecchi, Cell, 22: 479-488 (1980)), electroporation (see, e.g., Shigekawa et al., BioTechniques, 6: 742-751 (1988)), liposome mediated gene transfer (see, e.g., Mannino et al., BioTechniques, 6: 682-690 (1988)), lipid mediated transduction (see, e.g., Feigner et al., Proc. Natl. Acad. Sci. USA, 84: 7413-7417 (1987)), and nucleic acid delivery using high velocity microprojectiles (see, e.g., Klein et al., Nature, 327: 70-73 (1987)). [0277] In an embodiment, the vectors provided by the present disclosure can be prepared using standard techniques described in, for example, Sambrook et al., supra, and Ausubel et al., supra. Constructs of expression vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell, Replication systems can be derived, e.g., from ColEl, 2 µ plasmid, λ, SV40, bovine papilloma virus, and the like. [0278] The vector may comprise regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host cell (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate, and taking into consideration whether the vector is DNA- or RNA-based. The expression vector may comprise restriction sites to facilitate cloning. [0279] The vector can include one or more marker genes, which allow for selection of transformed or transfected host cells. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like. Suitable marker genes for the inventive expression vectors include, for instance, neomycin/G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes. [0280] The vectors can be designed for either transient expression, for stable expression, or for both, of the polynucleotides comprised therein. Also, the vectors can be made for constitutive expression or for inducible expression of the polynucleotides comprised therein. 338699-2168 Pharmaceutical Compositions [0281] In some embodiments, the present disclosure provides compositions comprising the 5T4 binding agents and/or multi-specific binding agents described herein. In some embodiments, the present disclosure provides pharmaceutical compositions comprising the 5T4 binding agents and/or multi-specific binding agents described herein and a pharmaceutically-acceptable excipient. [0282] Typically, formulations include all physiologically acceptable compositions including derivatives and/or prodrugs, solvates, stereoisomers, racemates, or tautomers thereof with any physiologically acceptable carriers, diluents, and/or excipients. A “therapeutic composition” or “pharmaceutical composition” (used interchangeably herein) is a composition capable of being administered to a subject for the treatment of a particular disease or disorder. [0283] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. [0284] As used herein “pharmaceutically acceptable carrier, diluent or excipient” includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, and/or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans and/or domestic animals. Exemplary pharmaceutically acceptable carriers include, but are not limited to, to sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffins, silicones, bentonites, silicic acid, zinc oxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and any other compatible substances employed in pharmaceutical formulations. Except insofar as any conventional media and/or agent is incompatible with the agents of the present disclosure, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions. 338699-2168 [0285] “Pharmaceutically acceptable salt” includes both acid and base addition salts. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2- disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, ptoluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. [0286] Wetting agents, emulsifiers, and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions. [0287] Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl 338699-2168 palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. [0288] Further guidance regarding formulations that are suitable for various types of administration can be found in Remington’s Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a brief review of methods for drug delivery, see, Langer, Science 249:1527-1533 (1990). Method of producing 5T4 binding agents [0289] In some embodiments, the present disclosure provides a method of producing the 5T4 binding agents and multi-specific binding agents comprising the same comprising introducing a polynucleotide encoding the 5T4 binding agent or a vector comprising the same into a host cell, culturing the host cell under conditions suitable for expression of the 5T4 binding agent, and purifying the expressed 5T4 binding agent from the culture supernatant. [0290] Exemplary host cells suitable for use in the manufacture of the binding agents described herein are known in the art, for example, a host cell can be a eukaryotic cell, e.g., plant, animal, fungi, or algae, or can be a prokaryotic cell, e.g., bacteria or protozoa. The host cell can be a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human. The host cell can be an adherent cell or a suspended cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for instance, DK5α E. coli cells, Chinese hamster ovarian cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For purposes of amplifying or replicating the expression vector, the host cell may be a prokaryotic cell, e.g., a DH5α cell. For the purposes of producing a binding agent, the host cell may be a mammalian cell. In some embodiments, the present disclosure provides a host cell comprising a polynucleotide described herein or vector comprising the same. [0291] Suitable methods of making antibodies, including single domain antibodies, are known in the art. For instance, standard hybridoma methods are described in, e.g., Köhler and Milstein, Eur. J. Immunol.5, 511-519 (1976), Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and C.A. Janeway et al. (eds,), Immumobiology, 5th Ed., Garland Publishing, New York, NY (2001). Alternatively, other methods, such as EBV-hybridoma methods (Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984), and Roder et al., Methods Enzymol., 121, 140-67 (1986)), and bacteriophage vector expression systems (see, e.g., Huse et al, Science, 246, 1275-81 (1989)) are known in the art. Further, methods of producing antibodies in non-human animals are described in, e.g., U.S. Patents 5,545,806, 5,569,825, and 5,714,352, U.S. Patent Application Publication No.2002/0197266, and U.S. Patent No.7,338,929. 338699-2168 [0292] Antibodies can be produced by transgenic mice that are transgenic for specific heavy and light chain immunoglobulin genes. Such methods are known in the art and described in, for example U.S. Patents 5,545,806 and 5,569,825, and Janeway et al., supra. [0293] Methods for generating humanized antibodies an antigen-binding fragments thereof are well known in the art and are described in detail in, for example, Janeway et al., supra, U.S. Patents 5,225,539, 5,585,089 and 5,693,761, European Patent No. 0239400 Bl, and United Kingdom Patent No.2188638. Humanized antibodies can also be generated using the antibody resurfacing technology described in U.S. Patent 5,639,641 and Pedersen et al., J. Mol. Biol., 235, 959-973 (1994). [0294] Multi-specific binding agents disclosed herein are typically produced recombinantly, i.e., by expression of polynucleotides encoding the multi-specific binding agents in suitable host cells, followed by purification of the produced recombinant multi-specific binding agent from the cell culture. Polynucleotides can be produced by standard molecular biological techniques well-known in the art. The polynucleotides are typically introduced into the host cell using an expression vector. Suitable nucleic acid constructs and expression vectors are known in the art. Host cells suitable for the recombinant expression of antibodies are well- known in the art, and include CHO (Chinese Hamster Ovary), HEK-293, Expi293F, PER-C6, NS/0 and Sp2/0 cells. [0295] Accordingly, in a further embodiment, disclosed herein are polynucleotides that encode a binding agent, such as multi-specific binding agent disclosed herein. In one embodiment, the polynucleotide is a DNA construct. In another embodiment, the polynucleotide is an RNA construct. The polynucleotide typically further comprises a promoter. [0296] In a further embodiment, disclosed herein are expression vectors comprising polynucleotides that encode multi-specific binding agents as disclosed herein. The expression vector typically further comprises a promoter. [0297] In a further embodiment, disclosed herein are host cells, such as a non-human host cell, for example a CHO cell, that comprise one or more polynucleotides that encode a multi- specific binding agent as disclosed herein or an expression vector comprising a polynucleotide encoding a multi-specific binding agent as disclosed herein. Method of Treatment [0298] In some embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof comprising administering a 5T4 binding agent described herein, a multi-specific binding agent comprising the same, or a pharmaceutical composition comprising the same. 338699-2168 [0299] In some embodiments, treatment comprises delivering an effective amount of a 5T4 binding agent or multi-specific binding agent comprising the same, or a pharmaceutical composition thereof, to a subject in need thereof. In some embodiments, treating refers to the treatment of a cancer in a mammal, e.g., in a human, including (a) inhibiting the cancer, i.e., arresting cancer development or preventing cancer progression; (b) relieving the cancer, i.e., causing regression of the cancer state or relieving one or more symptoms of the cancer; and (c) curing the cancer, i.e., remission of one or more cancer symptoms. In some embodiments, treatment may refer to a short-term (e.g., temporary and/or acute) and/or a long-term (e.g., sustained) reduction in one or more cancer symptoms. In some embodiments, treatment results in an improvement or remediation of the symptoms of the cancer. The improvement is an observable or measurable improvement, or may be an improvement in the general feeling of well-being of the subject. [0300] An “effective amount” or “an amount effective to treat” refers to a dose that is adequate to prevent or treat cancer in an individual. In some embodiments, an effective amount is also one in which any toxic or detrimental effects of the antibody are outweighed by the therapeutically beneficial effects. The effective amount of a 5T4 binding agent or multi-specific binding agent, or a pharmaceutical composition comprising the same administered to a particular subject will depend on a variety of factors, several of which will differ from patient to patient including the disorder being treated and the severity of the disorder; activity of the specific agent(s) employed; the age, body weight, general health, sex and diet of the patient; the timing of administration, route of administration; the duration of the treatment; the ability of the binding agent or multi-specific binding agent comprising the same to elicit a desired response in the individual; drugs used in combination; the judgment of the prescribing physician; and like factors known in the medical arts. The size of the dose will also be determined by the existence, nature, and extent of any adverse side- effects that might accompany the administration of a particular active, and the desired physiological effect. It will be appreciated by one of skill in the art that various diseases or disorders could require prolonged treatment involving multiple administrations, perhaps using various rounds of administration. [0301] The cancer can be any cancer, including any of acute lymphocytic cancer, acute myeloid leukemia (AML), alveolar rhabdomyosarcoma, B-cell chronic lymphoproliferative disorders, bladder cancer (e.g., bladder carcinoma), blastic plasmacytoid dendritic cell neoplasm, bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, extrahepatic bile duct , cancer of the joints, cancer of the head and neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, 338699-2168 chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, leukemia, liver cancer, lung cancer (e.g., non-small cell lung carcinoma and lung adenocarcinoma), lymphoma, mesothelioma, mastocytoma, melanoma, multiple myeloma, myelodysplastic syndrome, nasopharynx cancer, non-Hodgkin lymphoma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), Burkitt’s lymphoma, ovarian cancer, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, stomach cancer, small intestine cancer, soft tissue cancer, synovial sarcoma, gastric cancer, testicular cancer, thyroid cancer, and ureter cancer. [0302] Exemplary expression of 5T4 in certain isolated patient samples is shown in FIG.1A. In some embodiments, the cancer is colorectal cancer (CRC). In some embodiments, the colorectal cancer is metastatic colorectal cancer (mCRC). In some embodiments, the colorectal cancer is primary colorectal cancer (pCRC). In some embodiments, the cancer is clear cell renal cell cancer. In some embodiments the cancer is glioblastoma multiforme (GBM). In some embodiments, the cancer is cervical cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is urothelial cell carcinoma. In some embodiments, the cancer is melanoma. [0303] In some embodiments, the cancer is characterized by the expression of 5T4. For example, in some embodiments, the cancer is selected from colorectal cancer, primary colorectal cancer, metastatic colorectal cancer, clear cell renal cell cancer, glioblastoma multiforme (GBM), cervical cancer, cervical squamous cell carcinoma, cervical adenocarcinoma, gastric cancer, head and neck cancer, urothelial cell carcinoma, melanoma, kidney cancer, breast cancer, colon cancer, prostate cancer, non-small lung cancer, lung cancer, pancreatic cancer, mesothelioma, pre-B acute lymphoblastic leukemia, hepatocellular, and ovarian cancer. [0304] “Treatment” or “treating” refers to the administration of an effective amount of a binding agent or multi-specific binding agent comprising the same according to the present disclosure with the purpose of easing, ameliorating, arresting, eradicating (curing) or preventing symptoms or disease states. [0305] In some embodiments, the subject may be a neonate, a juvenile, or an adult. Of particular interest are mammalian subjects. Mammalian species that may be treated with the present methods include canines and felines; equines; bovines; ovines; etc. and primates, 338699-2168 particularly humans. Animal models, particularly small mammals (e.g., mice, rats, guinea pigs, hamsters, rabbits, etc.) may be used for experimental investigations. [0306] In some embodiments, the present disclosure provides methods of expanding Vγ9Vδ2 T cells comprising contacting the Vγ9Vδ2 T cells with a 5T4 binding agent described herein, a multi-specific binding agent comprising the same, or a pharmaceutical composition comprising the same. In some embodiments, the contacting is in vitro, in vivo, or ex vivo. Expansion of Vγ9Vδ2 T cells may be measured by any method known in the art (e.g., labeling with a suitable agent such as carboxyfluorescein succinimidyl ester followed by quantification of fluorescence by e.g., flow cytometry). [0307] Administration of the 5T4 binding agent or multi-specific binding agent, or of a pharmaceutical composition comprising the same, can occur by injection, irrigation, inhalation, consumption, electro-osmosis, hemodialysis, iontophoresis, and other methods known in the art. In some embodiments, administration route is local or systemic. In some embodiments administration route is intraarterial, intracranial, intradermal, intraduodenal, intramammary, intrameningeal, intraperitoneal, intrathecal, intratumoral, intravenous, intravitreal, ophthalmic, parenteral, spinal, subcutaneous, ureteral, urethral, vaginal, intramuscular, inhalation, oral, or intrauterine. In some embodiments, the administration will be parenteral. [0308] In some embodiments, the administration route is by infusion (e.g., continuous or bolus). Examples of methods for local administration, that is, delivery to the site of injury or disease, include through an Ommaya reservoir, e.g. for intrathecal delivery (See e.g., US Patent Nos.5,222,982 and 5,385,582, incorporated herein by reference); by bolus injection, e.g. by a syringe, e.g. into a joint; by continuous infusion, e.g. by cannulation, such as with convection (See e.g., US Patent Application Publication No. 2007-0254842, incorporated herein by reference); or by implanting a device upon which the binding agents have been reversibly affixed (see e.g. US Patent Application Publication Nos.2008-0081064 and 2009- 0196903, incorporated herein by reference). In some embodiments, the administration route is by topical administration or direct injection. In some embodiments, the binding agents described herein may be provided to the subject alone or with a suitable substrate or matrix. [0309] In some embodiments, at least 0.01 µg/kg of the 5T4 binding agent or multi-specific binding agent, or of a pharmaceutical composition comprising the same, is administered to a subject. In some embodiments, between about 0.01 µg/kg and about 100mg/kg, between about 0.1 µg/kg and about 50 mg/kg, between about 0.01 mg/kg and about 20 mg/kg, or between about 0.01 mg/kg and about 10 mg/kg of the 5T4 binding agent or multi-specific binding agent, or of a pharmaceutical composition comprising the same, is administered to a subject. In some embodiments, at least about 0.01 mg/kg, at least about 0.03 mg/kg, at least about 0.05 mg/kg, at least about 0.1 mg/kg, about 0.3 mg/kg, about 0.5 mg/kg, about 1 mg/kg, 338699-2168 about 3 mg/kg, about 5 mg/kg, about 8 mg/kg, or about 10 mg/kg of the 5T4 binding agent or multi-specific binding agent, or of a pharmaceutical composition comprising the same, is administered to the subject. In some embodiments, at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11.0, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or at least 500 nM of the 5T4 binding agent or multi-specific binding agent, or of a pharmaceutical composition comprising the same, is administered to a subject. [0310] The number of administrations of treatment to a subject may vary. In some embodiments, administration to the subject may be a one-time event. In some embodiments, such treatment may require an on-going series of repeated treatments. In some embodiments, multiple administrations may be required before an effect is observed. The exact protocols depend upon the disease or condition, the stage of the disease and parameters of the individual subject being treated. [0311] In some embodiments, the effective amount of a 5T4 binding agent, a multi-specific binding agent comprising the same, or a pharmaceutical composition comprising the same may be the amount required to result in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more fold decrease in tumor mass or volume, decrease in the number of tumor cells, or decrease in the number of metastases. In some embodiments, the effective amount of a 5T4 binding agent, a multi-specific binding agent comprising the same, or a pharmaceutical composition comprising the same may be the amount required to achieve an increase in life expectancy, an increase in progression-free or disease-free survival, or amelioration of various physiological symptoms associated with the disease being treated. [0312] In some embodiments, the methods of treatment provided herein further comprise the administration of one or more additional therapeutic agents. The one or more additional therapeutic agents can be administered prior to, simultaneously with, or after the 5T4 binding agent or multi-specific binding agent comprising the same, or composition comprising the same described herein. In some embodiments, the additional therapeutic agent is IL-2, IL-15, or variants thereof. [0313] In some embodiments, the additional therapeutic agent is IL-2. In some embodiments, the IL-2 is Aldesleukin/Proleukin. Other suitable forms of IL-2 include NKTR-214 (Bempegaldesleukin/Nektar), ALK 4230 (nemvaleukin/Aalkermes-Reliant), SAR444245 – THOR 707/Synthorx/Sanofi) and XTX-202 (Xilio). In some embodiments, the IL-2 is a variant IL-2. In some embodiments, the variant IL-2 binds to the IL-2Rbeta and gamma chains, but 338699-2168 does not bind or demonstrates reduced/abrogated binding to the IL-2Ralpha chain (e.g., NL201 from Neoleukin, Silva et al. De novo design of potent and selective mimics of IL-2 and IL-15. Nature 565, 186–191 (2019), and MDNA11 and MDNA19 from Medicenna). In some embodiments, the IL-2 and/or variants thereof is pegylated. [0314] In some embodiments, the additional therapeutic agent is IL-15. Suitable forms of IL- 15 are known in the art. See e.g., Int J Mol Sci.2022 Jul; 23(13): 7311, which is herein incorporated by reference in its entirety. In some embodiments, the IL-15 is a heterodimer of IL-15 and IL-15 receptor alpha (hetIL-15), which is also referred to as NIZ985. In some embodiments, the IL-15 is N-803, formerly known as ALT-803, which is an IL-15 variant complexed with a human IL-15Rα sushi domain-Fc fusion protein. In some embodiments, the IL-15 is SOT101, also referred to as Nanrilkefusp alfa, which is a human fusion protein comprising the cytokine IL-15 and the high-affinity binding sushi+ domain of IL-15 receptor alpha (IL-15Rα). In some embodiments, the IL-15 is NKTR-255, which is a polyethylene glycol- conjugate of rhIL-15. In some embodiments, the IL-15 and/or variants thereof is pegylated. [0315] In some embodiments, the 5T4 binding agent or multi-specific binding agent comprising the same is administered as monotherapy. However, 5T4 binding agents or multi- specific binding agents disclosed herein may also be administered in combination therapy, i.e., combined with other therapeutic agents relevant for the disease or condition to be treated. In some embodiments, the combination therapy comprises administration of a 5T4 binding agent or multi-specific binding agent comprising the same and an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti PD-1 antibody (e.g., Pembrolizumab (Keytruda®), nivolumab (Opdivo®)), an anti-PDL1 antibody (e.g., Atezolizumab (Tecentriq®), Avelumab (Bavencio®), Durvalumab (Imfinzi®)), or an anti- CTLA4 antibody (e.g., Ipilimumab (Yervoy®)). [0316] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application herein is not, and should not be, taken as acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world. EXAMPLES Example 1: Generation of anti-5T4 VHH [0317] Lama glama were immunized with either MCF7 cells or A-431 cells followed by 2 injections of recombinant human 5T4. Whole blood samples were collected from the 338699-2168 immunized animals for peripheral blood lymphocyte (PBL) isolation to generate phage libraries. [0318] Briefly, total RNA was extracted from the PBLs, transcribed into cDNA, purified, and used as a template for immunoglobulin heavy-chain-encoding cDNA amplification. The resultant cDNA was run on an agarose gel and the cDNA encoding heavy-chain-only immunoglobulins (binding agents) (~ 700 bp) was excised from the gel. The purified cDNA was used as a template for a nested PCR that introduced a 5` flanking restriction site and amplified only binding agent-encoding cDNA. The purified PCR product was digested with appropriate restriction enzymes, after which the digested products were ligated in frame with geneIII into the pQ81 phagemid vector. [0319] The ligated library was transformed into TG1 E. coli by electroporation for display on filamentous bacteriophage. Two consecutive rounds of selection were performed, followed by screening single clones for binding to plate-bound recombinant human 5T4. Those clones that scored positive for binding were sequenced and a selection of clones having different sequences were then tested for binding to recombinant human 5T4 by ELISA. Sixteen different clones were identified: LV1138 (SEQ ID NO: 101), LV1139 (SEQ ID NO: 92), LV1140 (SEQ ID NO: 108), LV1141 (SEQ ID NO: 114), LV1142 (SEQ ID NO: 118), LV1143 (SEQ ID NO: 128), LV1144 (SEQ ID NO: 136), LV1145 (SEQ ID NO: 140), LV1146 (SEQ ID NO: 144), LV1147 (SEQ ID NO: 148), LV1148 (SEQ ID NO: 152), LV1149 (SEQ ID NO: 156), LV1150 (SEQ ID NO: 160), LV1151 (SEQ ID NO: 164), LV1152 (SEQ ID NO: 168), and LV1292 (SEQ ID NO: 172). The binding profiles are shown in FIG.3A-FIG.3C. Example 2: Affinity determination of VHHs for binding to human 5T4 using biolayer interferometry (BLI) [0320] To determine the kinetics of 5T4-binding for all of the 5T4 binding agents, biotinylated recombinant purified human 5T4 protein (Sino Biological) was loaded onto SAX biosensors (Sartorius) at a concentration of 2.5 µg/mL for an Octet Red96e (Sartorius) instrument. The sensors were then dipped in different concentrations of the various 5T4 binding agents diluted in 10x kinetic buffer (10xKB) provided by the supplier. From the obtained sensorgrams, the kinetic association and dissociation rate constants were determined by curve fitting. The binding agents tested were LV1138, LV1139, LV1140, LV1141, LV1142, LV1143, LV1144, LV1145, LV1146, LV1147, LV1148, LV1149, LV1150, LV1151, and LV1152. [0321] Table 10 shows the binding affinities (KD) of the binding agents determined in two independent runs. The remainder of the binding agents did not show binding to (biotinylated) 5T4. 338699-2168 Table 10: Binding affinities (KD) of binding agents for human 5T4 determined in 2 independent BLI runs. Binding Run 1 Run 2 agent K on (1/Ms) K dis (1/s) KD (nM) K on (1/Ms) K dis (1/s) K D (nM) LV1138 2.53E+05 9.22E-04 3.65 3.03E+05 9.91E-04 3.27 LV1139 4.61E+05 7.33E-04 1.59 6.26E+05 7.51E-04 1.2 LV1140 3.17E+05 3.38E-03 10.7 3.40E+05 3.83E-03 11.25 LV1141 2.60E+05 3.16E-03 12.2 3.20E+05 3.48E-03 10.88 LV1142 2.33E+05 7.75E-04 3.32 2.86E+05 1.09E-03 3.8 LV1143 3.34E+05 2.90E-03 8.66 4.09E+05 4.04E-03 9.87 LV1144 3.87E+05 1.23E-02 31.9 4.86E+05 1.34E-02 27.56 LV1150 3.53E+05 1.00E-03 2.84 3.97E+05 9.13E-04 2.3 Example 3: Competition for binding to human 5T4 [0322] Competition for binding to recombinant human 5T4 between the 5T4-binding agents was determined. SAX biosensors were loaded with biotinylated human 5T4 protein as described in Example 2. The eight different binding agents identified in the previous example (LV1138, LV1139, LV1140, LV1141, LV1142, LV1143, LV1144, LV1150) were used in concentrations based on the affinities as indicated above. A fixed amount of the first binding agent was allowed to bind to the 5T4 loaded sensor, after which the sensor was exposed to a mixture of the same amount of the first binding agent and a fixed amount of a second binding agent, followed by dissociation in 10X KB. [0323] The results showed the following: (a) LV1138, LV1139, and LV1150 compete with each other for binding to human 5T4, (b) LV1140 and LV1141 compete with each other for binding to human 5T4, and (c) LV1142, LV1143, and LV1144 compete with each other for binding to human 5T4. Example 4: Synthetic gene synthesis, production, and purification of multi-specific binding agents [0324] The sequences of four 5T4 binding agents (LV1138, LV1139, LV1140, and LV1142) were then re-formatted into multi-specific binding agents by linking the 5T4 binding agent to a Vδ2 binding agent (5C8 – SEQ ID NO:5) in the following orientation, from N- to C-terminus: 5T4 binding agent – linker– Vδ2 binding agent. The linker between the two VHHs was a glycine(G)-serine(S) stretch with the sequence G4S (SEQ ID NO: 341). The cDNAs encoding 338699-2168 these proteins were prepared by synthetic gene assembly and cloned into a proprietary expression vector. The proteins were then expressed by transient transfection of the modified expression vector into HEK293E-253 cells. The multi-specific binding agents were then purified from the conditioned cell culture supernatant using HiTrap Fibro PrismA columns according to the supplier’s protocol. After elution from the column, the multi-specific binding agents were buffer-exchanged to PBS. Purified multi-specific binding agents were always >95% pure as determined by SDS-PAGE analysis using Coomassie staining and contained very low levels of endotoxin (≤0.05 EU/mg). Example 5: Binding of 5T4xVδ2 multi-specific binding agents to human, cynomolgus monkey and mouse 5T4 expressing cells using cell-based ELISA [0325] Protein sequences of human (Uniprot number Q13641), cynomolgus monkey (Uniprot number Q4R8Y9) and mouse (Uniprot number Q9Z0L0) 5T4 were reverse-translated into cDNA and then codon-optimized for expression in human cells. Regulatory elements were added to each cDNA, namely an N-terminal Kozak sequence and C-terminal stop codon (including BamH1 and Age1 restriction sites for cloning) and the cDNA was made into a synthetic gene. cDNAs were then cloned into a suitable vector and the sequences were verified. Expression of the proteins was performed by transient transfection of the resulting plasmids in CHO-K1 cells. Expression of human/cynomolgus 5T4 was confirmed using commercially available control antibody 5T4-AF647(R&D systems) in flow cytometry, and expression of mouse 5T4 was confirmed by expression of the eGFP-tag incorporated in the expression vector. [0326] Cells from each transfection were seeded in 96F CellBIND plates (Corning) and cultured overnight at 37°C/5% CO2 in a humidified atmosphere and subsequently incubated with serial dilutions of each 5T4xVδ2 multi-specific binding agent, ranging from 0.006 or 4 nM to 500 nM for one hour at 37°C/5% CO2. Plates were washed and cells were incubated with HRP-labeled rabbit anti-camelid VHH cocktail (GenScript) for one hour at 37°C/5% CO2. After washing, 3,3’,5,5’-tetramethylbenzidine (TMB) substrate was added, followed by 20 minute incubation in the dark at room temperature. The reaction was stopped by the addition of Stop buffer (1M H2SO4) and the optical densities were measured with a multi-mode plate reader Spectramax iD5, at wavelengths 450 nm and 610 nm. [0327] FIG. 4A-FIG. 4C show that all four 5T4xVδ2 multi-specific binding agents bound to CHO-K1 cells expressing human 5T4. The EC50 for each of the multi-specific binding agents to human 5T4 is shown in Table 11. None of the multi-specific VHHs specifically bound to non- transfected CHO-K1 cells (not shown), or to CHO-K1 cells expressing mouse or cynomolgus monkey 5T4. 338699-2168 Table 11: EC50 values for the indicated 5T4 multi-specific binding agents to human 5T4 Multi-specific binding agent EC50 LV1138xVδ2 (SEQ ID NO: 364) 3.13 nM LV1139xVδ2 (SEQ ID NO: 362) 1.51 nM LV1140xVδ2 (SEQ ID NO: 365) 6.11 nM LV1142xVδ2 (SEQ ID NO: 363) 3.03 nM Example 6: Binding of 5T4xVδ2 multi-specific binding agents to 5T4 expressing human tumor cell lines [0328] Based on the results in Example 5, the 5T4 binders were further characterized. Using flow cytometry, binding of the selected 5T4xVδ2 multi-specific binding agents to 5T4 endogenously expressed on human tumor cell lines was determined. [0329] First, 5T4 expression on these tumor cell lines (U-251 and A-431) was confirmed using a commercially available monoclonal 5T4 specific antibody (5T4-AF647(R&D systems)). [0330] Next, cells were incubated with a concentration range of each multi-specific binding agent, ranging from 0.003 to 316 nM and the binding of the 5T4 binding agent was detected using a rabbit anti-camelid iFluor 647-labeled antibody cocktail (Genscript). Staining was visualized using a FACS Celesta (Becton and Dickinson). [0331] FIG.5A and FIG.5B show that LV1139xVδ2 and LV1142xVδ2 bound to both cell lines with high affinity as seen in Table 12. LV1138xVδ2 and LV1140xVδ2 also bound to the tumor cell lines, yet with lower affinity. Table 12: EC50 values for the LV1139xVδ2 and LV1142xVδ25T4 multi-specific binding agents to 5T4-expressing tumor cells Tumor cell line EC50 LV1139xVδ2 EC50 LV1142xVδ2 U-251 2.5 nM 1.8 nM A-431 1.9 nM 1.7 nM Example 7: PBMC isolation and generation of human donor-derived Vγ9Vδ2-T cell cultures [0332] Buffy coats were obtained from blood supply service Sanquin and used for isolation of peripheral blood mononuclear cells (PBMC). PBMC were isolated using LymphoprepTM density gradient centrifugation. Vγ9Vδ2-T cells were then isolated from healthy donor-derived PBMC by magnetic-activated cell sorting (MACS) using a FITC-labeled anti-TCR Vδ2 mouse 338699-2168 monoclonal antibody (Mab) in combination with goat anti-mouse IgG microbeads. Purified Vγ9Vδ2-T cells were stimulated with a feeder cell mix consisting of irradiated PBMC from two (other) healthy donors and an Epstein Barr Virus transformed B cell line (JY) resuspended in Roswell Park Memorial Institute (RPMI) medium supplemented with 10 IU/mL IL-7, 10 ng/mL IL-15 and 50 ng/ml PHA. Expanded Vγ9Vδ2-T cell cultures were tested for purity (and only used when >90% pure) before being used for experiments. Example 8: Binding of 5T4xVδ2 multi-specific binding agents to Vγ9Vδ2 T cells [0333] Binding of the 5T4xVδ2 bispecific binding agents to human Vγ9Vδ2 T cells was determined by flow cytometry as described above. FIG. 6 shows that all 45T4xVδ2 multi- specific binding agents bound to Vγ9Vδ2 T cells with similar EC50s of 1-1.8 nM. Example 9: Vγ9Vδ2-T cell degranulation and cytotoxicity of tumor cell lines induced by 5T4xVδ2 multi-specific binding agents [0334] To test the 5T4-multi-specific binding agents for Vγ9Vδ2-T cell activation and tumor cytotoxicity, U-251 or A-431 tumor target cells and Vγ9Vδ2 T cells from normal donors (n=8; 4 unique donors for each tumor cell line experiment) were mixed in a 1:1 ratio in culture medium with labeled anti-CD107a antibody. This mixture was seeded in 96-well culture plates (with 50,000 target/Vγ9Vδ2 T cells per well) and serial dilutions of 5T4xVδ2 multi-specific binding agents were added. Tumor cells and Vγ9Vδ2-T cells alone with, and without, the highest concentration of 5T4xVδ2 multi-specific binding agents were also prepared as controls. After incubation for 24 hours at 37°C/5% CO2, cells were resuspended, washed, and transferred to 96-well U bottom FACS plates. Remaining adherent cells were detached using trypsin/0.5% EDTA, washed and transferred to the corresponding wells of the FACS plates. The plates were washed and the cells were resuspended in FACS buffer (PBS/1% BSA) and incubated with labeled anti-CD3 (Biolegend) and anti-Vγ9 (Beckman Coulter) antibodies for 30 minutes at 4°C. Cells were washed and labeled 7-AAD was added. Staining was visualized using a FACS Celesta (Becton and Dickinson). [0335] FIGs.7A-FIG. 8H show that all four 5T4xVδ2 multi-specific binding agents induced similar levels of Vγ9Vδ2 T cell degranulation as determined by CD107a expression in all of the donor Vγ9Vδ2-T cells. Surprisingly, despite differences in binding to tumor cells (See Example 6), all four 5T4xVδ2 bispecific VHHs induced a comparable level of lysis of tumor cells with similar range of EC50s (See Table 13 and Table 14). 338699-2168 Table 13: EC50 values for Vγ9Vδ2-T cell degranulation and cytotoxicity of U-251 cells induced by 5T4 multi-specific binding agents (nM) LV1138xVδ2 LV1139xVδ2 LV1140xVδ2 LV1142xVδ2 Donor Degranulation 0.0046 0.0030 0.0115 0.0064 1 Cytotoxicity 0.0032 0.0039 0.0070 0.0059 Donor Degranulation 0.0050 0.0030 0.0095 0.0070 2 Cytotoxicity 0.0037 0.0019 0.0074 0.0055 Donor Degranulation 0.0056 0.0050 0.0162 0.0119 3 Cytotoxicity 0.0036 0.0033 0.0106 0.0045 Donor Degranulation 0.0037 0.0037 0.0124 0.0047 4 Cytotoxicity 0.0051 0.0060 0.0199 0.0059 Table 14: EC50 values for Vγ9Vδ2-T cell degranulation and cytotoxicity of A-431 cells induced by 5T4 multi-specific binding agents LV1138xVδ2 LV1139xVδ2 LV1140xVδ2 LV1142xVδ2 Donor Degranulation 0.0065 0.0040 0.0146 0.0047 1 Cytotoxicity 0.0005 0.0006 0.0017 0.0004 Donor Degranulation 0.0016 0.0016 0.0047 0.0014 2 Cytotoxicity 0.0007 0.0006 0.0017 0.0005 Donor Degranulation 0.0016 0.0013 0.0035 0.0020 3 Cytotoxicity 0.0005 0.0004 0.0014 0.0005 Donor Degranulation 0.0033 0.0027 0.0076 0.0041 4 Cytotoxicity 0.0010 0.0001 0.0012 0.0003 Example 10: 5T4xVδ2 multi-specific binding agents induce Vγ9Vδ2-T cell degranulation and tumor cell lysis of patient derived tumor cells [0336] Tumor tissue was obtained from patients with squamous cell (n=1) or adeno- carcinoma (n=1) of the cervix, metastatic colorectal cancer (n=3) or glioblastoma multiforme (n=1). After enzymatic and mechanical dissociation, the presence of Vγ9Vδ2-T cells and the expression of 5T4 on tumor cells was determined by flow cytometry. All of the samples contained Vγ9Vδ2-T cells (varying between 0.18 to 2.42 of total T cells present in the sample) and 5T4-expressing tumor cells (data not shown). [0337] To test the 5T4-multi-specific binding agents for Vγ9Vδ2-T cell activation and tumor cytotoxicity against patient-derived target cells, dissociated tumor-suspensions were co- cultured with expanded allogeneic Vγ9Vδ2-T cells in a 1:2 effector:target (E:T) ratio for 18 hours or 7 days at 37°C in the absence or presence of 10 nM of the 5T4-multi-specific binding agent. Vγ9Vδ2-T cell degranulation and cytotoxicity was determined as described above. [0338] Only LV1139xVδ2 was tested in cervical adenocarcinoma and LV1139xVδ2 and LV1142xVδ2 were tested in metastatic colorectal cancer samples due to limited number of tumor cells obtained from the patients. 338699-2168 [0339] FIG. 9A-FIG. 9D show that Vγ9Vδ2 T cell degranulation as measured by CD107a expression was induced by all of the tested 5T4-multi-specific binding agents in patient- derived cervical squamous cell carcinoma (FIG. 9A), cervical adenocarcinoma (FIG. 9B), metastatic colorectal cancer (FIG.9C), and glioblastoma multiforme (FIG.9D). [0340] FIG.10A-FIG.10E show that tumor cell lysis was induced by all of the tested 5T4- multi-specific binding agents in patient-derived cervical squamous cell carcinoma at 18 hours (FIG.10A) and 7 days (FIG.10B), cervical adenocarcinoma at 18 hours (FIG.10C) and 7 days (FIG.10D), and in glioblastoma multiforme at 18 hours (FIG.10E). Example 11: Humanization of the anti-5T4 VHHs [0341] The amino acid sequence of the llama-derived 5T4 binding agents (LV1138, LV1139, LV1140 and LV1142) were aligned to the human V gene database and the closest match to a human germline sequence were identified. 5T4 Percentage identity Percentage identity Human germline VHH (full sequence, including (framework only, IMGT CDR1/2) numbering) LV1138 79% 83% IGHV3-23*04 LV1139 82% 89% IGHV3-23*04 LV1140 86% 90% IGHV3-48*03 LV1142 74% 78% IGHV3-66*01 [0342] Two or three humanized variants of each of the 5T4 binding agents were designed based on sequence differences in the framework regions between the human and llama- derived sequence (SEQ ID NOs: 94, 95, 96, 102, 103, 104, 109, 110, 119, 120, and 121). Framework residues close to the CDRs were not changed. Example 12: Expression of 5T4 in Cancers [0343] FIG.1A shows the expression of 5T4 on freshly isolated patient tumor samples and FIG.2 shows the expression of 5T4 in non-malignant urothelial cells as measured by the mean fluorescence index (MFI) (geometric (g)MFI stained/gMFI isotype). FIG. 1B shows the percentage of Vγ9+Vδ2+ T cells out of total CD3+ T cells in different cancer tissues. (m)/(p)CRC = (metastatic)/(primary) colorectal cancer; ccRCC = clear cell renal cell cancer; GBM = glioblastoma multiforme; H&N = head & neck cancer; UCC = urothelial cell carcinoma; AC = adenocarcinoma; SCC = squamous cell carcinoma. 338699-2168 Example 13: 5T4xVδ2 multi-specific binding agents induce Vγ9Vδ2 T cell degranulation and tumor cell lysis in 5T4+ patient-derived tumors. [0344] Experiments were performed to evaluate the activity of LAVA-378 (SEQ ID NO: 362, comprising the LV11395T4 binding agent and the 5C8 Vδ2 binding agent.) against 5T4+ solid tumors (CRC= colorectal cancer (Degranulation n=2); mCRC = metastatic colorectal cancer (Degranulation n=3); H&N = head & neck cancer (n=3); Melanoma (n=5) GBM = glioblastoma multiforme (Degranulation n=3, Cytotoxicity n=1); UCC = urothelial cell carcinoma (Degranulation n=9, Cytotoxicity n=7); Cervix (n=2); ccRCC = clear cell renal cell cancer (n=6)). The tumor samples were incubated overnight with Vγ9Vδ2 T cells and with 10 nM of LAVA-378. Control conditions without LAVA-378 were also included. [0345] As shown in Fig. 11A-FIG. 11B, there was an increase in both Vγ9Vδ2 T cell degranulation (as measured by percent increase in CD107a expression (FIG.11A)) and tumor cell lysis (FIG. 11B) in tumor samples incubated with allogeneic Vγ9Vδ2 T cells in the presence of LAVA-378 as opposed to the tumor samples that were only incubated with allogeneic Vγ9Vδ2 T cells. A multiple paired T test was used to test for significance. Tumor plus Vγ9Vδ2-T cells with compound was compared to tumor plus Vγ9Vδ2-T cells without compound.*=, P ≤ 0.05, **=, P ≤ 0.01, ***= P ≤ 0.001, ****= P ≤ 0.0001. [0346] Non-cancerous and cancerous urothelial cells were incubated with Vγ9Vδ2 T cells and LAVA-378. As shown in FIG.12A and FIG.12B, the addition of LAVA-378 led to an increase in urothelial cancer cell lysis but did not affect non-malignant urothelial cell lysis. The multiple paired T test was used to test for significance. Tumor plus Vγ9Vδ2-T cells with compound was compared to tumor plus Vγ9Vδ2-T cells without compound. ***= P ≤ 0.001. [0347] In urothelial cell carcinomas, the presence of LAVA-378 led to an increase in tumor cell lysis even without the addition of expanded Vγ9Vδ2-T cells (FIG.13). The multiple paired T test was used to test for significance. Tumor with compound was compared to tumor without compound. **= P ≤ 0.01. Example 14: 5T4xVδ2 multi-specific binding agents induce Vγ9Vδ2 T cell degranulation and tumor cell lysis in 5T4+ patient-derived tumors. [0348] Additional experiments were performed to evaluate the activity of LAVA-378 and LAVA-380 (SEQ ID NO: 363, comprising the LV11425T4 binding agent and the 5C8 Vδ2 biding agent) against metastatic CRC patient samples. The tumor samples were incubated overnight with Vγ9Vδ2 T cells at an effector: target ration of 1:2 and with 10 nM of LAVA-378, 10 nM of LAVA-380 or no additional compounds. FIG.14A shows the 5T4 expression on the patient samples. FIG.14B shows that both LAVA-378 and LAVA-380 induced Vγ9Vδ2 T cell degranulation. 338699-2168 Example 15: In vivo testing of the 5T4xVδ2 multi-specific binding agents [0349] The anti-tumor effect of the multi-specific binding agents are tested in vivo. Mice are inoculated with 5T4+ solid tumor cells. The mice are then split into different treatment groups: 1) Vγ9Vδ2-T cells; 2) Vγ9Vδ2-T cells + 0.1 mg/kg LAVA-1433, comprising SEQ ID NOs: 367 and 381 (LV1139 (E1D) x 5C8 var1 (E1D-Y105F-R109A); 3) Vγ9Vδ2-T cells + 0.5 mg/kg LAVA-1433; 4) Vγ9Vδ2-T cells + 1 mg/kg LAVA-1433; 5) Vγ9Vδ2-T cells + 2 mg/kg LAVA- 1433; 6) 0.1 mg/kg LAVA-1433; 7) 0.5 mg/kg LAVA-1433; 8) 1 mg/kg LAVA-1433; 9) 2 mg/kg LAVA-1433; and 10) vehicle. Survival is analyzed. After 6 weeks, mice are euthanized and tumor volume and mass are measured. Example 16: Modifications to binding agents to prevent pyroglutamate formation [0350] Pyroglutamate formation at the first amino acid position in proteins increases heterogeneity and can reduce the stability of the proteins at 2-8℃, therefore requiring storage at -20℃ as opposed to storage at 2-8℃. To prevent pyroglutamate formation, a multi-specific binding agent was generated wherein aspartic acid, which cannot form pyroglutamate, was substituted for glutamic acid at the first N-terminal amino acid position. Pyroglutamination increases the hydrophobicity of a protein. Therefore, reverse phase-HPLC was used to determine the presence of pyroglutamination, as reverse phase-HPLC separates proteins based on hydrophobicity. More hydrophilic protein species elute at shorter retention times and more hydrophobic species elute at longer retention times. [0351] Briefly, Chinese hamster ovary (CHO) cells were transfected with vectors coding either LAVA-1395 (forms pyroglutamate) or LAVA-1433 (cannot form pyroglutamate) and subsequently cultured in liquid medium for protein expression. The proteins were then purified and analyzed by reversed phase-HPLC. Protein samples were loaded onto a liquid chromatography system equipped with a reversed-phase column. Organic solvents were used for binding and eluting of proteins. Results are shown in FIG.15A and FIG.15B. The main species of LAVA-1395 and LAVA-1433 exhibit essentially identical hydrophobicities as demonstrated by their highly similar retention times. However, the LAVA-1395 chromatogram (Fig.15A) displays a small peak that elutes around 30 seconds after the main species. This peak carries mainly the N-terminal modification pyroglutamate. The N-terminal aspartate that is present in LAVA-1433 cannot undergo such a modification since ring formation is hampered by the shorter sidechains. Consequently, no hydrophobic pyroglutamation peak is detected for LAVA-1433 (FIG.15B).

Claims

338699-2168 CLAIMS 1. A single domain antibody that binds 5T4 comprising: a) a complementarity determining region (CDR)1 comprising an amino acid sequence selected from SEQ ID NOs: 88, 98, 105, 111, 115, 122, 123, 133, 137, 141, 145, 149, 153, 157, 161, 165, and 169; b) a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: : 89, 99, 106, 112, 116, 124, 125, 134, 138, 142, 146, 150, 154, 158, 162, 166 and 170; and c) a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 90, 100, 107, 113, 117, 126, 127, 135, 139, 143, 147, 151, 155, 159, 163, 137 and 171. 2. The single domain antibody of claim 1, wherein the CDR1, CDR2 and CDR3 sequences are selected from the combination disclosed in TABLE 4A. 3. The single domain antibody of any one of claims 1-2, comprising an amino acid sequence that is at least 90%, or at least 95%, identical to any one of SEQ ID NOs: 92, 93, 94, 95, 96, 97, 101, 102, 103, 104, 108, 109, 110, 114, 118, 119, 120, 121, 128, 129, 130, 131, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, and 172. 4. The single domain antibody of any one of claims 1-3, comprising SEQ ID NOs: 92, 93, 94, 95, 96, 97, 101, 102, 103, 104, 108, 109, 110, 114, 118, 119, 120, 121, 128, 129, 130, 131, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, and 172. 5. The single domain antibody of any one of claims 1-4, consisting of SEQ ID NOs: 92, 93, 94, 95, 96, 97, 101, 102, 103, 104, 108, 109, 110, 114, 118, 119, 120, 121, 128, 129, 130, 131, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, and 172. 6. The single domain antibody of claim 1 or claim 2, wherein the CDR1 comprises SEQ ID NO: 88, the CDR2 comprises SEQ ID NO: 89, and the CDR3 comprises SEQ ID NO: 90. 7. The single domain antibody of claim 6, comprising an amino acid sequence that is at least 90% or at least 95% identical to any one of SEQ ID NOs: 92, 93, 94, 95 and 96. 8. The single domain antibody of claim 1 or claim 2, wherein the CDR1 comprises SEQ ID NO: 88, the CDR2 comprises SEQ ID NO: 89, and the CDR3 comprises SEQ ID NO: 91. 338699-2168 9. The single domain antibody of claim 8, comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 97. 10. The single domain antibody of claim 1 or claim 2, wherein the CDR1 comprises SEQ ID NO: 98, the CDR2 comprises SEQ ID NO: 99, and the CDR3 comprises SEQ ID NO: 100. 11. The single domain antibody of claim 10, comprising an amino acid sequence that is at least 90% or at least 95% identical to any one of SEQ ID NOs: 101, 102, 103, and 104. 12. The single domain antibody of claim 1 or claim 2, wherein the CDR1 comprises SEQ ID NO: 105, the CDR2 comprises SEQ ID NO: 106, and the CDR3 comprises SEQ ID NO: 107. 13. The single domain antibody of claim 12, comprising an amino acid sequence that is at least 90% or at least 95% identical to any one of SEQ ID NOs: 108, 109, and 110. 14. The single domain antibody of claim 1 or claim 2, wherein the CDR1 comprises SEQ ID NO: 111, the CDR2 comprises SEQ ID NO: 112, and the CDR3 comprises SEQ ID NO: 113. 15. The single domain antibody of claim 14, comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 114. 16. The single domain antibody of claim 1 or claim 2, wherein the CDR1 comprises SEQ ID NO: 115, the CDR2 comprises SEQ ID NO: 116, and the CDR3 comprises SEQ ID NO: 117. 17. The single domain antibody of claim 16, comprising an amino acid sequence that is at least 90% or at least 95% identical to any one of SEQ ID NOs: 118, 119, 120, and 121. 18. The single domain antibody of claim 1 or claim 2, wherein the CDR1 comprises SEQ ID NO: 122, the CDR2 comprises SEQ ID NO: 124, and the CDR3 comprises SEQ ID NO: 126. 19. The single domain antibody of claim 18, comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 128. 20. The single domain antibody of claim 1 or claim 2, wherein the CDR1 comprises SEQ ID NO: 123, the CDR2 comprises SEQ ID NO: 124, and the CDR3 comprises SEQ ID NO: 126. 338699-2168 21. The single domain antibody of claim 20, comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 129. 22. The single domain antibody of claim 1 or claim 2, wherein the CDR1 comprises SEQ ID NO: 122, the CDR2 comprises SEQ ID NO: 125, and the CDR3 comprises SEQ ID NO: 126. 23. The single domain antibody of claim 22, comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 130. 24. The single domain antibody of claim 1 or claim 2, wherein the CDR1 comprises SEQ ID NO: 122, the CDR2 comprises SEQ ID NO: 124, and the CDR3 comprises SEQ ID NO: 127. 25. The single domain antibody of claim 24, comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 131. 26. The single domain antibody of claim 1 or claim 2, wherein the CDR1 comprises SEQ ID NO: 123, the CDR2 comprises SEQ ID NO: 125, and the CDR3 comprises SEQ ID NO: 127. 27. The single domain antibody of claim 26, comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 132. 28. The single domain antibody of claim 1 or claim 2, wherein the CDR1 comprises SEQ ID NO: 133, the CDR2 comprises SEQ ID NO: 134, and the CDR3 comprises SEQ ID NO: 135. 29. The single domain antibody of claim 28, comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 136. 30. The single domain antibody of claim 1 or claim 2, wherein the CDR1 comprises SEQ ID NO: 137, the CDR2 comprises SEQ ID NO: 138, and the CDR3 comprises SEQ ID NO: 139. 31. The single domain antibody of claim 30, comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 140. 338699-2168 32. The single domain antibody of claim 1 or claim 2, wherein the CDR1 comprises SEQ ID NO: 141, the CDR2 comprises SEQ ID NO: 142, and the CDR3 comprises SEQ ID NO: 143. 33. The single domain antibody of claim 32, comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 144. 34. The single domain antibody of claim 1 or claim 2, wherein the CDR1 comprises SEQ ID NO: 145, the CDR2 comprises SEQ ID NO: 146, and the CDR3 comprises SEQ ID NO: 147. 35. The single domain antibody of claim 34, comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 148. 36. The single domain antibody of claim 1 or claim 2, wherein the CDR1 comprises SEQ ID NO: 149, the CDR2 comprises SEQ ID NO: 150, and the CDR3 comprises SEQ ID NO: 151. 37. The single domain antibody of claim 36, comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 152. 38. The single domain antibody of claim 1 or claim 2, wherein the CDR1 comprises SEQ ID NO: 153, the CDR2 comprises SEQ ID NO: 154, and the CDR3 comprises SEQ ID NO: 155. 39. The single domain antibody of claim 38, comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 156. 40. The single domain antibody of claim 1 or claim 2, wherein the CDR1 comprises SEQ ID NO: 157, the CDR2 comprises SEQ ID NO: 158, and the CDR3 comprises SEQ ID NO: 159. 41. The single domain antibody of claim 40, comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 160. 42. The single domain antibody of claim 1 or claim 2, wherein the CDR1 comprises SEQ ID NO: 161, the CDR2 comprises SEQ ID NO: 162, and the CDR3 comprises SEQ ID NO: 163. 338699-2168 43. The single domain antibody of claim 42, comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 164. 44. The single domain antibody of claim 1 or claim 2, wherein the CDR1 comprises SEQ ID NO: 165, the CDR2 comprises SEQ ID NO: 166, and the CDR3 comprises SEQ ID NO: 167. 45. The single domain antibody of claim 44, comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 168. 46. The single domain antibody of claim 1 or claim 2, wherein the CDR1 comprises SEQ ID NO: 169, the CDR2 comprises SEQ ID NO: 170, and the CDR3 comprises SEQ ID NO: 171. 47. The single domain antibody of claim 46, comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 172. 48. The single domain antibody of claim 1 or claim 2, wherein the CDR1 comprises SEQ ID NO: 88, the CDR2 comprises SEQ ID NO: 89, and the CDR3 comprises SEQ ID NO: 90. 49. The single domain antibody of claim 1, 2 or 48, comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 93. 50. The single domain antibody of claim 1, 2, 48, or 49, comprising or consisting of the amino acid sequence of SEQ ID NO: 93. 51. A polynucleotide encoding the single domain antibody of any one of claims 1-50. 52. A vector comprising the polynucleotide of claim 51. 53. A host cell comprising the polynucleotide of claim 51 or the vector of claim 52. 54. A method of producing the single domain antibody of any one of claims 1-50, comprising: a) introducing the vector of claim 52 into a host cell under conditions permitting expression of the polynucleotide encoding the single domain antibody; and b) isolating the single domain antibody from the culture supernatant. 338699-2168 55. A pharmaceutical composition comprising the single domain antibody of any one of claims 1-50 and a pharmaceutically acceptable carrier. 56. A binding agent comprising the single domain antibody of any one of claims 1-50. 57. A multi-specific binding agent comprising: a) a first antigen-binding domain that specifically binds to human 5T4 comprising the single domain antibody of any one of claims 1-50; and b) a second antigen-binding domain that specifically binds to an antigen expressed on an immune cell. 58. The multi-specific binding agent of claim 57, wherein the antigen expressed on the immune cell is selected from CD3, CD2, an αβ T cell receptor, a γδ T cell receptor (TCR). 59. The multi-specific binding agent of claim 58, wherein the γδ T cell receptor is a Vδ2 TCR. 60. The multi-specific binding agent of claim 58, wherein the Vδ2 TCR is a human Vδ2 T cell receptor. 61. The multi-specific binding agent of claim 60, wherein the human Vδ2 T cell receptor is a Vγ9Vδ2 T cell receptor. 62. The multi-specific binding agent of any one of claims 57-61, wherein the first and second antigen-binding domains are present in the same polypeptide chain. 63. The multi-specific binding agent of any one of claims 57-62, wherein the first antigen- binding domain and second antigen-binding domain are covalently linked via a peptide linker. 64. The multi-specific binding agent of any one of claims 57-63, comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NOs: 362 or 363. 65. The multi-specific binding agent of any one of claims 57-64, comprising or consisting of the amino acid sequence of SEQ ID NOs: 362 or 363. 338699-2168 66. The multi-specific binding agent of any one of claims 57-60, wherein the first and second antigen-binding domains are present on different polypeptide chains. 67. The multi-specific binding agent of any one of claims 57-59 or 65, further comprising an Fc domain comprising a first and a second Fc domain monomer. 68. The multi-specific binding agent of claim 67, comprising: a) a first polypeptide comprising the first antigen-binding domain that binds to a human 5T4 protein and the first Fc domain monomer; and b) a second polypeptide comprising the second antigen-binding domain that binds to a human Vδ2 T cell receptor and the second Fc domain monomer. 69. The multi-specific binding agent of claim 68, wherein the human Vδ2 T cell receptor is a Vγ9Vδ2 T cell receptor. 70. The multi-specific binding agent of any one of claims 67-69, wherein the antigen- binding domain that binds to the Vγ9Vδ2 T cell receptor comprises SEQ ID NO: 22. 71. The multi-specific binding agent of any one of claims 67-70, wherein the antigen- binding domain that binds to the Vγ9Vδ2 T cell receptor consists of SEQ ID NO: 22. 72. The multi-specific binding agent of any one of claims 67-71, wherein the multi-specific binding agent induces proliferation of Vγ9Vδ2 T cells. 73. The multi-specific binding agent of any one of claims 67-72, wherein the first and second Fc domain monomers comprise a mutation at position 234 and/or 235 according to the EU numbering system. 74. The multi-specific binding agent of any one of claims 67-73, wherein the first and second Fc domain monomers comprise an L234F and an L235E substitution. 75. The multi-specific binding agent of any one of claims 67-74, a) wherein the first Fc domain monomer comprises a T366W substitution and the second Fc polypeptide comprises T366S, L368A and Y407V substitutions, or vice versa, according to the EU numbering system; and/or 338699-2168 b) wherein the cysteine residue at position 220 according to the EU numbering system in the first or second Fc domain monomers have been deleted or substituted. 76. A multi-specific binding agent comprising: a) a first antigen-binding domain that binds to human 5T4, where the first antigen- binding domain comprises a complementarity determining region (CDR)1 comprising SEQ ID NO: 88, a CDR2 comprising SEQ ID NO: 89, and a CDR3 comprising SEQ ID NO: 90; and b) a second antigen-binding domain that binds to Vδ2, where the second antigen- binding domain comprises a CDR1 comprising SEQ ID NO: 18, a CDR2 comprising SEQ ID NO: 19, and a CDR3 comprising SEQ ID NO: 20. 77. The multi-specific binding agent of claim 76, wherein the first antigen-binding domain comprises SEQ ID NO: 93 and the second antigen-binding domain comprises SEQ ID NO: 22. 78. The multi-specific binding agent of any one of claims 76 or 77, wherein the first and second antigen-binding domains are comprised in the same polypeptide chain. 79. A multi-specific binding agent comprising a) a first polypeptide comprising i. a first antigen-binding domain that binds to a human 5T4 protein and comprises a complementarity determining region (CDR)1 comprising SEQ ID NO: 88, a CDR2 comprising SEQ ID NO: 89, and a CDR3 comprising SEQ ID NO: 90; and ii. a first Fc domain monomer b) a second polypeptide comprising i. a second antigen-binding domain that binds to a human Vδ2 T cell receptor and comprises a CDR1 comprising SEQ ID NO: 18, a CDR2 comprising SEQ ID NO: 19, and a CDR3 comprising SEQ ID NO: 20; and ii. a second Fc domain monomer. 80. The multi-specific binding agent of claim 79, wherein the first Fc domain monomer comprises SEQ ID NO: 358 and the second Fc domain monomer comprises SEQ ID NO: 359. 338699-2168 81. The multi-specific binding agent of claim 79, wherein the first Fc domain monomer comprises SEQ ID NO: 359 and the second Fc domain monomer comprises SEQ ID NO: 358. 82. The multi-specific binding agent of any one of claims 79-81, wherein the first antigen- binding domain comprises SEQ ID NO: 369. 83. The multi-specific binding agent of any one of claims 79-81, wherein the second antigen-binding domain comprises SEQ ID NO: 381. 84. The multi-specific binding agent of any one of claims 79-83, wherein a) the first polypeptide comprises SEQ ID NO: 369; and b) the second polypeptide comprises SEQ ID NO: 381. 85. A polynucleotide encoding the multi-specific binding agent of any one of claims 57- 84. 86. An expression vector comprising the polynucleotide of claim 85. 87. A host cell comprising the polynucleotide of claim 85 or the expression vector of claim 86. 88. The host cell of claim 87, wherein the cell is a CHO cell. 89. A method of producing the multi-specific binding agent of any one of claims 57-84, comprising culturing the host cell of claim 87 or 88 under conditions sufficient to express the multi-specific binding agent from the polynucleotide or expression vector therein and purifying the expressed multi-specific binding agent from the culture supernatant. 90. A pharmaceutical composition comprising the single domain antibody of any one of claim 1-50, the binding agent of claim 56, or the multi-specific binding agent according to any one of claims 57-84, and a pharmaceutically-acceptable excipient. 91. A method of treatment a cancer in a subject in need thereof, comprising administration to the subject the single domain antibody of any one of claim 1-50, the binding agent of claim 338699-2168 56, the multi-specific binding agent according to any one of claims 57-84, or the pharmaceutical composition of claim 90. 92. The method of claim 91, wherein the subject is human. 93. The method of claim 91 or 92, wherein the cancer expresses 5T4. 94. The method of any one of claims 91-93, wherein the subject is suffering from a cervical, colorectal, head and neck, glioblastoma, melanoma, urothelial, gastric, renal cell carcinoma, lung cancer, pancreatic cancer, mesothelioma, pre-B acute lymphoblastic leukemia, or hepatocellular cancer. 95. A method of expanding Vγ9Vδ2 T cells comprising contacting the Vγ9Vδ2 T cells with single domain antibody of any one of claims 1-50, the binding agent of claim 56, the multi- specific binding agent according to any one of claims 57-84, or the pharmaceutical composition of claim 90.
PCT/EP2024/069004 2023-07-07 2024-07-05 5t4 binding agents and methods of use Pending WO2025012118A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363525435P 2023-07-07 2023-07-07
US63/525,435 2023-07-07

Publications (2)

Publication Number Publication Date
WO2025012118A2 true WO2025012118A2 (en) 2025-01-16
WO2025012118A3 WO2025012118A3 (en) 2025-04-10

Family

ID=91898897

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2024/069004 Pending WO2025012118A2 (en) 2023-07-07 2024-07-05 5t4 binding agents and methods of use

Country Status (1)

Country Link
WO (1) WO2025012118A2 (en)

Citations (61)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2188638A (en) 1986-03-27 1987-10-07 Gregory Paul Winter Chimeric antibodies
US5222982A (en) 1991-02-11 1993-06-29 Ommaya Ayub K Spinal fluid driven artificial organ
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5385582A (en) 1991-02-11 1995-01-31 Ommaya; Ayub K. Spinal fluid driven artificial organ
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5569825A (en) 1990-08-29 1996-10-29 Genpharm International Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5585089A (en) 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
US5624821A (en) 1987-03-18 1997-04-29 Scotgen Biopharmaceuticals Incorporated Antibodies with altered effector functions
US5639641A (en) 1992-09-09 1997-06-17 Immunogen Inc. Resurfacing of rodent antibodies
US5714352A (en) 1996-03-20 1998-02-03 Xenotech Incorporated Directed switch-mediated DNA recombination
WO2001058957A2 (en) 2000-02-11 2001-08-16 Lexigen Pharmaceuticals Corp. Enhancing the circulating half-life of antibody-based fusion proteins
US6277375B1 (en) 1997-03-03 2001-08-21 Board Of Regents, The University Of Texas System Immunoglobulin-like domains with increased half-lives
WO2002006919A2 (en) 2000-07-18 2002-01-24 Aegis Analytical Corporation System, method and computer program product for mapping data of multi-database origins
US20020197266A1 (en) 2000-02-08 2002-12-26 Waldemar Debinski Immunotherapy using interleukin 13 receptor subunit alpha 2
WO2004016750A2 (en) 2002-08-14 2004-02-26 Macrogenics, Inc. FcϜRIIB-SPECIFIC ANTIBODIES AND METHODS OF USE THEREOF
WO2004029207A2 (en) 2002-09-27 2004-04-08 Xencor Inc. Optimized fc variants and methods for their generation
WO2004035752A2 (en) 2002-10-15 2004-04-29 Protein Design Labs, Inc. ALTERATION OF FcRn BINDING AFFINITIES OR SERUM HALF-LIVES OF ANTIBODIES BY MUTAGENESIS
US6737056B1 (en) 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
WO2004063351A2 (en) 2003-01-09 2004-07-29 Macrogenics, Inc. IDENTIFICATION AND ENGINEERING OF ANTIBODIES WITH VARIANT Fc REGIONS AND METHODS OF USING SAME
WO2004074455A2 (en) 2003-02-20 2004-09-02 Applied Molecular Evolution Fc REGION VARIANTS
WO2004099249A2 (en) 2003-05-02 2004-11-18 Xencor, Inc. Optimized fc variants and methods for their generation
WO2005040217A2 (en) 2003-10-17 2005-05-06 Cambridge University Technical Services Limited Antibodies having a mutated amino acid residue at position 268 (ch2 region) in constant regions
WO2005070963A1 (en) 2004-01-12 2005-08-04 Applied Molecular Evolution, Inc Fc region variants
WO2005092925A2 (en) 2004-03-24 2005-10-06 Xencor, Inc. Immunoglobulin variants outside the fc region
WO2006010834A1 (en) 2004-06-25 2006-02-02 Centre National De La Recherche Scientifique Non-integrative and non-replicative lentivirus, preparation and uses thereof
WO2006020114A2 (en) 2004-08-04 2006-02-23 Applied Molecular Evolution, Inc. Variant fc regions
WO2006064136A1 (en) 2004-12-16 2006-06-22 Centre National De La Recherche Scientifique (Cnrs) Production of antibody formats and immunological applications of said formats
WO2007098420A2 (en) 2006-02-17 2007-08-30 Syntonix Pharmaceuticals, Inc. Peptides that block the binding of igg to fcrn
US20070254842A1 (en) 2006-04-25 2007-11-01 The Regents Of The University Of California Administration of growth factors for the treatment of cns disorders
US20080081064A1 (en) 2006-09-28 2008-04-03 Surmodics, Inc. Implantable Medical Device with Apertures for Delivery of Bioactive Agents
WO2009058383A2 (en) 2007-10-31 2009-05-07 Domantis Limited Ligand
WO2009080254A1 (en) 2007-12-21 2009-07-02 F. Hoffmann-La Roche Ag Bivalent, bispecific antibodies
US20090196903A1 (en) 2008-01-29 2009-08-06 Kliman Gilbert H Drug delivery devices, kits and methods therefor
WO2009127691A1 (en) 2008-04-17 2009-10-22 Ablynx N.V. Peptides capable of binding to serum proteins and compounds, constructs and polypeptides comprising the same
WO2010059315A1 (en) 2008-11-18 2010-05-27 Merrimack Pharmaceuticals, Inc. Human serum albumin linkers and conjugates thereof
WO2010111625A1 (en) 2009-03-27 2010-09-30 Zymogenetics, Inc. Compositions and methods for using multispecific-binding proteins comprising an antibody-receptor combination
WO2010134666A1 (en) 2009-05-20 2010-11-25 주식회사 파멥신 Dual targeting antibody of novel form, and use thereof
WO2011051489A2 (en) 2009-10-30 2011-05-05 Novozymes Biopharma Dk A/S Albumin variants
WO2012023053A2 (en) 2010-08-16 2012-02-23 Novimmune S.A. Methods for the generation of multispecific and multivalent antibodies
WO2012059486A1 (en) 2010-11-01 2012-05-10 Novozymes Biopharma Dk A/S Albumin variants
WO2012150319A1 (en) 2011-05-05 2012-11-08 Novozymes Biopharma Dk A/S Albumin variants
WO2013041687A1 (en) 2011-09-23 2013-03-28 Amgen Research (Munich) Gmbh Bispecific binding molecules for 5t4 and cd3
WO2013075066A2 (en) 2011-11-18 2013-05-23 Eleven Biotherapeutics, Inc. Proteins with improved half-life and other properties
WO2013135896A1 (en) 2012-03-16 2013-09-19 Novozymes Biopharma Dk A/S Albumin variants
WO2013157953A1 (en) 2012-04-20 2013-10-24 Merus B.V. Methods and means for the production of ig-like molecules
WO2014072481A1 (en) 2012-11-08 2014-05-15 Novozymes Biopharma Dk A/S Albumin variants
WO2014081202A1 (en) 2012-11-21 2014-05-30 주식회사 파멥신 Dual-target antibody targeting vegfr-2 and dll4, and pharmaceutical composition comprising same
US8784799B2 (en) 2000-06-01 2014-07-22 The University Of North Carolina At Chapel Hill Duplexed parvovirus vectors
US8809058B2 (en) 1995-06-07 2014-08-19 The University Of North Carolina At Chapel Hill Helper virus-free AAV production
US8889641B2 (en) 2009-02-11 2014-11-18 The University Of North Carolina At Chapel Hill Modified virus vectors and methods of making and using the same
US9012224B2 (en) 2004-12-15 2015-04-21 The University Of North Carolina At Chapel Hill Chimeric vectors
WO2015156673A1 (en) 2014-04-10 2015-10-15 Stichting Vu-Vumc IMMUNOGLOBULINS BINDING HUMAN Vγ9Vδ2 T CELL RECEPTORS
US9169494B2 (en) 2010-01-12 2015-10-27 The University Of North Carolina At Chapel Hill Restrictive inverted terminal repeats for viral vectors
US9169492B2 (en) 2010-02-05 2015-10-27 The University Of North Carolina At Chapel Hill Compositions and methods for enhanced parvovirus transduction
WO2016097408A1 (en) 2014-12-19 2016-06-23 Biotecnol Limited Fusion protein comprising three binding domains to 5t4 and cd3
US20200319181A1 (en) 2003-05-30 2020-10-08 Merus N.V. Antibody producing non-human animals
WO2021032963A1 (en) 2019-08-16 2021-02-25 GammaDelta Therapeutics Limited Therapeutic uses of anti-tcr delta variable 1 antibodies
WO2021048423A1 (en) 2019-09-12 2021-03-18 Genmab A/S Bispecific antibodies binding to 5t4 and cd3 for use in treatment of cancer
WO2022008646A1 (en) 2020-07-08 2022-01-13 LAVA Therapeutics N.V. Antibodies that bind psma and gamma-delta t cell receptors
WO2022122973A1 (en) 2020-12-10 2022-06-16 LAVA Therapeutics N.V. Antibodies that bind gamma-delta t cell receptors
WO2023242319A1 (en) 2022-06-15 2023-12-21 LAVA Therapeutics N.V. Variant antibodies that bind gamma-delta t cell receptors

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TW202506736A (en) * 2018-10-11 2025-02-16 美商因荷布瑞克斯生物科學公司 5t4 single domain antibodies and therapeutic compositions thereof
EP4298125A1 (en) * 2021-02-26 2024-01-03 LAVA Therapeutics N.V. Antibodies that bind cd123 and gamma-delta t cell receptors
WO2024173376A2 (en) * 2023-02-13 2024-08-22 LAVA Therapeutics N.V. Combination therapy comprising multispecific gamma delta tcr antibodies

Patent Citations (64)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
EP0239400B1 (en) 1986-03-27 1994-08-03 Medical Research Council Recombinant antibodies and methods for their production
GB2188638A (en) 1986-03-27 1987-10-07 Gregory Paul Winter Chimeric antibodies
US5624821A (en) 1987-03-18 1997-04-29 Scotgen Biopharmaceuticals Incorporated Antibodies with altered effector functions
US5693761A (en) 1988-12-28 1997-12-02 Protein Design Labs, Inc. Polynucleotides encoding improved humanized immunoglobulins
US5585089A (en) 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5569825A (en) 1990-08-29 1996-10-29 Genpharm International Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5222982A (en) 1991-02-11 1993-06-29 Ommaya Ayub K Spinal fluid driven artificial organ
US5385582A (en) 1991-02-11 1995-01-31 Ommaya; Ayub K. Spinal fluid driven artificial organ
US5639641A (en) 1992-09-09 1997-06-17 Immunogen Inc. Resurfacing of rodent antibodies
US8809058B2 (en) 1995-06-07 2014-08-19 The University Of North Carolina At Chapel Hill Helper virus-free AAV production
US5714352A (en) 1996-03-20 1998-02-03 Xenotech Incorporated Directed switch-mediated DNA recombination
US6277375B1 (en) 1997-03-03 2001-08-21 Board Of Regents, The University Of Texas System Immunoglobulin-like domains with increased half-lives
US6737056B1 (en) 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
US20020197266A1 (en) 2000-02-08 2002-12-26 Waldemar Debinski Immunotherapy using interleukin 13 receptor subunit alpha 2
US7338929B2 (en) 2000-02-08 2008-03-04 The Penn State Research Foundation Cancer immunotherapy
WO2001058957A2 (en) 2000-02-11 2001-08-16 Lexigen Pharmaceuticals Corp. Enhancing the circulating half-life of antibody-based fusion proteins
US8784799B2 (en) 2000-06-01 2014-07-22 The University Of North Carolina At Chapel Hill Duplexed parvovirus vectors
WO2002006919A2 (en) 2000-07-18 2002-01-24 Aegis Analytical Corporation System, method and computer program product for mapping data of multi-database origins
WO2004016750A2 (en) 2002-08-14 2004-02-26 Macrogenics, Inc. FcϜRIIB-SPECIFIC ANTIBODIES AND METHODS OF USE THEREOF
WO2004029207A2 (en) 2002-09-27 2004-04-08 Xencor Inc. Optimized fc variants and methods for their generation
WO2004035752A2 (en) 2002-10-15 2004-04-29 Protein Design Labs, Inc. ALTERATION OF FcRn BINDING AFFINITIES OR SERUM HALF-LIVES OF ANTIBODIES BY MUTAGENESIS
WO2004063351A2 (en) 2003-01-09 2004-07-29 Macrogenics, Inc. IDENTIFICATION AND ENGINEERING OF ANTIBODIES WITH VARIANT Fc REGIONS AND METHODS OF USING SAME
WO2004074455A2 (en) 2003-02-20 2004-09-02 Applied Molecular Evolution Fc REGION VARIANTS
WO2004099249A2 (en) 2003-05-02 2004-11-18 Xencor, Inc. Optimized fc variants and methods for their generation
US20200319181A1 (en) 2003-05-30 2020-10-08 Merus N.V. Antibody producing non-human animals
WO2005040217A2 (en) 2003-10-17 2005-05-06 Cambridge University Technical Services Limited Antibodies having a mutated amino acid residue at position 268 (ch2 region) in constant regions
WO2005070963A1 (en) 2004-01-12 2005-08-04 Applied Molecular Evolution, Inc Fc region variants
WO2005092925A2 (en) 2004-03-24 2005-10-06 Xencor, Inc. Immunoglobulin variants outside the fc region
WO2006010834A1 (en) 2004-06-25 2006-02-02 Centre National De La Recherche Scientifique Non-integrative and non-replicative lentivirus, preparation and uses thereof
WO2006020114A2 (en) 2004-08-04 2006-02-23 Applied Molecular Evolution, Inc. Variant fc regions
US9012224B2 (en) 2004-12-15 2015-04-21 The University Of North Carolina At Chapel Hill Chimeric vectors
WO2006064136A1 (en) 2004-12-16 2006-06-22 Centre National De La Recherche Scientifique (Cnrs) Production of antibody formats and immunological applications of said formats
WO2007098420A2 (en) 2006-02-17 2007-08-30 Syntonix Pharmaceuticals, Inc. Peptides that block the binding of igg to fcrn
US20070254842A1 (en) 2006-04-25 2007-11-01 The Regents Of The University Of California Administration of growth factors for the treatment of cns disorders
US20080081064A1 (en) 2006-09-28 2008-04-03 Surmodics, Inc. Implantable Medical Device with Apertures for Delivery of Bioactive Agents
WO2009058383A2 (en) 2007-10-31 2009-05-07 Domantis Limited Ligand
WO2009080254A1 (en) 2007-12-21 2009-07-02 F. Hoffmann-La Roche Ag Bivalent, bispecific antibodies
US20090196903A1 (en) 2008-01-29 2009-08-06 Kliman Gilbert H Drug delivery devices, kits and methods therefor
WO2009127691A1 (en) 2008-04-17 2009-10-22 Ablynx N.V. Peptides capable of binding to serum proteins and compounds, constructs and polypeptides comprising the same
WO2010059315A1 (en) 2008-11-18 2010-05-27 Merrimack Pharmaceuticals, Inc. Human serum albumin linkers and conjugates thereof
US8889641B2 (en) 2009-02-11 2014-11-18 The University Of North Carolina At Chapel Hill Modified virus vectors and methods of making and using the same
WO2010111625A1 (en) 2009-03-27 2010-09-30 Zymogenetics, Inc. Compositions and methods for using multispecific-binding proteins comprising an antibody-receptor combination
WO2010134666A1 (en) 2009-05-20 2010-11-25 주식회사 파멥신 Dual targeting antibody of novel form, and use thereof
WO2011051489A2 (en) 2009-10-30 2011-05-05 Novozymes Biopharma Dk A/S Albumin variants
US9169494B2 (en) 2010-01-12 2015-10-27 The University Of North Carolina At Chapel Hill Restrictive inverted terminal repeats for viral vectors
US9169492B2 (en) 2010-02-05 2015-10-27 The University Of North Carolina At Chapel Hill Compositions and methods for enhanced parvovirus transduction
WO2012023053A2 (en) 2010-08-16 2012-02-23 Novimmune S.A. Methods for the generation of multispecific and multivalent antibodies
WO2012059486A1 (en) 2010-11-01 2012-05-10 Novozymes Biopharma Dk A/S Albumin variants
WO2012150319A1 (en) 2011-05-05 2012-11-08 Novozymes Biopharma Dk A/S Albumin variants
WO2013041687A1 (en) 2011-09-23 2013-03-28 Amgen Research (Munich) Gmbh Bispecific binding molecules for 5t4 and cd3
WO2013075066A2 (en) 2011-11-18 2013-05-23 Eleven Biotherapeutics, Inc. Proteins with improved half-life and other properties
WO2013135896A1 (en) 2012-03-16 2013-09-19 Novozymes Biopharma Dk A/S Albumin variants
WO2013157953A1 (en) 2012-04-20 2013-10-24 Merus B.V. Methods and means for the production of ig-like molecules
WO2014072481A1 (en) 2012-11-08 2014-05-15 Novozymes Biopharma Dk A/S Albumin variants
WO2014081202A1 (en) 2012-11-21 2014-05-30 주식회사 파멥신 Dual-target antibody targeting vegfr-2 and dll4, and pharmaceutical composition comprising same
WO2015156673A1 (en) 2014-04-10 2015-10-15 Stichting Vu-Vumc IMMUNOGLOBULINS BINDING HUMAN Vγ9Vδ2 T CELL RECEPTORS
WO2016097408A1 (en) 2014-12-19 2016-06-23 Biotecnol Limited Fusion protein comprising three binding domains to 5t4 and cd3
WO2021032963A1 (en) 2019-08-16 2021-02-25 GammaDelta Therapeutics Limited Therapeutic uses of anti-tcr delta variable 1 antibodies
WO2021048423A1 (en) 2019-09-12 2021-03-18 Genmab A/S Bispecific antibodies binding to 5t4 and cd3 for use in treatment of cancer
WO2022008646A1 (en) 2020-07-08 2022-01-13 LAVA Therapeutics N.V. Antibodies that bind psma and gamma-delta t cell receptors
WO2022122973A1 (en) 2020-12-10 2022-06-16 LAVA Therapeutics N.V. Antibodies that bind gamma-delta t cell receptors
WO2023242319A1 (en) 2022-06-15 2023-12-21 LAVA Therapeutics N.V. Variant antibodies that bind gamma-delta t cell receptors

Non-Patent Citations (56)

* Cited by examiner, † Cited by third party
Title
"Antibodies: A Laboratory Manual", 1988, CSH PRESS
"Current Protocols in Immunology", 1992, GREENE PUBLISHING ASSOC, AND WILEY INTERSCIENCE
"Immumobiology", 2001, GARLAND PUBLISHING
"Remington's Pharmaceutical Sciences", 1985, MACE PUBLISHING COMPANY
ACADEMIC INSTITUTION, PEARCE ET AL., BIOCHEM MOL BIOL INT., vol. 42, no. 6, September 1997 (1997-09-01), pages 1179
ACADEMIC, LAWRENCE, FEBS LETT., vol. 425, no. 3, 3 April 1998 (1998-04-03), pages 479 - 84
AL-LAZIKANI ET AL., JMB, vol. 273, 1997, pages 927 - 948
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
ALTSCHUL ET AL., METHODS IN ENZYMOLOGY, vol. 266, 1996, pages 460 - 480
ALTSCHUL ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402
AMATO ET AL., CLIN CANCER RES, vol. 16, no. 22, 2010, pages 5539
BITTER ET AL., METHODS IN ENZYMOLOGY, vol. 153, 1987, pages 516 - 544
BLANKENSHIP JW ET AL., AACR 100TH ANNUAL MEETING 2009
CAPECCHI, CELL, vol. 22, 1980, pages 479 - 488
CHU ET AL., GENE, vol. 13, 1981, pages 97
CURR OPIN MOL THER, vol. 9, 2007, pages 327
DEVEREUX ET AL., NUCL. ACID RES., vol. 12, 1984, pages 387 - 395
DIMASI ET AL., J MOL BIOL., vol. 393, no. 3, 30 October 2009 (2009-10-30), pages 672 - 92
DONDELINGER ET AL.: "Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface/Residue Definition", FRONT IMMUNOL., vol. 9, 16 October 2018 (2018-10-16), pages 2278
DOPPALAPUDI, V.R. ET AL., BIOORG. MED. CHEM. LETT., vol. 17, 2007, pages 501 - 506
E. MEYERSW. MILLER, COMPUT. APPL. BIOSCI, vol. 4, 1988, pages 11 - 17
ELI LILLY, LEWIS ET AL., NAT BIOTECHNOL., vol. 32, no. 2, February 2014 (2014-02-01), pages 191 - 8
ELLERMAN, METHODS, vol. 154, 2019, pages 102
ENGELBERTS ET AL., EBIOMEDICINE, vol. 52, 2020, pages 102625
FEIGNER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 84, 1987, pages 7413 - 7417
GENENTECH, BOSTROM ET AL., SCIENCE, vol. 323, 2009, pages 1610 - 1614
GENENTECH, WRANIK ET AL., J. BIOL. CHEM., vol. 287, no. 52, 2012, pages 43331 - 9
GRAHAM ET AL., VIROLOGY, vol. 52, 1973, pages 456 - 467
HAMERS-CASTERMAN ET AL., NATURE, vol. 363, 1993, pages 446
HASKARDARCHER, J. IMMUNOL. METHODS, vol. 74, no. 2, 1984, pages 361 - 67
HMILA ET AL., FASEB J., 2010
HUANG ET AL., MOL. CELL. BIOL., vol. 5, pages 3864
HUEHLS ET AL., IMMUNOL CELL BIOL, vol. 93, 2015, pages 290
HUSE ET AL., SCIENCE, vol. 246, 1989, pages 1275 - 81
INT J MOL SCI., vol. 23, no. 13, July 2022 (2022-07-01), pages 7311
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, NATIONAL TECHNICAL INFORMATION SERVICE
KARLIN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 5873 - 5787
KLEIN ET AL., NATURE, vol. 327, 1987, pages 70 - 73
KOHLERMILSTEIN, EUR. J. IMMUNOL., vol. 5, 1976, pages 511 - 519
KRAH ET AL., IMMUNOPHARMACOL IMMUNOTOXICOL, vol. 38, 2016, pages 21
LANGER, SCIENCE, vol. 249, 1990, pages 1527 - 1533
LEFRANC ET AL.: "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains", DEV COMP IMMUNOL., vol. 27, no. 1, January 2003 (2003-01-01), pages 55 - 77, XP055585227, DOI: 10.1016/S0145-305X(02)00039-3
LIU ET AL., GENES DEV., vol. 9, 1995, pages 1766
MULLER, METH. ENZYMOL., vol. 92, 1983, pages 589 - 601
NEEDLEMANWUNSCH, J MOL. BIOL., vol. 48, 1970, pages 443
NEOLEUKIN, SILVA ET AL.: "De novo design of potent and selective mimics of IL-2 and IL-15", NATURE, vol. 565, 2019, pages 186 - 191
PEARSONLIPMAN, PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 2444
PEDERSEN ET AL., J. MOL. BIOL., vol. 235, 1994, pages 959 - 973
RIDGWAY ET AL.: "Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization", PROTEIN ENG, vol. 9, 1996, pages 617, XP002610995, DOI: 10.1093/protein/9.7.617
RODER ET AL., METHODS ENZYMOL., vol. 121, 1986, pages 140 - 67
SCHULZ, G. E.SCHIRMER, R. H.: "Principles of Protein Structure", 1979, SPRINGER-VERLAG
SHIGEKAWA ET AL., BIOTECHNIQUES, vol. 6, 1988, pages 682 - 690
SMITHWATERMAN, ADV. APPL. MATH., vol. 2, 1981, pages 482
ZHU ET AL., IMMUNOL CELL BIOL., vol. 88, no. 6, August 2010 (2010-08-01), pages 667 - 75
ZUFFEREY ET AL., J. VIROL., vol. 73, 1999, pages 2886
ZYNGENIA, LAFLEUR ET AL., MABS., vol. 5, no. 2, pages 208 - 18

Also Published As

Publication number Publication date
WO2025012118A3 (en) 2025-04-10

Similar Documents

Publication Publication Date Title
US20250197487A1 (en) Anti-pd-1/vegfa bifunctional antibody, pharmaceutical composition thereof and use thereof
JP7115758B2 (en) Tandem diabodies for CD16A-directed NK cell engagement
CN104203981A (en) Bispecific antibody molecule
JP7676652B2 (en) B7-H3 antibodies and uses thereof
US20230322952A1 (en) Multispecific antibodies that bind both mait and tumor cells
US20250206823A1 (en) Antigen targeting, anti-cd16a, and immune effector cell activating trifunctional fusion protein, and application thereof
JP7395465B2 (en) Chimeric antigen receptor and CAR-T cells that bind to CXCR5
WO2020259536A1 (en) Monoclonal antibody-cytokine fusion protein dimer and application thereof
JP7495409B2 (en) Improved anti-FLT3 antigen binding proteins
CA3233075A1 (en) Interleukin-2 mutant and fusion protein thereof
EP4400518A1 (en) Antigen-targeting, anti-cd16a and immune effector cell-activating trifunctional fusion protein, and use thereof
CN115894701A (en) Antibodies that bind CD3and uses thereof
WO2023208014A1 (en) Antibodies binding bcma and cd3 and uses thereof
CN118562015A (en) Bispecific antibody aiming at CD20/CD3 as well as preparation method and application thereof
WO2025012118A2 (en) 5t4 binding agents and methods of use
WO2023217062A1 (en) Chimeric antigen receptor and use thereof
WO2024200573A1 (en) Nectin-4 binding agents and methods of use
RU2830982C2 (en) Improved anti-flt3 antigen-binding proteins
TW202413430A (en) Anti-CD37/anti-CD3 bispecific antibody
WO2025061032A1 (en) Bispecific antibody targeting human ror1 and cd3
WO2024199468A1 (en) Bispecific antibody, pharmaceutical composition, and use
JP2025539875A (en) Fusion Polypeptides and Uses Thereof
WO2025256579A1 (en) Targeted il12 fusion protein, and preparation and treatment method therefor
WO2023066322A1 (en) Fusion polypeptide and use thereof
TW202305011A (en) Specific binding protein targeting pd-1 and ox40

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24740844

Country of ref document: EP

Kind code of ref document: A2