WO2023081644A1 - Matériels et méthodes de traitement avec un arnm codant des molécules de liaison multispécifiques - Google Patents

Matériels et méthodes de traitement avec un arnm codant des molécules de liaison multispécifiques Download PDF

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WO2023081644A1
WO2023081644A1 PCT/US2022/079055 US2022079055W WO2023081644A1 WO 2023081644 A1 WO2023081644 A1 WO 2023081644A1 US 2022079055 W US2022079055 W US 2022079055W WO 2023081644 A1 WO2023081644 A1 WO 2023081644A1
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binding
seq
binding domain
amino acid
antigen
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William Gillis OLSEN
Kathryn Strobel KWANT
Ulrike Gisela PFLUECKHAHN
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Engage Biologics Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • 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
    • C07K16/2851—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 lectin superfamily, e.g. CD23, CD72
    • 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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • 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
    • 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
    • C07K16/303—Liver or Pancreas
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C07K2317/50—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
    • 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
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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
    • C07K2319/00—Fusion polypeptide

Definitions

  • Antibodies are known to have powerful therapeutic effects and are currently used for the treatment of a range of diseases including cancer and autoimmune diseases.
  • therapeutic antibodies including multispecific immune cell engaging therapies, are produced by recombinant technology, purified, formulated and then administered to patients in need of antibody therapy.
  • antibody production, purification and formulation are highly complex and expensive.
  • antibody therapy often requires high does and frequent administration.
  • High doses of systemically administered antibodies, particularly multispecific immune cell engaging antibodies often lead to toxicity.
  • Systemically administered antibodies also suffer from poor tumor-penetrance, limiting anti-tumor efficacy.
  • therapies employing antibodies are also reported to be inhibited by shed antigen in circulation. Therefore, novel approaches are needed to improve antibody -based therapies including multispecific immune cell engaging therapies. 3.
  • Autocrine-like secretion and binding refers to the secretion by a target cell of a multispecific immune cell engager with a binding domain capable of binding to an antigen present on the surface of the target cell.
  • mRNA-induced autocrine-like secretion and binding of immune engagers eliminates the substantial barriers to tumor penetration which traditional antibody therapies suffer from.
  • Traditional antibody-based therapeutics when delivered as proteins (e.g., intravenously or subcutaneously), often have poor tumor penetrance which can limit efficacy in solid tumors.
  • Antibodies are excluded from tumors in part because of high hydrostatic pressure in tumors which reduces convection (see Thurber et al., 2008, Adv Drug Deliv Rev, 60(12)1421- 1434).
  • Antibodies may also be excluded from the center of tumors due to rapid antigen binding and internalization at the outer surface of the tumor (i.e., the binding site barrier effect; see Singh et al., 2020, AAPS J, 22(2), 28). These effects prevent entry of traditional antibodies into deeper portions of tumors and can hinder efficacy.
  • mRNA may be delivered to target cells by numerous means which are not hindered by the same physical barriers as traditional antibodies, including a delivery vehicle (e.g., lipid nanoparticle), tissue-targeted delivery vehicle, target-antigen targeted delivery vehicle, and/or direct (e.g., intratumoral) injection.
  • a delivery vehicle e.g., lipid nanoparticle
  • tissue-targeted delivery vehicle e.g., lipid nanoparticle
  • target-antigen targeted delivery vehicle e.g., intratumoral
  • a tumor therapeutic for delivery to a target cell comprising mRNA encoding a multispecific binding molecule, wherein the multispecific binding molecule comprises (1) a first binding domain capable of binding to a first antigen present on an immune cell, and (2) a second binding domain capable of binding to a second antigen present on the target cell, wherein the multispecific binding molecule is expressed and secreted by the target target cell, wherein the multispecific binding molecule binds to the second antigen on the target cell in an autocrine-like fashion, and wherein the multispecific binding molecule binds to the first antigen on an immune cell, inducing the immune cell to kill the target cell.
  • a method of treating a disease or disorder in a subject comprising administering to a target tissue of said subject one or more mRNA(s) encoding a multispecific binding molecule, wherein the multispecific binding molecule comprises (1) a first binding domain capable of binding to a first antigen present on an immune cell, and (2) a second binding domain capable of binding to a second antigen present on a target cell in said target tissue.
  • the disease or disorder is cancer.
  • the cancer is liver cancer.
  • the cancer is lung cancer.
  • the cancer is skin cancer.
  • a composition for use in treatment of cancer comprising one or more mRNA(s) encoding multispecific binding molecule(s) with binding affinity for one or more target cells and one or more immune cells, to achieve the selective, immune-cell-mediated elimination of target cells.
  • the target cells are tumor cells.
  • the cancer is liver cancer.
  • the cancer is lung cancer.
  • the cancer is skin cancer.
  • the immune cells are peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • the immune cells are T cells.
  • the T cells are Pan T cells.
  • the T cells are CD8+ T cells.
  • the immune cells are NK cells.
  • the immune cells are macrophages.
  • the immune cells are tissue-resident macrophages, such as a Kupffer cells or a peritoneal, lung, splenic, or bone marrow resident macrophages.
  • the multispecific binding molecule is expressed at a higher level in the target tissue compared to a non-target tissue of said subject.
  • the target cell is transfected with the mRNA(s) encoding the multispecific binding molecule, the target cell expresses and secretes the multispecific binding molecule, and the multispecific binding molecule binds to the target cell and the immune cell inducing the target cell elimination.
  • the first binding domain comprises a VH and a VL region, and/or a single domain antigen binding fragment.
  • the second binding domain comprises a VH and a VL region, and/or a single domain antigen binding fragment.
  • the multispecific binding molecule comprises one polypeptide; wherein the polypeptide comprises the first binding domain and the second binding domain.
  • the polypeptide is encoded by a single mRNA.
  • the polypeptide further comprises a secretion signal.
  • the secretion signal is N-terminal secretion signal and directs the target cell to secrete the polypeptide.
  • the polypeptide further comprises a C-terminal His tag.
  • the first binding domain and the second binding domain are connected via a linker.
  • the first antigen is CD3.
  • the first binding domain comprises: (i) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 6, and a VL comprising the amino acid sequence of SEQ ID NO: 7, or (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and a VL comprising the amino acid sequence of SEQ ID NO: 16, or (iii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 21, and a VL comprising the amino acid sequence of SEQ ID NO: 22, (iv) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:35, and a VL comprising the amino acid sequence of SEQ ID NO:36, or (v) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:48, and a VL comprising the amino acid sequence of SEQ ID NO:
  • the first antigen is CD 16.
  • the first binding domain is a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 39.
  • the second antigen is GPC3.
  • the second binding domain is a single domain antigen binding fragment comprising (i) the amino acid sequence of SEQ ID NO: 10 or (ii) the amino acid sequence of SEQ ID NO: 30.
  • the second binding domain comprises: (i) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 11, and a VL comprising the amino acid sequence of SEQ ID NO: 12 or (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 27, and a VL comprising the amino acid sequence of SEQ ID NO: 28.
  • the second antigen is EpCAM.
  • the second binding domain is a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 41.
  • the second binding domain comprises: (i) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 4, and a VL comprising the amino acid sequence of SEQ ID NO: 2, or (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 14, and a VL comprising the amino acid sequence of SEQ ID NO: 13.
  • the second antigen is uPAR.
  • the second binding domain comprises an amino terminal fragment (ATF) of uPA comprising the amino acid sequence of SEQ ID NO: 17.
  • the second antigen is an NKG2D ligand.
  • the NKG2D ligand is selected from MICA, MICB, RAET1E, RAET1G, RAET1L/ULBP6, ULBP1, ULBP2, ULBP3 or ULBP4.
  • the second binding domain comprises an extracellular domain of NKG2D comprising the amino acid sequence of SEQ ID NO: 24.
  • the second antigen is EGFR.
  • the second binding domain is a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 50.
  • the multispecific binding molecule comprises one polypeptide; wherein the polypeptide comprises (1) the first binding domain binding to the first antigen present on an immune cell, and (2) the second binding domain binding to the second antigen present on a target cell; wherein the first binding domain is a scFv comprising a VH and a VL region, and the second binding domain is a scFv comprising a VH and a VL region.
  • the first antigen is CD3.
  • the multispecific binding molecule comprises one polypeptide; wherein the polypeptide comprises (1) the first binding domain binding to the first antigen present on an immune cell, and (2) the second binding domain binding to the second antigen present on a target cell; wherein the first binding domain is a scFv comprising a VH and a VL region, and the second binding domain is a single domain antigen binding fragment.
  • the first antigen is CD3.
  • the multispecific binding molecule further comprises a third binding domain capable of binding to a third antigen present on a target cell in said target tissue.
  • the second binding domain and the third binding domain bind to a same antigen.
  • the second binding domain and the third binding domain bind to two different antigens.
  • the third binding domain comprises a VH and a VL region, and/or a single domain antigen binding fragment.
  • the multispecific binding molecule comprises two polypeptides; wherein the first polypeptide comprises the VH region of the first binding domain and the second binding domain, and wherein the second polypeptide comprises the VL region of the first binding domain and the third binding domain.
  • the first polypeptide is encoded by a first mRNA
  • the second polypeptide is encoded by a second mRNA.
  • the first mRNA and the second mRNA are administered together.
  • the first polypeptide and the second polypeptide are encoded by a single mRNA.
  • the first antigen is CD3.
  • the first antigen is CD 16.
  • the second antigen is GPC3.
  • the second antigen is EpCAM.
  • the second antigen is uPAR.
  • the second antigen is NKG2DL.
  • the second antigen is EGFR.
  • the third antigen is GPC3.
  • the third antigen is EpCAM.
  • the third antigen is uPAR.
  • the third antigen is NKG2DL.
  • the third antigen is EGFR.
  • the multispecific binding molecule(s) bind said immune cell and said target cell inducing said target cell elimination.
  • the multispecific binding molecule is expressed at a higher level in the target tissue compared to a non-target tissue of said subject.
  • the target cell is transfected with the mRNA(s) encoding the multispecific binding molecule, the target cell expresses and secretes the multispecific binding molecule, and the multispecific binding molecule binds to the target cell.
  • a non-target cell is transfected with the mRNA(s) encoding the multispecific binding molecule, the non-target cell expresses and secretes the multispecific binding molecule, and the multispecific binding molecule binds to the target cell.
  • the method comprises (1) delivery of the mRNA to the target cell, and (2) the multispecific binding molecule is expressed and secreted by the target cell, and (3) the multispecific binding molecule binds to the second antigen on the target cell in an autocrine-like fashion, and (4) the multispecific binding molecule binds to the first antigen on an immune cell, inducing the immune cell to kill the target cell.
  • the mRNA is administered to the subject via a delivery vehicle.
  • the delivery vehicle is a lipid vehicle.
  • the delivery vehicle is a lipid nanoparticle.
  • the lipid nanoparticle comprises a cationic or ionizable lipid.
  • the lipid nanoparticle comprises an ionizable lipid and one or more non-cationic lipids.
  • said non-cationic lipids comprise a phospholipid and cholesterol.
  • said delivery vehicle comprises a polyethylene glycol conjugate such as DMG-PEG.
  • said delivery vehicle preferentially transfects hepatocytes.
  • delivery of mRNAs to target cells and tissues is accomplished by passive targeting means without relying upon the use of additional excipients or means to enhance recognition of the transfer vehicle by target cells.
  • delivery of mRNAs to target cells and tissues is accomplished active targeting means which involves the use of additional excipients, referred to herein as “targeting ligands” that may be bound (either covalently or non-covalently) to the transfer vehicle to encourage localization of such transfer vehicle at certain target cells or target tissues.
  • the targeting ligand is cholesterol.
  • cholesterol increases LDL-receptor mediated uptake of the mRNA by liver cancer cells, relative to non-cancer cells outside the liver.
  • the mRNAs and/or compositions comprising the mRNAs are administered via intravenous injection. In some embodiments, the mRNAs and/or compositions comprising the mRNAs are administered via direct injection. In some embodiments, the mRNAs and/or compositions comprising the mRNAs are administered via intratumoral injection.
  • the target tissue is liver. In some embodiments, the target tissue is lung. In some embodiments, the target tissue is spleen. In some embodiments, the target tissue is a solid tumor.
  • the target cell is a cancer cell. In some embodiments, the target cell is a senescent cell. In some embodiments, the target cell is an activated fibroblast. In some embodiments, the target cell is a tumor-associated fibroblast. In some embodiments, the target cell is a tumor-associated macrophage. In some embodiments, the target cell is a pro-tumorigenic cell. In some embodiments, the target cell is a tumor-microenvironment cell. In some embodiments, the target cell is a pro-inflammatory cell.
  • the method provided herein exhibits reduced inhibition from binding to shed antigen compared to traditional recombinant multispecific antibody therapy. [0042] In some embodiments, the method provided herein reduces on-target off-tumor toxicity compared to traditional recombinant multispecific antibody therapy.
  • the method provided herein achieves increased target cell killing compared with equivalent amounts of traditional recombinant multispecific antibody therapy.
  • the method provided herein reduces systemic antibody exposure compared to traditional recombinant multispecific antibody therapy.
  • the method achieves high local cell surface concentrations of the multispecific binding molecule encoded by the mRNA compared to traditional recombinant multispecific antibody therapy. [0046] In some embodiments, the method reduces or eliminates barriers to tumor penetration compared to traditional recombinant multispecific antibody therapy.
  • FIG. 1 Illustration of target cell transfection and “autocrine-like” multispecific antibody binding.
  • mRNA encoding a bispecific T cell engager is encapsulated in a lipid nanoparticle (LNP) (1.1).
  • LNP lipid nanoparticle
  • This LNP is administered to an animal and transfects a target tissue (e.g., liver) containing a target cell (e.g., hepatocyte) (1.2).
  • the mRNA is translated into the bispecific antibody (1.3).
  • the bispecific antibody is secreted by, and binds to the surface of, the target cell (1.4).
  • the CD3 binding domain binds a cytotoxic T cell, enabling T-cell mediated cellular cytotoxicity and the selective elimination of the target cell (1.5).
  • FIG. 2. Schematic of traditional bi-scFv and “split” CD3 binding domain constructs
  • FIG. 3. Example of “split” immune cell engaging domain. Two bispecific antibodies, each of which includes either the VH or VL of a CD3 scFv. A cytotoxic T cell (3.1) with CD3 domain (3.2) are shown. Further, a target cell (3.3) with target antigen domains (3.4 and 3.5) are shown. The VH fragment of an anti-CD3 binding domain (3.6) is linked to the VH (3.7) and VL (3.8) fragment of a scFv with affinity for a target antigen (3.4).
  • VL fragment of an anti-CD3 binding domain (3.9) is linked to the VH (3.10) and VL (3.11) fragments of a scFv with affinity for a target antigen (3.5).
  • VH (3.6) and VL (3.9) fragments of the anti- CD3 binding domain are in close proximity, they form a complete CD3 binding domain and are able to bind to CD3 (3.2), thus affecting T-cell mediated cytotoxic target cell elimination.
  • Target antigens (3.4 and 3.5) may represent the same antigen or different antigens.
  • Target antigens (3.4 and 3.5) may further represent two epitopes on the same antigen.
  • Target antigen binding domains may be scFvs (as shown by, e.g., 3.7 and 3.8 or 3.10 and 3.11, respectively), or they may be single domain antibodies (sdAb) such as VHH or VNar, or any other suitable binding agent.
  • sdAb single domain antibodies
  • Certain target antigens may be shed from the cell surface (11.1) and bind to antibody -based therapies such as CAR-T (11.2) or bispecific antibodies (11.3); said binding of shed antigen may inhibit antibody -based therapies from binding to the target antigen on target cell surfaces (11.4), and thus inhibit the desired therapeutic activity.
  • antibody -based therapies such as CAR-T (11.2) or bispecific antibodies (11.3); said binding of shed antigen may inhibit antibody -based therapies from binding to the target antigen on target cell surfaces (11.4), and thus inhibit the desired therapeutic activity.
  • mRNA encoding multispecific antibodies are delivered to the target cell (11.5), expressed (11.6), and secreted to the cell surface (11.7). Said mRNA-encoded multispecific antibodies are shown herein to exhibit resistance to shed antigen inhibition and robust levels of T cell dependent cytotoxicity (11.8).
  • FIG. 12 Diagram of workflow for comparison of TDCC assay by recombinant bispecific antibody in solution vs target cell secretion via mRNA delivery.
  • FIG. 13 TDCC assay of recombinant bispecific GPC3 targeting antibody delivered in solution compared with target cell secretion via mRNA delivery. Experimental conditions, including construct number and binding domains, are displayed on the X axis. Percent viability (as measured by luciferase signal intensity and normalized to average signal intensity in GFP treated control) is shown on the Y axis.
  • FIG. 14 TDCC assay of recombinant bispecific EpCAM targeting antibody delivered in solution compared with target cell secretion via mRNA delivery. Experimental conditions, including construct number and binding domains, are displayed on the X axis. Percent viability (as measured by luciferase signal intensity and normalized to average signal intensity in GFP treated control) is shown on the Y axis.
  • FIG. 15 TDCC assay of recombinant bispecific GPC3 targeting constructs with split CD3 binding domains delivered in solution compared with target cell secretion via mRNA delivery. Experimental conditions, including construct number and binding domains, are displayed on the X axis. Percent viability (as measured by luciferase signal intensity and normalized to average signal intensity in GFP treated control) is shown on the Y axis.
  • FIG. 16 TDCC assay of recombinant bispecific EpCAM targeting constructs with split CD3 binding domains delivered in solution compared with target cell secretion via mRNA delivery. Experimental conditions, including construct number and binding domains, are displayed on the X axis. Percent viability (as measured by luciferase signal intensity and normalized to average signal intensity in GFP treated control) is shown on the Y axis.
  • FIG. 17 Diagram of Cell Surface Concentration Gradient Hypothesis.
  • recombinant antibody is introduced into the system (17.1), diffusion drives the concentration of the antibody throughout the system (17.2).
  • This model may represent an in vitro system (e.g., a well plate) or in vivo system (e.g., a human). For a given antibody concentration throughout the system, a certain level of bound (17.3) and unbound or off-target binding antibody (17.4) will be achieved.
  • mRNA encoding bispecific antibodies is delivered to a target cell (17.5)
  • the mRNA is translated (17.6) and secreted (17.7) continuously onto the cell surface. This may create a high cell-surface concentration (17.8) relative to the cell-surface concentration produced with an equivalent amount of recombinant antibody.
  • FIG. 20 shows cancer cell killing by PBMCs as a function of dose of mRNA-encoded immune cell engager.
  • FIG. 21 shows cancer cell killing by T cells as a function of dose of mRNA-encoded immune cell engager.
  • Immune cell engagers utilized a range of signal sequences and anti-CD3 domains.
  • FIG. 22 shows expression levels as measured by HiBit assay following HepG2 transfection with mRNA encoding immune cell engagers containing different domain orientations and secretion signals.
  • FIG. 23 shows expression levels as measured by HiBit assay following A431 transfection with mRNA encoding immune cell engagers containing different domain orientations and secretion signals.
  • FIG. 24 shows cancer cell killing by T cells as a function of dose of mRNA-encoded immune cell engager or dose of supernatant containing protein immune cell engager.
  • FIG. 25 shows the protein concentration of immune cell engager Cl 03 produced in the supernatant of A431 cells 48 hours after transfection with different amounts of mRNA.
  • FIGs. 26A-E shows cancer cell killing by mRNA-encoded immune cell engagers using different subsets of immune effector cells from the same donor (Donor 11).
  • FIGs. 27A-B show A431 cancer cell killing by mRNA-encoded immune cell engagers where the CD3 targeting domain is a traditional scFv (FIG. 27A) or where the CD3 targeting domain is split into two separate molecules, each containing an anti-tumor antigen binder and either the variable heavy or the variable light chain of anti-CD3 (FIG. 27B).
  • FIGs. 28A-B show HepG2 cancer cell killing by mRNA-encoded immune cell engagers where the CD3 targeting domain is a traditional scFv (FIG. 28A) or where the CD3 targeting domain is split into two separate molecules, each containing an anti-tumor antigen binder and either the variable heavy or the variable light chain of anti-CD3 (FIG. 28B).
  • FIG. 29 shows cancer cell killing by mRNA-encoded immune cell engagers with different CD3 binders, tumor associated antigen binders, domain orientations, and linkers.
  • FIG. 30 shows luciferase activity in mice treated with LNP encapsulated mRNA encoding luciferase (dose in mg mRNA/kg b.w., from left to right: 0.6, 0.2, 0.07, 0)
  • FIG. 31 shows luciferase activity in the organs of mice treated with LNP encapsulated mRNA encoding luciferase.
  • FIG. 32 shows the quantification of luciferase activity (luminescence) in the livers of mice treated with LNP encapsulated mRNA encoding luciferase as a function of mRNA dose.
  • FIG. 33 shows bioluminescent imaging of mice orthotopically engrafted with luciferase labeled liver cancer cells and treated with either control (GFP) mRNA or C080 mRNA encoding an exemplary immune cell engager.
  • GFP control
  • FIG. 34 shows the tumor size over time of mice engrafted with luciferase labeled liver cancer cells and treated with either control (GFP) mRNA or C073 (anti-CD3, anti-GPC3 immune cell engager) mRNA encoding an exemplary immune cell engager.
  • GFP control
  • C073 anti-CD3, anti-GPC3 immune cell engager
  • FIG. 35 shows bioluminescent imaging of mice engrafted with luciferase labeled liver cancer cells and treated with either control (GFP) mRNA or C023 A mRNA encoding an exemplary immune cell engager.
  • FIG. 36 shows the tumor size over time of mice engrafted with luciferase labeled liver cancer cells and treated with either control (GFP) mRNA or C023 A mRNA encoding an exemplary immune cell engager.
  • FIG. 37 In vitro model system of tumor and peripheral compartments to compare protein immune cell engagers and mRNA encoded immune cell engagers delivered to target tissue.
  • FIGs. 38A-B show cell killing compared to concentration of immune cell engaging protein in the “periphery” (FIG. 38A) or total mass of immune cell engaging protein in the system (FIG. 38B). The data show tumor cell killing at much lower systemic concentrations (FIG. 38A) and total protein mass (FIG. 38B) for mRNA-induced local protein production compared systemically administered protein.
  • FIG. 39 shows bioluminescent imaging of a mouse engrafted subcutaneously with liver cancer cells which were intratumorally injected with lipid nanoparticles encapsulating luciferase mRNA.
  • the data demonstrate significant protein production following intratumoral injection of mRNA.
  • FIG. 40 shows bioluminescent imaging of mice engrafted subcutaneously with luciferase labeled liver cancer cells and treated with either control (GFP) mRNA or C080 mRNA encoding an exemplary immune cell engager.
  • the data show tumor shrinkage in 2 of 3 treated mice compared with 0 of 3 control mice.
  • the present disclosure relates to materials and methods of treatment of a subject in need thereof with one or more mRNA(s) encoding a multispecific binding molecule with binding affinity for one or more target cells and one or more immune effector cells, to achieve the selective, immune-cell-mediated elimination of target cells.
  • the terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, H. G. W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).
  • antibody immunoglobulin
  • immunoglobulin is used interchangeably herein, and is used in the broadest sense and specifically covers, for example, monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, and multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity), formed from at least two intact antibodies, recombinantly produced antibodies, single domain (e.g., VHH) antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), synthetic antibodies, chimeric antibodies, humanized antibodies, or human versions of antibodies having full-length heavy and/or light chains.
  • monoclonal antibodies including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies
  • multispecific antibodies e.g., bispecific antibodies so long as they exhibit the desired biological activity
  • single domain e.g., VHH
  • monospecific antibodies e.g
  • VHH as used herein refers to a domain antibody derived from a variable region of a heavy chain only antibody.
  • Exemplary single domain antibodies include, but are not limited to, antibodies naturally devoid of light chains such as those from Camelidae species (e.g., llama), single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies.
  • Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit, and bovine.
  • VHH can also be derived from other species besides Camelidae that may produce heavy chain antibodies naturally devoid of light chain.
  • Non-limiting examples of antibody fragments include antigen-binding regions and/or effector regions of the antibody, e.g., Fab, Fab’, F(ab’)2, Fv, scFv, (scFv)2, single chain antibody molecule, dual variable domain antibody, single variable domain, linear antibody, V region, a multispecific antibody formed from antibody fragments, F(ab)2, Fd, Fc, diabody, di-diabody, disulfide-linked Fvs (dsFv), single-domain antibody (e.g., nanobody) or other fragments (e.g., fragments consisting of the variable regions of the heavy and light chains that are non-covalently coupled).
  • variable region domain may be any suitable arrangement of immunoglobulin heavy (VH) and/or light (VL) variable domains.
  • VH immunoglobulin heavy
  • VL light
  • antibodies also include tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, and an antibody heavy chain monomer.
  • the V region domain may be dimeric and contain VHH- VHH, VH-VH, VH-VL, or VL-VL dimers that bind to the antigen.
  • the VH and VL may be covalently coupled either directly or through a linker to form a single chain Fv (scFv).
  • an antibody can be human, humanized, chimeric and/or affinity matured, as well as an antibody from other species, for example, mouse and rabbit, etc.
  • antibody is intended to include a polypeptide product of B cells within the immunoglobulin class of polypeptides that is able to bind to a specific molecular antigen and is composed of two identical pairs of polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids, and each carboxy -terminal portion of each chain includes a constant region. See, e.g., Antibody Engineering (Borrebaeck, ed., 2d ed. 1995); and Kuby, Immunology (3d ed. 1997).
  • the specific molecular antigen can be bound by an antibody provided herein, including a polypeptide or an epitope.
  • Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, camelized antibodies or their humanized variants, intrabodies, and anti -idiotypic (anti-Id) antibodies.
  • the term “antibody” as used herein also comprises any binding molecule having a Fc region and a functional fragment (e.g., an antigen-binding fragment) of any of the above, which refers to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived.
  • antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for example, antigen-binding domains or molecules that contain an antigen-binding site that binds to an antigen (e.g., one or more CDRs of an antibody).
  • an antigen e.g., one or more CDRs of an antibody.
  • antibody fragments can be found in, for example, Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol, Biology and Biotechnology: A Comprehensive Desk Reference (Myers, ed., 1995); Huston, et al., 1993, Cell Biophysics 22: 189-224; Pliickthun and Skerra, 1989, Meth. Enzymol. 178:497-515; and Day, Advanced Immunochemistry (2d ed. 1990).
  • the antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) of immunoglobulin molecule.
  • Antibodies may be agonistic antibodies or antagonistic antibodies.
  • An “antigen” is a structure to which an antibody can selectively bind.
  • a target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound.
  • the target antigen is a polypeptide.
  • an antigen is associated with a cell, for example, is present on or in a cell.
  • An “intact” antibody is one comprising an antigen binding site as well as a constant domain (CL) and at least heavy chain constant regions, CHI, CH2 and CH3. The constant regions may include human constant regions or amino acid sequence variants thereof.
  • an intact antibody has one or more effector functions.
  • binding refers to an interaction between molecules including, for example, to form a complex. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and/or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interactions between a single antigen-binding site on an antibody and a single epitope of a target molecule, such as an antigen, is the affinity of the antibody or functional fragment for that epitope.
  • the ratio of dissociation rate (k O ff) to association rate (kon) of a binding molecule (e.g., an antibody) to a monovalent antigen (k o ff/k on ) is the dissociation constant KD, which is inversely related to affinity.
  • KD dissociation constant
  • the value of KD varies for different complexes of antibody and antigen and depends on both k on and k O ff.
  • the dissociation constant KD for an antibody provided herein can be determined using any method provided herein or any other method well known to those skilled in the art.
  • the affinity at one binding site does not always reflect the true strength of the interaction between an antibody and an antigen.
  • the terms such as “bind to,” “that specifically bind to,” and analogous terms are also used interchangeably herein and refer to antibodies of antigen binding domains that specifically bind to an antigen, such as a polypeptide.
  • An antibody or antigen binding domain that binds to or specifically binds to an antigen may be cross-reactive with related antigens.
  • an antibody or antigen binding domain that binds to or specifically binds to an antigen does not cross-react with other antigens.
  • an antibody or antigen binding domain that binds to or specifically binds to an antigen can be identified, for example, by immunoassays, Octet®, Biacore®, or other techniques known to those of skill in the art.
  • an antibody or antigen binding domain binds to or specifically binds to an antigen when it binds to an antigen with higher affinity than to any cross- reactive antigen as determined using experimental techniques, such as radioimmunoassays (RIA) and enzyme linked immunosorbent assays (ELISAs).
  • RIA radioimmunoassays
  • ELISAs enzyme linked immunosorbent assays
  • a specific or selective reaction will be at least twice background signal or noise and may be more than 10 times background. See, e.g., Fundamental Immunology 332-36 (Paul, ed., 2d ed.
  • the extent of binding of an antibody or antigen binding domain to a “non-target” protein is less than about 10% of the binding of the antibody or antigen binding domain to its particular target antigen, for example, as determined by fluorescence activated cell sorting (FACS) analysis or RIA.
  • FACS fluorescence activated cell sorting
  • specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target.
  • An antibody or antigen binding domain that binds to an antigen includes one that is capable of binding the antigen with sufficient affinity such that the antibody is useful, for example, as a diagnostic or therapeutic agent in targeting the antigen.
  • an antibody or antigen binding domain that binds to an antigen has a dissociation constant (KD) of less than or equal to 1000 nM, 800 nM, 500 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM.
  • KD dissociation constant
  • an antibody or antigen binding domain binds to an epitope of an antigen that is conserved among the antigen from different species (e.g., between human and cynomolgus macaque species).
  • Binding affinity generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., a binding protein such as an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen).
  • the affinity of a binding molecule X for its binding partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein.
  • the “KD” or “KD value” may be measured by assays known in the art, for example by a binding assay.
  • the KD may be measured in a RIA, for example, performed with the Fab version of an antibody of interest and its antigen (Chen, et al., J. Mol Biol, 1999, 293:865-81).
  • the KD or KD value may also be measured by using biolayer interferometry (BLI) or surface plasmon resonance (SPR) assays by Octet®, using, for example, an Octet®Red96 system, or by Biacore®, using, for example, a Biacore® 2000 or a Biacore® 3000.
  • An “on-rate” or “rate of association” or “association rate” or “k on ” may also be determined with the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques described above using, for example, the Octet®Red96, the Biacore® 2000, or the Biacore® 3000 system.
  • the antibodies can comprise “chimeric” sequences in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison, et al., Proc. Natl. Acad. Sci. USA, 1984, 81 :6851-55).
  • the antibodies can comprise portions of “humanized” forms of nonhuman (e.g., murine) antibodies that are chimeric antibodies that include human immunoglobulins (e.g., recipient antibody) in which the native CDR residues are replaced by residues from the corresponding CDR of a nonhuman species (e.g., donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and capacity.
  • a nonhuman species e.g., donor antibody
  • humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • a humanized antibody heavy or light chain can comprise substantially all of at least one or more variable regions, in which all or substantially all of the CDRs correspond to those of a nonhuman immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the antibodies can comprise portions of a “fully human antibody” or “human antibody,” wherein the terms are used interchangeably herein and refer to an antibody that comprises a human variable region and, for example, a human constant region. In specific embodiments, the terms refer to an antibody that comprises a variable region and constant region of human origin. “Fully human” antibodies, in certain embodiments, can also encompass antibodies which bind polypeptides and are encoded by nucleic acid sequences which are naturally occurring somatic variants of human germline immunoglobulin nucleic acid sequence. The term “fully human antibody” includes antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences as described by Kabat, et al. (see Kabat, et al.
  • a “human antibody” is one that possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • Human antibodies can be produced using various techniques known in the art, including phage-display libraries (Hoogenboom and Winter, J. Mol. Biol., 1991, 227:381; Marks, et al., 1991, J. Mol.
  • Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., mice see, e.g., Jakobovits, Curr. Opin. Biotechnol., 1995, 6(5): 561-66; Briiggemann and Taussing, Curr. Opin. Biotechnol., 1997, 8(4):455-58; and U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSETM technology). See also, for example, Li, et al., Proc. Natl. Acad. Sci. USA, 2006, 103:3557-62, regarding human antibodies generated via a human B-cell hybridoma technology.
  • the antibodies can comprise portions of a “recombinant human antibody,” wherein the phrase includes human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse or cow) that is transgenic and/or transchromosomal for human immunoglobulin genes (see e.g., Taylor, L. D., et al., Nucl.
  • human antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant human antibodies can have variable and constant regions derived from human germline immunoglobulin sequences (See Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).
  • such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • the antibodies can comprise a portion of a “monoclonal antibody,” wherein the term as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts, and each monoclonal antibody will typically recognize a single epitope on the antigen.
  • a “monoclonal antibody,” as used herein is an antibody produced by a single hybridoma or other cell. The term “monoclonal” is not limited to any particular method for making the antibody.
  • the monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma methodology first described by Kohler et al., 1975, Nature 256:495, or may be made using recombinant DNA methods in bacterial or eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567).
  • the “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson, et al. , Nature, 1991, 352:624-28 and Marks, et al. , J. Mol. BioL, 1991, 222:581-97, for example.
  • a typical 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • the 4-chain unit is generally about 150,000 daltons.
  • Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype.
  • Each H and L chain also has regularly spaced intrachain disulfide bridges.
  • Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the a and y chains and four CH domains for p and a isotypes.
  • VH variable domain
  • CH constant domains
  • Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end.
  • VL variable domain
  • CL constant domain
  • the VL is aligned with the VH
  • the CL is aligned with the first constant domain of the heavy chain (CHI).
  • Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
  • the pairing of a VH and VL together forms a single antigen-binding site.
  • Fab refers to an antibody region that binds to antigens.
  • a conventional IgG usually comprises two Fab regions, each residing on one of the two arms of the Y-shaped IgG structure.
  • Each Fab region is typically composed of one variable region and one constant region of each of the heavy and the light chain. More specifically, the variable region and the constant region of the heavy chain in a Fab region are VH and CHI regions, and the variable region and the constant region of the light chain in a Fab region are VL and CL regions.
  • the VH, CHI, VL, and CL in a Fab region can be arranged in various ways to confer an antigen binding capability according to the present disclosure.
  • VH and CHI regions can be on one polypeptide, and VL and CL regions can be on a separate polypeptide, similarly to a Fab region of a conventional IgG.
  • VH, CHI, VL and CL regions can all be on the same polypeptide and oriented in different orders as described in more detail the sections below.
  • the term “variable region,” “variable domain,” “V region,” or “V domain” refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and are used in the binding and specificity of each particular antibody for its particular antigen.
  • variable region of the heavy chain may be referred to as “VH.”
  • variable region of the light chain may be referred to as “VL .”
  • the term “variable” refers to the fact that certain segments of the variable regions differ extensively in sequence among antibodies. The V region mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable regions. Instead, the V regions consist of less variable (e.g., relatively invariant) stretches called framework regions (FRs) of about 15-30 amino acids separated by shorter regions of greater variability (e.g., extreme variability) called “hypervariable regions” that are each about 9-12 amino acids long.
  • FRs framework regions
  • variable regions of heavy and light chains each comprise four FRs, largely adopting a P sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases form part of, the P sheet structure.
  • the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991)).
  • the constant regions are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC).
  • the variable regions differ extensively in sequence between different antibodies.
  • the variable region is a human variable region.
  • variable region residue numbering according to Kabat or “amino acid position numbering as in Kabat”, and variations thereof, refer to the numbering system used for heavy chain variable regions or light chain variable regions of the compilation of antibodies in Kabat, et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, an FR or CDR of the variable domain.
  • a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 and three inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after residue 82.
  • the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.
  • the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat, et al., supra).
  • the “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (c.g, the EU index reported in Kabat, et al., supra).
  • the “EU index as in Kabat” refers to the residue numbering of the human IgGl EU antibody. Other numbering systems have been described, for example, by AbM, Chothia, Contact, IMGT, and AHon.
  • the term “heavy chain” when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids, and a carboxy -terminal portion includes a constant region.
  • the constant region can be one of five distinct types, (e.g., isotypes) referred to as alpha (a), delta (6), epsilon (a), gamma (y), and mu (p), based on the amino acid sequence of the heavy chain constant region.
  • the distinct heavy chains differ in size: a, 6, and y contain approximately 450 amino acids, while p and a contain approximately 550 amino acids.
  • IgA immunoglobulin A
  • IgD immunoglobulin D
  • IgE immunoglobulin G
  • IgM immunoglobulin M
  • subclasses of IgG namely IgGl, IgG2, IgG3, and IgG4.
  • the term “light chain” when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids, and a carboxy -terminal portion includes a constant region.
  • the approximate length of a light chain is 211 to 217 amino acids.
  • K kappa
  • X lambda
  • CDR refers to one of three hypervariable regions (Hl, H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH P-sheet framework, or one of three hypervariable regions (LI, L2 or L3) within the non-framework region of the antibody VL P-sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences.
  • CDR regions are well known to those skilled in the art and have been defined by well- known numbering systems.
  • CDRs Kabat Complementarity Determining Regions
  • Chothia refers instead to the location of the structural loops (see, e.g., Chothia and Lesk, J. Mol. BioL, 1987, 196:901-17).
  • the end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35 A and 35B are present, the loop ends at 34).
  • the AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software (see, e.g., Antibody Engineering Vol. 2 (Kontermann and Diibel, eds., 2d ed. 2010)).
  • IMGT ImMunoGeneTics
  • IG immunoglobulins
  • TCR T-cell receptors
  • MHC major histocompatibility complex
  • CDR complementary determining region
  • individual CDRs e.g., “CDR-H1, CDR-H2”
  • the scheme for identification of a particular CDR or CDRs is specified, such as the CDR as defined by the Kabat, Chothia, or Contact method. In other cases, the particular amino acid sequence of a CDR is given.
  • Hypervariable regions may comprise “extended hypervariable regions” as follows: 24- 36 or 24-34 (LI), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 or 26-35A (Hl), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in the VH.
  • constant region or “constant domain” refers to a carboxy terminal portion of the light and heavy chain which is not directly involved in binding of the antibody to antigen but exhibits various effector function, such as interaction with the Fc receptor.
  • the term refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable region, which contains the antigen binding site.
  • the constant region may contain the CHI, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.
  • framework or “FR” refers to those variable region residues flanking the CDRs.
  • FR residues are present, for example, in chimeric, humanized, human, domain antibodies, diabodies, linear antibodies, and bispecific antibodies. FR residues are those variable domain residues other than the hypervariable region residues or CDR residues.
  • the FR regions in VH are VH FR1, VH FR2, VH FR3, and VH FR4 (or FR Hl, FR H2, FR H3 and FR H4).
  • the FR regions in VL are VL FR1, VL FR2, VL FR3 and VL FR4 (or FR LI, FR L2, FR L3 and FR L4).
  • Fc region herein is used to define a C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is often defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxylterminus thereof.
  • the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody.
  • a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.
  • a “functional Fc region” possesses an “effector function” of a native sequence Fc region.
  • exemplary “effector functions” include Clq binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor), etc.
  • effector functions generally require the Fc region to be combined with a binding region or binding domain (e.g., an antibody variable region or domain) and can be assessed using various assays known to those skilled in the art.
  • a “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification (e.g., substituting, addition, or deletion).
  • the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, for example, from about one to about ten amino acid substitutions, or from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of a parent polypeptide.
  • the variant Fc region herein can possess at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, or at least about 90% homology therewith, for example, at least about 95% homology therewith.
  • multispecific antibody refers to an antibody that comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope.
  • the first and second epitopes do not overlap or do not substantially overlap.
  • the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein).
  • a multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain.
  • a multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.
  • bispecific antibody refers to a multispecific antibody that binds no more than two epitopes or two antigens.
  • a bispecific antibody is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.
  • the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein).
  • a bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a second epitope.
  • a bispecific antibody comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope.
  • a bispecific antibody comprises a scFv, or fragment thereof, having binding specificity for a first epitope, and a scFv, or fragment thereof, having binding specificity for a second epitope.
  • variant when used in relation to an antigen or an antibody may refer to a peptide or polypeptide comprising one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid sequence substitutions, deletions, and/or additions as compared to a native or unmodified sequence.
  • identity refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences.
  • Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNAStar, Inc.) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • a “modification” of an amino acid residue/position refers to a change of a primary amino acid sequence as compared to a starting amino acid sequence, wherein the change results from a sequence alteration involving said amino acid residue/position.
  • typical modifications include substitution of the residue with another amino acid (e.g., a conservative or non-conservative substitution), insertion of one or more (e.g., generally fewer than 5, 4, or 3) amino acids adjacent to said residue/position, and/or deletion of said residue/position.
  • an “epitope” is a term in the art and refers to a localized region of an antigen to which an antibody can specifically bind.
  • An epitope can be a linear epitope or a conformational, non-linear, or discontinuous epitope.
  • an epitope can be contiguous amino acids of the polypeptide (a “linear” epitope) or an epitope can comprise amino acids from two or more non-contiguous regions of the polypeptide (a “conformational,” “non-linear” or “discontinuous” epitope).
  • a linear epitope may or may not be dependent on secondary, tertiary, or quaternary structure.
  • an antibody binds to a group of amino acids regardless of whether they are folded in a natural three dimensional protein structure.
  • an antibody requires amino acid residues making up the epitope to exhibit a particular conformation (e.g., bend, twist, turn or fold) in order to recognize and bind the epitope.
  • polypeptide and peptide and “protein” are used interchangeably herein and refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification.
  • polypeptides containing one or more analogs of an amino acid including but not limited to, unnatural amino acids, as well as other modifications known in the art. It is understood that, because the polypeptides of this disclosure may be based upon antibodies or other members of the immunoglobulin superfamily, in certain embodiments, a “polypeptide” can occur as a single chain or as two or more associated chains.
  • secretion signal As used herein, the term “secretion signal,” “signal peptide,” “sequence for secretion” or similar terms refer to a signal sequence that allows a sufficient passage through the secretory pathway and/or secretion of the binding agent into the extracellular environment.
  • the secretion signal sequence is cleavable and is removed from the mature binding agent.
  • the secretion signal sequence preferably is chosen with respect to the cell or organism wherein the binding agent is produced in.
  • vector refers to a substance that is used to carry or include a nucleic acid sequence, including for example, a nucleic acid sequence encoding an antibody as described herein, in order to introduce a nucleic acid sequence into a host cell.
  • Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences.
  • Selection control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art.
  • both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors.
  • the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter.
  • nucleic acid molecules into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the nucleic acid molecules are expressed in a sufficient amount to produce a desired product and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.
  • nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA
  • immunoblotting for expression of gene products or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product.
  • the term “host” as used herein refers to an animal, such as a mammal (e.g., a human).
  • the term “host cell” as used herein refers to a particular subject cell that may be transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.
  • An “isolated nucleic acid” is a nucleic acid, for example, an RNA, DNA, or a mixed nucleic acids, which is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence.
  • An “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule.
  • an “isolated” nucleic acid molecule, such as a cDNA molecule can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • nucleic acid molecules encoding an antibody as described herein are isolated or purified.
  • the term embraces nucleic acid sequences that have been removed from their naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.
  • a substantially pure molecule may include isolated forms of the molecule.
  • Polynucleotide refers to polymers of nucleotides of any length and includes DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs.
  • Oligonucleotide refers to short, generally single-stranded, synthetic polynucleotides that are generally, but not necessarily, fewer than about 200 nucleotides in length.
  • oligonucleotide and polynucleotide are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.
  • a cell that produces an antibody of the present disclosure may include a parent hybridoma cell, as well as bacterial and eukaryotic host cells into which nucleic acids encoding the antibodies have been introduced.
  • the left-hand end of any singlestranded polynucleotide sequence disclosed herein is the 5’ end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5’ direction.
  • the direction of 5’ to 3’ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5’ to the 5’ end of the RNA transcript are referred to as “upstream sequences”; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3 ’ to the 3 ’ end of the RNA transcript are referred to as “downstream sequences.”
  • the term “expression” is used according to the present disclosure in its most general meaning and comprises the production of RNA and/or peptides or proteins, e.g., by transcription and/or translation.
  • expression or “translation” relates in particular to the production of peptides or proteins. It also comprises partial expression of nucleic acids. Moreover, expression can be transient or stable. According to the present disclosure, the term expression also includes an “aberrant expression” or “abnormal expression”.
  • the term “specifically expressed” means that a protein is essentially only expressed in a specific tissue or organ.
  • a tumor antigen specifically expressed in gastric mucosa means that said protein is primarily expressed in gastric mucosa and is not expressed in other tissues or is not expressed to a significant extent in other tissue or organ types.
  • a tumor antigen may also be specifically expressed under normal conditions in more than one tissue type or organ, such as in 2 or 3 tissue types or organs, but preferably in not more than 3 different tissue or organ types. In this case, the tumor antigen is then specifically expressed in these organs. For example, if a tumor antigen is expressed under normal conditions preferably to an approximately equal extent in lung and stomach, said tumor antigen is specifically expressed in lung and stomach.
  • abnormal expression means that expression is altered, preferably increased, compared to a reference, e.g., a state in a subject not having a disease associated with aberrant or abnormal expression of a certain protein, e.g., a tumor antigen.
  • An increase in expression refers to an increase by at least 10%, in particular at least 20%, at least 50% or at least 100%, or more. In one embodiment, expression is only found in a diseased tissue, while expression in a healthy tissue is repressed.
  • ADCC antibody-dependent cell- mediated cytotoxicity, which describes the cell-killing ability of effector cells as described herein, in particular lymphocytes, which preferably requires the target cell being marked by an antibody.
  • ADCC preferably occurs when antibodies bind to antigens on tumor cells and the antibody Fc domains engage Fc receptors (FcR) on the surface of immune effector cells.
  • FcR Fc receptors
  • Several families of Fc receptors have been identified, and specific cell populations characteristically express defined Fc receptors.
  • ADCC can be viewed as a mechanism to directly induce a variable degree of immediate tumor destruction that leads to antigen presentation and the induction of tumor-directed T-cell responses.
  • in vivo induction of ADCC will lead to tumor- directed T-cell responses and host-derived antibody responses.
  • CDC complement-dependent cytotoxicity, and is another cell-killing method that can be directed by antibodies.
  • IgM is the most effective isotype for complement activation.
  • IgGl and IgG3 are also both very effective at directing CDC via the classical complement-activation pathway.
  • the formation of antigen-antibody complexes results in the uncloaking of multiple Clq binding sites in close proximity on the CH2 domains of participating antibody molecules such as IgG molecules (Clq is one of three subcomponents of complement Cl).
  • these uncloaked Clq binding sites convert the previously low-affinity Clq-IgG interaction to one of high avidity, which triggers a cascade of events involving a series of other complement proteins and leads to the proteolytic release of the effector-cell chemotactic/activating agents C3a and C5a.
  • the complement cascade ends in the formation of a membrane attack complex, which creates pores in the cell membrane that facilitate free passage of water and solutes into and out of the cell.
  • cell surface is used in accordance with its normal meaning in the art, and thus includes the outside of the cell which is accessible to binding by proteins, antibodies, ligands, and other molecules.
  • a uPA is expressed on the surface of cells if it is located at the surface of said cells and is accessible to binding by a uPAR-specific binding agent such as an antibody or uPA fragment added to the cells.
  • the term “cell” or “host cell” preferably relates to an intact cell, i.e., a cell with an intact membrane that has not released its normal intracellular components such as enzymes, organelles, or genetic material.
  • An intact cell preferably is a viable cell, i.e., a living cell capable of carrying out its normal metabolic functions.
  • said term relates according to the present disclosure to any cell which can be transfected with an exogenous nucleic acid.
  • the cell when transfected with an exogenous nucleic acid and transferred to a recipient can express the nucleic acid in the recipient.
  • the term “cell” includes bacterial cells; other useful cells are yeast cells, fungal cells or mammalian cells.
  • Suitable bacterial cells include cells from gram -negative bacterial strains such as strains of Escherichia coli, Proteus, and Pseudomonas, and gram -positive bacterial strains such as strains of Bacillus, Streptomyces, Staphylococcus, and Lactococcus.
  • Suitable fungal cell include cells from species of Trichoderma, Neurospora, and Aspergillus.
  • Suitable yeast cells include cells from species of Saccharomyces (Tor example Saccharomyces cerevisiae), Schizosaccharomyces (for example Schizosaccharomyces pombe), Pichia (for example Pichia pastoris and Pichia methanolied), and Hansenula.
  • Suitable mammalian cells include for example CHO cells, BHK cells, HeLa cells, COS cells, 293 HEK and the like. However, amphibian cells, insect cells, plant cells, and any other cells used in the art for the expression of heterologous proteins can be used as well.
  • Mammalian cells are particularly preferred for adoptive transfer, such as cells from humans, mice, hamsters, pigs, goats, and primates.
  • the cells may be derived from a large number of tissue types and include primary cells and cell lines such as cells of the immune system, in particular antigen-presenting cells such as dendritic cells and T cells, stem cells such as hematopoietic stem cells and mesenchymal stem cells and other cell types.
  • An antigen-presenting cell is a cell that displays antigen in the context of major histocompatibility complex on its surface. T cells may recognize this complex using their T cell receptor (TCR).
  • TCR T cell receptor
  • extracellular portion refers to a part of a molecule such as a protein that is facing the extracellular space of a cell and preferably is accessible from the outside of said cell, e.g., by antigen-binding molecules such as antibodies or natural ligands located outside the cell.
  • antigen-binding molecules such as antibodies or natural ligands located outside the cell.
  • the term refers to one or more extracellular domains or a fragment thereof.
  • the term “reduce”, “decrease” or “inhibit” means an overall decrease or the ability to cause an overall decrease, preferably of 5% or greater, 10% or greater, 20% or greater, more preferably of 50% or greater, and most preferably of 75% or greater, in the level, e.g., in the level of expression or in the level of proliferation of cells.
  • the term “increase” or “enhance” preferably relates to an increase or enhancement by about at least 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or even more.
  • allogeneic is used to describe anything that is derived from different individuals of the same species. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical.
  • autologous transplant refers to a transplant of tissue or organs derived from the same subject. Such procedures are advantageous because they overcome the immunological barrier which otherwise results in rejection.
  • the term “risk” or “being at risk” refers to a subject that is identified as having a higher than normal chance of developing a disease compared to the general population.
  • a subject who has had, or who currently has, a disease is a subject who has an increased risk for developing a disease, as such a subject may continue to develop a disease.
  • Subjects who currently have, or who have had, a cancer also have an increased risk for cancer metastases.
  • mRNA or “messenger RNA” relates to a “transcript” which may be produced using DNA as template and encodes a peptide or protein.
  • mRNA typically comprises a 5' untranslated region (5'-UTR), a protein or peptide coding region, and a 3' untranslated region (3'-UTR).
  • mRNA has a limited half-life in cells and in vitro (resulting in “transient” expression).
  • mRNA is produced by in vitro transcription using a DNA template.
  • the RNA is obtained by in vitro transcription or chemical synthesis. The in vitro transcription methodology is known to the skilled person.
  • mRNA refers to a polynucleotide that encodes at least one polypeptide.
  • mRNA as used herein encompasses both modified and unmodified RNA.
  • mRNA may contain one or more coding and non-coding regions.
  • RNA encoding means that RNA, if present in the appropriate environment, preferably within a cell, can be expressed to produce a protein or peptide it encodes.
  • RNA relates to a molecule which comprises ribonucleotide residues and preferably being entirely or substantially composed of ribonucleotide residues.
  • “Ribonucleotide” relates to a nucleotide with a hydroxyl group at the 2'-position of a P-D- ribofuranosyl group.
  • the term includes double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
  • RNA molecules can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides.
  • RNA includes and preferably relates to mRNA.
  • the RNA is self-replicating RNA.
  • the term “self-replicating RNA” refers to RNA that is capable of selfamplification.
  • the self-replicating RNA is single stranded RNA of positive sense.
  • the self-replicating RNA is viral RNA or RNA derived from viral RNA.
  • the self-replicating RNA is alphaviral genomic RNA or is derived from alphaviral genomic RNA.
  • the self-replicating RNA is a viral gene expression vector.
  • the virus is Semliki forest virus.
  • the self-replicating RNA contains one or more transgenes at least one of said transgenes encoding the binding agent described herein.
  • the transgenes may partially or completely replace viral sequences such as viral sequences encoding structural proteins.
  • the self-replicating RNA is in vitro transcribed RNA.
  • the term “stability” of RNA relates to the “half-life” of RNA. “Halflife” relates to the period of time which is needed to eliminate half of the activity, amount, or number of molecules. In the context of the present invention, the half-life of an RNA is indicative for the stability of said RNA. The half-life of RNA may influence the “duration of expression” of the RNA. It can be expected that RNA having a long half-life will be expressed for an extended time period.
  • genotypeic is used to describe anything that is derived from individuals or tissues having identical genotypes, i.e., identical twins or animals of the same inbred strain, or their tissues.
  • pharmaceutically acceptable means being approved by a regulatory agency of the Federal or a state government, or listed in United States Pharmacopeia, European Pharmacopeia, or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.
  • Excipient means a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material.
  • Excipients include, for example, encapsulating materia residue 52a according to Kabat Is or additives such as absorption accelerators, antioxidants, binders, buffers, carriers, coating agents, coloring agents, diluents, disintegrating agents, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, perfumes, preservatives, propellants, releasing agents, sterilizing agents, sweeteners, solubilizers, wetting agents and mixtures thereof.
  • the term “excipient” can also refer to a diluent, adjuvant (e.g., Freunds’ adjuvant (complete or incomplete) or vehicle.
  • excipients are pharmaceutically acceptable excipients.
  • Examples of pharmaceutically acceptable excipients include buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (e.g., fewer than about 10 amino acid residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM.
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • each component is “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable excipients are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed.
  • a pharmaceutically acceptable excipient is an aqueous pH buffered solution.
  • excipients are sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.
  • Water is an exemplary excipient when a composition (e.g., a pharmaceutical composition) is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, particularly for injectable solutions.
  • An excipient can also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • Compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like.
  • compositions can include standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • Compositions, including pharmaceutical compounds may contain an antibody, for example, in isolated or purified form, together with a suitable amount of excipients.
  • T cell engager refers to a binding agent capable of binding to a cytotoxic cell by binding to, or "engaging," the CD3 receptor.
  • the T cell engager binds the epsilon chain of CD3.
  • said binding results in activation and/or proliferation of said cytotoxic cells (e.g., T cells), wherein said activated cytotoxic cells preferably release cytotoxic factors, e.g., perforins and granzymes, and initiate cytolysis and apoptosis of target cells.
  • CD3 refers to cluster of differentiation 3.
  • the CD3 complex denotes an antigen that is expressed on mature human T-cells, thymocytes and a subset of natural killer cells as part of the multimolecular T-cell receptor (TCR) complex.
  • the T-cell co-receptor is a protein complex and is composed of four distinct chains. In mammals, the complex contains a CD3 gamma chain, a CD3 delta chain, and two CD3 epsilon chains. These chains associate with a molecule known as the T-cell receptor (TCR) and the zeta-chain (CD247) to generate an activation signal in T lymphocytes.
  • TCR T-cell receptor
  • CD247 zeta-chain
  • the TCR, zeta-chain, and CD3 molecules together comprise the TCR complex.
  • CD3 is responsible for the signal transduction of the TCR.
  • activation of the TCR complex by binding of MHC-presented specific antigen epitopes results in the phosphorylation of immunoreceptor tyrosine-based activation motifs (IT AMs) by Src family kinases, triggering recruitment of further kinases which results in T cell activation including Ca2+ release.
  • IT AMs immunoreceptor tyrosine-based activation motifs
  • CD3 includes human CD3 and denotes an antigen that is expressed on human T cells as part of the multimolecular T cell receptor complex.
  • the human CD3 epsilon is indicated in GenBank Accession No. NM 000733.
  • the human CD3 gamma is indicated in GenBank Accession No. NM 000073.
  • the human CD3 delta is indicated in GenBank Accession No. NM_000732.
  • CD16 is also known as FcyRIII.
  • CD16 is a cluster of differentiation molecule found on the surface of natural killer cells, neutrophils, monocytes, macrophages, and certain T cells.
  • CD16 has been identified as Fc receptors FcyRIIIa (CD16a) and FcyRIIIb (CD 16b), which participate in signal transduction.
  • CD16 as used herein preferentially refers to FcyRIIIa (CD16a).
  • GPC3 refers to Glypican-3.
  • Glypican-3 (GPC-3) is described in Uniprot under ID P51654 and has a Reference Sequence of mRNA NM 001164617 and a Reference Sequence of protein NP 001158089.
  • EpCAM refers to Epithelial cell adhesion molecule.
  • Epithelial cell adhesion molecule (EpCAM) is described in Uniprot under ID Pl 6422 and has a Reference Sequence of mRNA NM_002354.2 and a Reference Sequence of protein NP_002345.2.
  • uPAR refers to urokinase plasminogen activator surface receptor, or CD87 (cluster of differentiation 87), as encoded by the PLAUR gene, and preferably refers to human uPAR unless otherwise specified.
  • ATF refers to the amino terminal fragment of urokinase plasminogen activator (uPA), and preferably refers to human uPA ATF unless otherwise specified.
  • the term “NKG2D” refers to the extracellular domain of NKG2-D type II integral membrane protein, as encoded by the KLRK1 gene, and preferably refers to human NKG2D unless otherwise specified. NKG2D is described in Uniprot under ID P26718.
  • the term “NKG2D Ligand” or “NKG2DL” refers to those ligands to which NGK2D binds with high affinity, including MICA, MICB, RAET1E, RAET1G, RAET1L/ULBP6, ULBP1, ULBP2, ULBP3 and ULBP4, and preferably refers to human ligands unless otherwise specified.
  • EGFR refers to the epidermal growth factor receptor. EGFR is described in Uniprot under ID P00533. EGFR is a cell surface protein that binds to epidermal growth factor, thus inducing receptor dimerization and tyrosine autophosphorylation leading to cell proliferation.
  • disease or “disease states” refers to any pathological state, including diseases of or associated with senescence or cancer, in particular those senescence associated diseases and forms of cancer described herein. Any reference herein to cancer or particular forms of cancer also includes cancer metastasis thereof. Any reference herein to cancer or particular forms of cancer also includes the pro-tumorigenic cancer microenvironment.
  • an effective amount or “therapeutically effective amount” as used herein refers to the amount of an antibody or pharmaceutical composition provided herein which is sufficient to result in the desired outcome.
  • a subject is a mammal, such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey and human).
  • the subject is a human.
  • the subject is a mammal, e.g., a human, diagnosed with a condition or disorder.
  • the subject is a mammal, e.g., a human, at risk of developing a condition or disorder.
  • administer refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body into a patient, such as by mucosal, intradermal, intravenous, intramuscular, subcutaneous delivery, and/or any other method of physical delivery described herein or known in the art.
  • the terms “treat,” “treatment” and “treating” refer to the reduction or amelioration of the progression, severity, and/or duration of a disease or condition resulting from the administration of one or more therapies. Treating may be determined by assessing whether there has been a decrease, alleviation and/or mitigation of one or more symptoms associated with the underlying disorder such that an improvement is observed with the patient, despite that the patient may still be afflicted with the underlying disorder.
  • Treating includes both managing and ameliorating the disease.
  • the terms “manage,” “managing,” and “management” refer to the beneficial effects that a subject derives from a therapy which does not necessarily result in a cure of the disease.
  • prevent refers to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptom(s).
  • the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include both A and B; A or B; A (alone); and B (alone).
  • the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • compositions and methods of treatment of a subject in need thereof with one or more mRNA(s) encoding multispecific binding molecule(s) with binding affinity for one or more target cells and one or more immune effector cells are provided herein.
  • the multispecific binding molecules provided herein comprise a binding domain capable of binding to an antigen present on immune effector cell(s).
  • the immune effector cells are peripheral blood mononuclear cells (PBMCs).
  • the immune effector cells are T cells.
  • the T cells are Pan T cells.
  • the T cells are CD8+ T cells.
  • the immune effector cells are NK cells.
  • the immune effector cells are macrophages.
  • the immune effector cells are tissue-resident macrophages, such as Kupffer cells or peritoneal, lung, splenic, or bone marrow resident macrophages.
  • the antigen is CD3. In some embodiments, the antigen is CD 16.
  • the multispecific binding molecules provided herein comprise a binding domain is capable of binding to a second antigen on a target cell.
  • the second antigen is an antigen expressed on a tumor cell.
  • the second binding domain is capable of binding to a tumor specific antigen (TSA) or a tumor associated antigen (TAA).
  • TSA tumor specific antigen
  • TAA tumor associated antigen
  • the antigen is GPC3.
  • the antigen is EpCAM.
  • the antigen is uPAR.
  • the antigen is NKG2DL.
  • the antigen is EGFR.
  • the multispecific binding molecules of the present disclosure may contain a third binding domains capable of binding to a third antigen expressed on a target cell.
  • the third antigen is an antigen expressed on a tumor cell.
  • the second binding domain and the third binding domain bind to a same antigen.
  • the second binding domain and the third binding domain bind to two different antigens.
  • the second binding domain and the third binding domain bind to two epitopes on the same antigen.
  • the multispecific binding molecule provided herein is a multispecific antibody.
  • the antibodies provided herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, human antibodies, humanized antibodies, chimeric antibodies, etc.
  • the antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to an antigen.
  • the immunoglobulin molecules provided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
  • an antibody provided herein is an IgG antibody, such as an IgGl antibody, IgG2 antibody or IgG4 antibody (e.g., IgG4 nullbody and variants of IgG4 antibodies).
  • the various multispecific binding molecules provided herein comprises a variant and/or derivative of antibodies include antibody fragments that retain the ability to specifically bind to an epitope.
  • the first binding domain and/or the second binding domain is a variant and/or derivative of antibodies include antibody fragments that retain the ability to specifically bind to an epitope.
  • Antibody fragments include but not limited to those described in Section 5.1 above.
  • Exemplary fragments include Fab fragments (an antibody fragment that contains the antigen-binding domain and comprises a light chain and part of a heavy chain bridged by a disulfide bond); Fab' (an antibody fragment containing a single anti -binding domain comprising an Fab and an additional portion of the heavy chain through the hinge region); F(ab')2 (two Fab' molecules joined by interchain disulfide bonds in the hinge regions of the heavy chains; the Fab' molecules may be directed toward the same or different epitopes); a bispecific Fab (a Fab molecule having two antigen binding domains, each of which may be directed to a different epitope); a single chain Fab chain comprising a variable region, also known as, a scFv (the variable, antigen-binding determinative region of a single light and heavy chain of an antibody linked together by, e.g.
  • Fab fragments an antibody fragment that contains the antigen-binding domain and comprises a light chain and part of a heavy chain bridge
  • a chain of 10-25 amino acids a disulfide-linked Fv, or dsFv (the variable, antigen-binding determinative region of a single light and heavy chain of an antibody linked together by a disulfide bond); a camelized VH (the variable, antigen-binding determinative region of a single heavy chain of an antibody in which some amino acids at the VH interface are those found in the heavy chain of naturally occurring camel antibodies); a bispecific scFv (a scFv or a dsFv molecule having two antigen-binding domains, each of which may be directed to a different epitope); a diabody (a dimerized scFv formed when the VH domain of a first scFv assembles with the VL domain of a second scFv and the VL domain of the first scFv assembles with the VH domain of the second scFv; the two antigen-binding regions of the diabody may be directed
  • an antibody provided herein comprises a single-chain Fv (“scFv”).
  • scFvs are antibody fragments comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
  • scFvs see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).
  • an antibody provided herein is a single domain antibody (sdAb).
  • the antibodies provided herein may be from any animal origin including birds and mammals (e.g., human, monkey, murine, donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken).
  • the antibodies provided herein are human or humanized monoclonal antibodies.
  • “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from mice that express antibodies from human genes.
  • the anti-CD3 binding molecules provided herein are those described in Section 7 below.
  • the anti-CD3 binding molecule provided herein is an anti-CD3 antibody.
  • the antibody provided herein comprises one or more CDR sequences. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering.
  • the anti-CD3 antibody is humanized. In some embodiments, the anti-CD3 antibody comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.
  • the anti-CD3 antibody provided herein comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 6. In some embodiments, the anti-CD3 antibody provided herein comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO:
  • the anti-CD3 antibody provided herein comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 21. In some embodiments, the anti-CD3 antibody provided herein comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 35. In some embodiments, the anti-CD3 antibody provided herein comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 48.
  • CDR sequences can be determined according to well- known numbering systems or a combination thereof. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering.
  • the anti-CD3 antibody provided herein comprises LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 7. In some embodiments, the anti-CD3 antibody provided herein comprises LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO:
  • the anti-CD3 antibody provided herein comprises LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 22. In some embodiments, the anti-CD3 antibody provided herein comprises LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 36. In some embodiments, the anti-CD3 antibody provided herein comprises LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 49.
  • CDR sequences can be determined according to well- known numbering systems or a combination thereof. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering.
  • the anti-CD3 antibody or antigen binding fragment provided herein comprises an HCDR1, an HCDR2, and an HCDR3 as set forth in SEQ ID NO: 6, and a LCDR1, a LCDR2, and a LCDR3 as set forth in SEQ ID NO: 7.
  • the anti- CD3 antibody or antigen binding fragment provided herein comprises an HCDR1, an HCDR2, and an HCDR3 as set forth in SEQ ID NO: 15, and a LCDR1, a LCDR2, and a LCDR3 as set forth in SEQ ID NO: 16.
  • the anti-CD3 antibody or antigen binding fragment provided herein comprises an HCDR1, an HCDR2, and an HCDR3 as set forth in SEQ ID NO: 21, and a LCDR1, a LCDR2, and a LCDR3 as set forth in SEQ ID NO: 22.
  • the anti-CD3 antibody or antigen binding fragment provided herein comprises an HCDR1, an HCDR2, and an HCDR3 as set forth in SEQ ID NO: 35, and a LCDR1, a LCDR2, and a LCDR3 as set forth in SEQ ID NO: 36.
  • the anti-CD3 antibody or antigen binding fragment provided herein comprises an HCDR1, an HCDR2, and an HCDR3 as set forth in SEQ ID NO: 48, and a LCDR1, a LCDR2, and a LCDR3 as set forth in SEQ ID NO: 49.
  • CDR sequences can be determined according to well-known numbering systems or a combination thereof.
  • the CDRs are according to IMGT numbering.
  • the CDRs are according to Kabat numbering.
  • the CDRs are according to AbM numbering.
  • the CDRs are according to Chothia numbering.
  • the CDRs are according to Contact numbering.
  • the anti-CD3 antibody or antigen binding fragment provided herein comprises a heavy chain variable region (VH) selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • VH heavy chain variable region
  • the anti-CD3 antibody or antigen binding fragment provided herein comprises a light chain variable region (VL) selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • VL light chain variable region
  • the anti-CD3 antibody or antigen binding fragment provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 6, and a VL comprising the amino acid sequence of SEQ ID NO: 7.
  • the anti-CD3 antibody or antigen binding fragment provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 15, and a VL comprising the amino acid sequence of SEQ ID NO: 16.
  • the anti-CD3 antibody or antigen binding fragment provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 21, and a VL comprising the amino acid sequence of SEQ ID NO: 22.
  • the anti-CD3 antibody or antigen binding fragment provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 35, and a VL comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, the anti-CD3 antibody or antigen binding fragment provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 48, and a VL comprising the amino acid sequence of SEQ ID NO: 49.
  • provided herein is an antibody that competes for binding to CD3 with any of the CD3 antibodies described herein. In another aspect, provided herein is an antibody that binds to the same epitope as any of the CD3 antibodies described herein. In another aspect, provided is a CD3 antibody that binds an epitope on CD3 that overlaps with the epitope on CD3 bound by a CD3 antibody described herein.
  • an antibody that competes for binding to CD3 with a CD3 reference antibody.
  • a CD3 antibody that binds to the same CD3 epitope as a CD3 reference antibody.
  • a CD3 antibody that binds an epitope on CD3 that overlaps with the epitope on CD3 bound by a CD3 reference antibody.
  • the antibody or antigen binding fragment provided herein binds CD3 with a KD of less than lOOOnM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD3 with a KD of less than lOOnM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD3 with a KD of less than 50nM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD3 with a KD of less than 40nM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD3 with a KD of less than 30nM.
  • the antibody or antigen binding fragment provided herein binds CD3 with a KD of less than 20nM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD3 with a KD of less than lOnM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD3 with a KD of less than 9 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD3 with a KD of less than 8 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD3 with a KD of less than 7 nM.
  • the antibody or antigen binding fragment provided herein binds CD3 with a KD of less than 6 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD3 with a KD of less than 5 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD3 with a KD of less than 4 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD3 with a KD of less than 3 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD3 with a KD of less than 2 nM.
  • the antibody or antigen binding fragment provided herein binds CD3 with a KD of less than 1 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD3 with a KD of less than 0.1 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD3 with a KD of less than 0.01 nM.
  • the KD or KD value may also be measured by any known methods in the art, for example, using biolayer interferometry (BLI) or surface plasmon resonance (SPR) assays by Octet®, using, for example, an Octet®Red96 system, or by Biacore®, using, for example, a Biacore®TM-2000 or a Biacore®TM-3000.
  • An “on-rate” or “rate of association” or “association rate” or “kon” may also be determined with the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques described above using, for example, the Octet®Red96, the Biacore®TM-2000, or the Biacore®TM-3000 system.
  • the KD is determined by a Biacore® assay.
  • CD3 is a human CD3.
  • CD3 is a cynomolgus macaque CD3.
  • CD3 is a rat CD3.
  • CD3 is mouse CD3.
  • the anti -CD 16 binding molecules provided herein are those described in Section 7 below.
  • the anti -CD 16 binding molecule provided herein is an anti-CD16 antibody.
  • the antibody provided herein comprises one or more CDR sequences.
  • CDR sequences can be determined according to well- known numbering systems.
  • the CDRs are according to IMGT numbering.
  • the CDRs are according to Kabat numbering.
  • the CDRs are according to AbM numbering.
  • the CDRs are according to Chothia numbering.
  • the CDRs are according to Contact numbering.
  • the anti -CD 16 antibody is humanized.
  • the antiCD 16 antibody comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.
  • the anti -CD 16 antibody provided herein comprises VHH CDR1, VHH CDR2, and VHH CDR3 as set forth in SEQ ID NO: 39.
  • CDR sequences can be determined according to well-known numbering systems or a combination thereof.
  • the CDRs are according to IMGT numbering.
  • the CDRs are according to Kabat numbering.
  • the CDRs are according to AbM numbering.
  • the CDRs are according to Chothia numbering.
  • the CDRs are according to Contact numbering.
  • the anti-CD16 antibody or antigen binding fragment provided herein comprises a single-domain antibody (sdAb) comprising a variable domain (such as VHH domain) having the amino acid sequence of SEQ ID NO: 39, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • sdAb single-domain antibody
  • VHH domain variable domain having the amino acid sequence of SEQ ID NO: 39, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • provided herein is an antibody that competes for binding to CD 16 with any of the CD 16 antibodies described herein. In another aspect, provided herein is an antibody that binds to the same epitope as any of the CD 16 antibodies described herein. In another aspect, provided is a CD 16 antibody that binds an epitope CD 16 that overlaps with the epitope on CD 16 bound by a CD 16 antibody described herein.
  • an antibody that competes for binding to CD 16 with a CD 16 reference antibody.
  • a CD 16 antibody that binds to the same CD 16 epitope as a CD 16 reference antibody.
  • a CD 16 antibody that binds an epitope on CD 16 that overlaps with the epitope on CD 16 bound by a CD 16 reference antibody.
  • the antibody or antigen binding fragment provided herein binds CD 16 with a KD of less than lOOOnM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD 16 with a KD of less than lOOnM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD 16 with a KD of less than 50nM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD 16 with a KD of less than 40nM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD 16 with a KD of less than 30nM.
  • the antibody or antigen binding fragment provided herein binds CD 16 with a KD of less than 20nM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD 16 with a KD of less than lOnM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD 16 with a KD of less than 9 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD 16 with a KD of less than 8 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD 16 with a KD of less than 7 nM.
  • the antibody or antigen binding fragment provided herein binds CD 16 with a KD of less than 6 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD 16 with a KD of less than 5 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD 16 with a KD of less than 4 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD 16 with a KD of less than 3 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD 16 with a KD of less than 2 nM.
  • the antibody or antigen binding fragment provided herein binds CD 16 with a KD of less than 1 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD 16 with a KD of less than 0.1 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds CD16 with a KD of less than 0.01 nM.
  • the KD or KD value may also be measured by any known methods in the art, for example, using biolayer interferometry (BLI) or surface plasmon resonance (SPR) assays by Octet®, using, for example, an Octet®Red96 system, or by Biacore®, using, for example, a Biacore®TM-2000 or a Biacore®TM-3000.
  • An “on-rate” or “rate of association” or “association rate” or “kon” may also be determined with the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques described above using, for example, the Octet®Red96, the Biacore®TM-2000, or the Biacore®TM-3000 system.
  • the KD is determined by a Biacore® assay.
  • CD 16 is a human CD 16.
  • CD 16 is a cynomolgus macaque CD 16.
  • CD 16 is a rat CD 16.
  • CD16 is mouse CD16.
  • the anti-GPC3 binding molecules provide herein are those described in Section 7 below.
  • the anti-GPC3 binding molecule provided herein is an anti-GPC3 antibody.
  • the antibody provided herein comprises one or more CDR sequences. CDR sequences can be determined according to well- known numbering systems. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering.
  • the anti- GPC3 antibody is humanized. In some embodiments, the anti- GPC3 antibody comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.
  • the anti-GPC3 antibody provided herein comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 11. In some embodiments, the anti-GPC3 antibody provided herein comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO:
  • CDR sequences can be determined according to well-known numbering systems or a combination thereof.
  • the CDRs are according to IMGT numbering.
  • the CDRs are according to Kabat numbering.
  • the CDRs are according to AbM numbering.
  • the CDRs are according to Chothia numbering.
  • the CDRs are according to Contact numbering.
  • the anti-GPC3 antibody provided herein comprises LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 12. In some embodiments, the anti-GPC3 antibody provided herein comprises LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO:
  • CDR sequences can be determined according to well-known numbering systems or a combination thereof.
  • the CDRs are according to IMGT numbering.
  • the CDRs are according to Kabat numbering.
  • the CDRs are according to AbM numbering.
  • the CDRs are according to Chothia numbering.
  • the CDRs are according to Contact numbering.
  • the anti-GPC3 antibody or antigen binding fragment provided herein comprises an HCDR1, an HCDR2, and an HCDR3 as set forth in SEQ ID NO: 11, and a LCDR1, a LCDR2, and a LCDR3 as set forth in SEQ ID NO: 12.
  • the anti-GPC3 antibody or antigen binding fragment provided herein comprises an HCDR1, an HCDR2, and an HCDR3 as set forth in SEQ ID NO: 27, and a LCDR1, a LCDR2, and a LCDR3 as set forth in SEQ ID NO: 28.
  • CDR sequences can be determined according to well-known numbering systems or a combination thereof. In some embodiments, the CDRs are according to IMGT numbering.
  • the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering.
  • the anti-GPC3 antibody or antigen binding fragment provided herein comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 11, or a fragment thereof or a variant of said amino acid sequence or fragment. In one embodiment, the anti-GPC3 antibody or antigen binding fragment provided herein comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 27, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the anti-GPC3 antibody or antigen binding fragment provided herein comprises a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 12, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the anti-GPC3 antibody or antigen binding fragment provided herein comprises a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 28, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the anti-GPC3 antibody or antigen binding fragment provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 11, and a VL comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-GPC3 antibody or antigen binding fragment provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 27, and a VL comprising the amino acid sequence of SEQ ID NO: 28.
  • the anti-GPC3 antibody provided herein comprises VHH CDR1, VHH CDR2, and VHH CDR3 as set forth in SEQ ID NO: 10. In some embodiments, the anti-GPC3 antibody provided herein comprises VHH CDR1, VHH CDR2, and VHH CDR3 as set forth in SEQ ID NO: 30.
  • CDR sequences can be determined according to well-known numbering systems or a combination thereof. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering.
  • an antibody binding to GPC3 comprising only one of a single domain antibody region such as a VHH region, or other single domain antibody region.
  • the anti-GPC3 antibody or antigen binding fragment provided herein comprises a single-domain antibody (sdAb) comprising a variable domain (such as VHH domain) having the amino acid sequence of SEQ ID NO: 10.
  • the anti- GPC3 antibody or antigen binding fragment provided herein comprises a single-domain antibody (sdAb) comprising a variable domain (such as VHH domain) having the amino acid sequence of SEQ ID NO: 30.
  • provided herein is an antibody that competes for binding to GPC3 with any of the GPC3 antibodies described herein. In another aspect, provided herein is an antibody that binds to the same epitope as any of the GPC3 antibodies described herein. In another aspect, provided is a GPC3 antibody that binds an epitope on GPC3 that overlaps with the epitope on GPC3 bound by a GPC3 antibody described herein.
  • an antibody that competes for binding to GPC3 with a GPC3 reference antibody.
  • a GPC3 antibody that binds to the same GPC3 epitope as a GPC3 reference antibody.
  • a GPC3 antibody that binds an epitope on GPC3 that overlaps with the epitope on GPC3 bound by a GPC3 reference antibody.
  • the antibody or antigen binding fragment provided herein binds GPC3 with a KD of less than lOOOnM. In some embodiments, the antibody or antigen binding fragment provided herein binds GPC3 with a KD of less than lOOnM. In some embodiments, the antibody or antigen binding fragment provided herein binds GPC3 with a KD of less than 50nM. In some embodiments, the antibody or antigen binding fragment provided herein binds GPC3 with a KD of less than 40nM. In some embodiments, the antibody or antigen binding fragment provided herein binds GPC3 with a KD of less than 30nM.
  • the antibody or antigen binding fragment provided herein binds GPC3 with a KD of less than 20nM. In some embodiments, the antibody or antigen binding fragment provided herein binds GPC3 with a KD of less than lOnM. In some embodiments, the antibody or antigen binding fragment provided herein binds GPC3 with a KD of less than 9 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds GPC3 with a KD of less than 8 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds GPC3 with a KD of less than 7 nM.
  • the antibody or antigen binding fragment provided herein binds GPC3 with a KD of less than 6 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds GPC3 with a KD of less than 5 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds GPC3 with a KD of less than 4 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds GPC3 with a KD of less than 3 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds GPC3 with a KD of less than 2 nM.
  • the antibody or antigen binding fragment provided herein binds GPC3 with a KD of less than 1 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds GPC3 with a KD of less than 0.1 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds GPC3 with a KD of less than 0.01 nM.
  • the KD or KD value may also be measured by any known methods in the art, for example, using biolayer interferometry (BLI) or surface plasmon resonance (SPR) assays by Octet®, using, for example, an Octet®Red96 system, or by Biacore®, using, for example, a Biacore®TM-2000 or a Biacore®TM-3000.
  • An “on-rate” or “rate of association” or “association rate” or “kon” may also be determined with the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques described above using, for example, the Octet®Red96, the Biacore®TM-2000, or the Biacore®TM-3000 system.
  • the KD is determined by a Biacore® assay.
  • GPC3 is a human GPC3.
  • GPC3 is a cynomolgus macaque GPC3.
  • GPC3 is a rat GPC3.
  • GPC3 is mouse GPC3.
  • the anti- EpCAM binding molecules provided herein are those described in Section 7 below.
  • the anti- EpCAM binding molecule provided herein is an anti- EpCAM antibody.
  • the antibody provided herein comprises one or more CDR sequences. CDR sequences can be determined according to well-known numbering systems. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering.
  • the anti-EpCAM antibody is humanized. In some embodiments, the anti- EpCAM antibody comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.
  • the anti-EpCAM antibody provided herein comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 4. In some embodiments, the anti-EpCAM antibody provided herein comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 14.
  • CDR sequences can be determined according to well-known numbering systems or a combination thereof. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering.
  • the anti-EpCAM antibody provided herein comprises LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 2. In some embodiments, the anti-EpCAM antibody provided herein comprises LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 13.
  • CDR sequences can be determined according to well-known numbering systems or a combination thereof. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering.
  • the anti- EpCAM antibody or antigen binding fragment provided herein comprises an HCDR1, an HCDR2, and an HCDR3 as set forth in SEQ ID NO: 4, and a LCDR1, a LCDR2, and a LCDR3 as set forth in SEQ ID NO: 2.
  • the anti- EpCAM antibody or antigen binding fragment provided herein comprises an HCDR1, an HCDR2, and an HCDR3 as set forth in SEQ ID NO: 14, and a LCDR1, a LCDR2, and a LCDR3 as set forth in SEQ ID NO: 13.
  • CDR sequences can be determined according to well- known numbering systems or a combination thereof. In some embodiments, the CDRs are according to IMGT numbering.
  • the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering.
  • the anti-EpCAM antibody or antigen binding fragment provided herein comprises a heavy chain variable region (VH) selected from the group consisting of SEQ ID NOs: 4 or 14, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • VH heavy chain variable region
  • the anti-EpCAM antibody or antigen binding fragment provided herein comprises a light chain variable region (VL) selected from the group consisting of SEQ ID NOs: 2 or 13, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the anti-EpCAM antibody or antigen binding fragment provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 4, and a VL comprising the amino acid sequence of SEQ ID NO: 2.
  • the antibody or antigen binding fragment provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO: 14, and a VL comprising the amino acid sequence of SEQ ID NO: 13.
  • the anti- EpCAM antibody provided herein comprises VHH CDR1, VHH CDR2, and VHH CDR3 as set forth in SEQ ID NO: 41. CDR sequences can be determined according to well-known numbering systems or a combination thereof.
  • the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering.
  • an antibody binding to EpCAM comprising only one of a single domain antibody region such as a VHH region, or other single domain antibody region.
  • the anti- EpCAM antibody or antigen binding fragment provided herein comprises a single-domain antibody (sdAb) comprising a variable domain (such as VHH domain) having the amino acid sequence of SEQ ID NO: 41.
  • the antibody or antigen binding fragment provided herein binds EpCAM with a KD of less than lOOOnM. In some embodiments, the antibody or antigen binding fragment provided herein binds EpCAM with a KD of less than lOOnM. In some embodiments, the antibody or antigen binding fragment provided herein binds EpCAM with a KD of less than 50nM. In some embodiments, the antibody or antigen binding fragment provided herein binds EpCAM with a KD of less than 40nM. In some embodiments, the antibody or antigen binding fragment provided herein binds EpCAM with a KD of less than 30nM.
  • the antibody or antigen binding fragment provided herein binds EpCAM with a KD of less than 20nM. In some embodiments, the antibody or antigen binding fragment provided herein binds EpCAM with a KD of less than lOnM. In some embodiments, the antibody or antigen binding fragment provided herein binds EpCAM with a KD of less than 9 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds EpCAM with a KD of less than 8 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds EpCAM with a KD of less than 7 nM.
  • the antibody or antigen binding fragment provided herein binds EpCAM with a KD of less than 6 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds EpCAM with a KD of less than 5 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds EpCAM with a KD of less than 4 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds EpCAM with a KD of less than 3 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds EpCAM with a KD of less than 2 nM.
  • the antibody or antigen binding fragment provided herein binds EpCAM with a KD of less than 1 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds EpCAM with a KD of less than 0.1 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds EpCAM with a KD of less than 0.01 nM.
  • the KD or KD value may also be measured by any known methods in the art, for example, using biolayer interferometry (BLI) or surface plasmon resonance (SPR) assays by Octet®, using, for example, an Octet®Red96 system, or by Biacore®, using, for example, a Biacore®TM-2000 or a Biacore®TM-3000.
  • An “on-rate” or “rate of association” or “association rate” or “kon” may also be determined with the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques described above using, for example, the Octet®Red96, the Biacore®TM-2000, or the Biacore®TM-3000 system.
  • the KD is determined by a Biacore® assay.
  • EpCAM is a human EpCAM.
  • EpCAM is a cynomolgus macaque EpCAM.
  • EpCAM is a rat EpCAM.
  • EpCAM is mouse EpCAM.
  • the anti-EGFR binding molecules provided herein are those described in Section 7 below.
  • the anti- EGFR binding molecule provided herein is an anti- EGFR antibody.
  • the antibody provided herein comprises one or more CDR sequences. CDR sequences can be determined according to well- known numbering systems. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering.
  • the anti- EGFR antibody is humanized. In some embodiments, the anti- EGFR antibody comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.
  • the anti- EGFR antibody provided herein comprises VHH CDR1, VHH CDR2, and VHH CDR3 as set forth in SEQ ID NO: 50.
  • CDR sequences can be determined according to well-known numbering systems or a combination thereof.
  • the CDRs are according to IMGT numbering.
  • the CDRs are according to Kabat numbering.
  • the CDRs are according to AbM numbering.
  • the CDRs are according to Chothia numbering.
  • the CDRs are according to Contact numbering.
  • an antibody binding to EGFR comprising only one of a single domain antibody region such as a VHH region, or other single domain antibody region.
  • the anti- EGFR antibody or antigen binding fragment provided herein comprises a single-domain antibody (sdAb) comprising a variable domain (such as VHH domain) having the amino acid sequence of SEQ ID NO: 50.
  • an antibody that competes for binding to EGFR with any of the EGFR antibodies described herein.
  • an antibody that binds to the same epitope as any of the EGFR antibodies described herein.
  • an EGFR antibody that binds an epitope on EGFR that overlaps with the epitope on EGFR bound by an EGFR antibody described herein.
  • an antibody that competes for binding to EGFR with an EGFR reference antibody.
  • an EGFR antibody that binds to the same EGFR epitope as an EGFR reference antibody.
  • an EGFR antibody that binds an epitope on EGFR that overlaps with the epitope on EGFR bound by an EGFR reference antibody.
  • the antibody or antigen binding fragment provided herein binds EGFR with a KD of less than lOOOnM. In some embodiments, the antibody or antigen binding fragment provided herein binds EGFR with a KD of less than lOOnM. In some embodiments, the antibody or antigen binding fragment provided herein binds EGFR with a KD of less than 50nM. In some embodiments, the antibody or antigen binding fragment provided herein binds EGFR with a KD of less than 40nM. In some embodiments, the antibody or antigen binding fragment provided herein binds EGFR with a KD of less than 30nM.
  • the antibody or antigen binding fragment provided herein binds EGFR with a KD of less than 20nM. In some embodiments, the antibody or antigen binding fragment provided herein binds EGFR with a KD of less than lOnM. In some embodiments, the antibody or antigen binding fragment provided herein binds EGFR with a KD of less than 9 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds EGFR with a KD of less than 8 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds EGFR with a KD of less than 7 nM.
  • the antibody or antigen binding fragment provided herein binds EGFR with a KD of less than 6 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds EGFR with a KD of less than 5 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds EGFR with a KD of less than 4 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds EGFR with a KD of less than 3 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds EGFR with a KD of less than 2 nM.
  • the antibody or antigen binding fragment provided herein binds EGFR with a KD of less than 1 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds EGFR with a KD of less than 0.1 nM. In some embodiments, the antibody or antigen binding fragment provided herein binds EGFR with a KD of less than 0.01 nM.
  • the KD or KD value may also be measured by any known methods in the art, for example, using biolayer interferometry (BLI) or surface plasmon resonance (SPR) assays by Octet®, using, for example, an Octet®Red96 system, or by Biacore®, using, for example, a Biacore®TM-2000 or a Biacore®TM-3000.
  • An “on-rate” or “rate of association” or “association rate” or “kon” may also be determined with the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques described above using, for example, the Octet®Red96, the Biacore®TM-2000, or the Biacore®TM-3000 system.
  • the KD is determined by a Biacore® assay.
  • EGFR is a human EGFR.
  • EGFR is a cynomolgus macaque EGFR.
  • EGFR is a rat EGFR.
  • EGFR is mouse EGFR.
  • the multispecific binding molecules provided herein comprise a binding domain capable of binding to an antigen present on immune effector cell(s).
  • the immune effector cells are peripheral blood mononuclear cells (PBMCs).
  • the immune effector cells are T cells.
  • the T cells are Pan T cells.
  • the T cells are CD8+ T cells.
  • the immune effector cells are NK cells.
  • the immune effector cells are macrophages.
  • the immune effector cells are tissue-resident macrophages, such as Kupffer cells or peritoneal, lung, splenic, or bone marrow resident macrophages.
  • the antigen is CD3. In some embodiments, the antigen is CD 16.
  • the multispecific binding molecules provided herein further comprise a second binding domain is capable of binding to a second antigen on a target cell.
  • the second antigen is an antigen expressed on a tumor cell.
  • the second binding domain is capable of binding to a tumor specific antigen (TSA) or a tumor associated antigen (TAA).
  • TSA tumor specific antigen
  • TAA tumor associated antigen
  • the second antigen is GPC3.
  • the second antigen is EpCAM.
  • the second antigen is uPAR.
  • the second antigen is NKG2DL.
  • the second antigen is EGFR.
  • the multispecific binding molecules of the present disclosure may contain a third binding domains capable of binding to a third antigen expressed on a target cell.
  • the third antigen is an antigen expressed on a tumor cell.
  • the second binding domain and the third binding domain bind to a same antigen.
  • the second binding domain and the third binding domain bind to two different antigens.
  • the second binding domain and the third binding domain bind to two epitopes on the same antigen.
  • the third antigen is GPC3.
  • the third antigen is EpCAM.
  • the third antigen is uPAR.
  • the third antigen is NKG2DL.
  • the third antigen is EGFR.
  • the multispecific binding molecule comprises one polypeptide; wherein the polypeptide comprises a first antigen binding domain comprising a first VH region and a first VL region, and/or a first single domain antigen binding fragment; and a second antigen binding domain comprising a second VH region and a second VL region, and/or a second single domain antigen binding; and wherein the first antigen binding domain and the second antigen binding domain bind to two different antigens.
  • the first antigen present on immune effector cell(s).
  • the immune effector cells are peripheral blood mononuclear cells (PBMCs).
  • the immune effector cells are T cells.
  • the T cells are Pan T cells. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the immune cells are NK cells. In some embodiments, the immune effector cells are macrophages. In some embodiments, the immune effector cells are tissue-resident macrohpages, such as Kupffer cells or peritoneal, lung, splenic, or bone marrow resident macrophages.
  • the first antigen is CD3. In some embodiments, the first antigen is CD16. In some embodiments, the second antigen expressed on a tumor cell. In some embodiments, the second antigen is GPC3. In some embodiments, the second antigen is EpCAM. In some embodiments, the second antigen is uPAR. In some embodiments, the second antigen is NKG2DL. In some embodiments, the second antigen is EGFR.
  • each of the first antigen binding domain and the second antigen binding domain is a Fab fragment, a scFv, or a single domain antigen binding fragment (sdAb).
  • the first antigen binding domain is a Fab fragment and the second antigen binding domain is a Fab fragment. In some embodiments, the first antigen binding domain is a Fab fragment and the second antigen binding domain is a scFv. In some embodiments, the first antigen binding domain is a Fab fragment and the second antigen binding domain is a single domain antigen binding fragment.
  • the first antigen binding domain is a scFv fragment and the second antigen binding domain is a Fab fragment. In some embodiments, the first antigen binding domain is a scFv fragment and the second antigen binding domain is a scFv. In some embodiments, the first antigen binding domain is a scFv fragment and the second antigen binding domain is a single domain antigen binding fragment.
  • the first antigen binding domain is a single domain antigen binding fragment and the second antigen binding domain is a Fab fragment. In some embodiments, the first antigen binding domain is a single domain antigen binding fragment and the second antigen binding domain is a scFv. In some embodiments, the first antigen binding domain is a single domain antigen binding fragment and the second antigen binding domain is a single domain antigen binding fragment.
  • the first binding domain can be at N-terminus or C-terminus of the second binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • multispecific binding molecules arrangement is CD3 sdAb- GPC3 sdAb. In some embodiments, multispecific binding molecules arrangement is GPC3 sdAb -CD3 sdAb. In some embodiments, multispecific binding molecules arrangement is CD3 scFv- GPC3 sdAb. In some embodiments, multispecific binding molecules arrangement is GPC3 sdAb-CD3 scFv. In some embodiments, multispecific binding molecules arrangement is CD3 sdAb-GPC3 scFv. In some embodiments, multispecific binding molecules arrangement is GPC3 scFv- CD3 sdAb.
  • multispecific binding molecules arrangement is CD3 scFv-GPC3 scFv. In some embodiments, multispecific binding molecules arrangement is GPC3 scFv-CD3 scFv. In some embodiments, multispecific binding molecules arrangement is CD3 sdAb- EpCAM sdAb. In some embodiments, multispecific binding molecules arrangement is EpCAM sdAb -CD3 sdAb. In some embodiments, multispecific binding molecules arrangement is CD3 scFv- EpCAM sdAb. In some embodiments, multispecific binding molecules arrangement is EpCAM sdAb-CD3 scFv. In some embodiments, multispecific binding molecules arrangement is CD3 sdAb- EpCAM scFv. In some embodiments, multispecific binding molecules arrangement is CD3 sdAb- EpCAM scFv.
  • multispecific binding molecules arrangement is EpCAM scFv- CD3 sdAb. In some embodiments, multispecific binding molecules arrangement is EpCAM scFv-CD3 scFv. In some embodiments, multispecific binding molecules arrangement is CD3 scFv- EpCAM scFv. In some embodiments, multispecific binding molecules arrangement is CD3 sdAb- EGFR sdAb. In some embodiments, multispecific binding molecules arrangement is EGFR sdAb -CD3 sdAb. In some embodiments, multispecific binding molecules arrangement is CD3 scFv- EGFR sdAb. In some embodiments, multispecific binding molecules arrangement is EGFR sdAb-CD3 scFv. In some embodiments, multispecific binding molecules arrangement is EGFR sdAb-CD3 scFv.
  • multispecific binding molecules arrangement is CD3 sdAb- EGFR scFv. In some embodiments, multispecific binding molecules arrangement is EGFR scFv- CD3 sdAb. In some embodiments, multispecific binding molecules arrangement is EGFR scFv-CD3 scFv. In some embodiments, multispecific binding molecules arrangement is CD3 scFv- EGFR scFv. In some embodiments, multispecific binding molecules arrangement is CD3 sdAb - uPAR (ATF). In some embodiments, multispecific binding molecules arrangement is uPAR (ATF) - CD3 sdAb.
  • multispecific binding molecules arrangement is uPAR (ATF) - CD3 scFv. In some embodiments, multispecific binding molecules arrangement is CD3 scFv - uPAR (ATF). In some embodiments, multispecific binding molecules arrangement is CD3 sdAb - NKG2D. In some embodiments, multispecific binding molecules arrangement is NKG2D - CD3 sdAb. In some embodiments, multispecific binding molecules arrangement is NKG2D - CD3 scFv. In some embodiments, multispecific binding molecules arrangement is CD3 scFv - NKG2D. The scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • multispecific binding molecules arrangement is CD 16 sdAb- GPC3 sdAb. In some embodiments, multispecific binding molecules arrangement is GPC3 sdAb - CD16 sdAb. In some embodiments, multispecific binding molecules arrangement is CD16 scFv-GPC3 sdAb. In some embodiments, multispecific binding molecules arrangement is GPC3 sdAb- CD 16 scFv. . In some embodiments, multispecific binding molecules arrangement is CD 16 sdAb-GPC3 scFv. In some embodiments, multispecific binding molecules arrangement is GPC3 scFv- CD16 sdAb.
  • multispecific binding molecules arrangement is CD 16 scFv-GPC3 scFv. In some embodiments, multispecific binding molecules arrangement is GPC3 scFv- CD 16 scFv. In some embodiments, multispecific binding molecules arrangement is CD16 sdAb- EpCAM sdAb. In some embodiments, multispecific binding molecules arrangement is EpCAM sdAb - CD16 sdAb. In some embodiments, multispecific binding molecules arrangement is CD16 scFv- EpCAM sdAb. In some embodiments, multispecific binding molecules arrangement is EpCAM sdAb- CD 16 scFv. In some embodiments, multispecific binding molecules arrangement is CD 16 sdAb- EpCAM scFv. In some embodiments, multispecific binding molecules arrangement is CD 16 sdAb- EpCAM scFv.
  • multispecific binding molecules arrangement is EpCAM scFv- CD16 sdAb. In some embodiments, multispecific binding molecules arrangement is EpCAM scFv- CD 16 scFv. In some embodiments, multispecific binding molecules arrangement is CD 16 scFv- EpCAM scFv. In some embodiments, multispecific binding molecules arrangement is CD 16 sdAb- EGFR sdAb. In some embodiments, multispecific binding molecules arrangement is EGFR sdAb - CD16 sdAb. In some embodiments, multispecific binding molecules arrangement is CD16 scFv- EGFR sdAb. In some embodiments, multispecific binding molecules arrangement is EGFR sdAb CD 16 scFv. In some embodiments, multispecific binding molecules arrangement is EGFR sdAb CD 16 scFv.
  • multispecific binding molecules arrangement is CD 16 sdAb- EGFR scFv. In some embodiments, multispecific binding molecules arrangement is EGFR scFv- CD16 sdAb. In some embodiments, multispecific binding molecules arrangement is EGFR scFv- CD 16 scFv. In some embodiments, multispecific binding molecules arrangement is CD 16 scFv- EGFR scFv. In some embodiments, multispecific binding molecules arrangement is CD 16 sdAb - uPAR (ATF). In some embodiments, multispecific binding molecules arrangement is uPAR (ATF) - CD16 sdAb.
  • multispecific binding molecules arrangement is uPAR (ATF) - CD 16 scFv. In some embodiments, multispecific binding molecules arrangement is CD 16 scFv - uPAR (ATF). In some embodiments, multispecific binding molecules arrangement is CD 16 sdAb - NKG2D. In some embodiments, multispecific binding molecules arrangement is NKG2D - CD16 sdAb. In some embodiments, multispecific binding molecules arrangement is NKG2D - CD 16 scFv. In some embodiments, multispecific binding molecules arrangement is CD 16 scFv - NKG2D. The scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • multispecific binding molecules arrangement is arranged, in the order CD3(VH)-CD3(VL) - GPC3 sdAb. In some embodiments, multispecific binding molecules arrangement is arranged, CD3(VL)-CD3(VH) - GPC3 sdAb. In some embodiments, multispecific binding molecules arrangement is GPC3 sdAb- CD3(VH)-CD3(VL). In some embodiments, multispecific binding molecules arrangement is GPC3 sdAb- CD3(VL)-CD3(VH).
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 10 or 30, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • multispecific binding molecules arrangement is arranged, in the order CD3(VH)-CD3(VL) - GPC3 (VH) - GPC3 (VL). In some embodiments, multispecific binding molecules arrangement is arranged, in the order CD3(VL)-CD3(VH) - GPC3 (VH) - GPC3 (VL). In some embodiments, multispecific binding molecules arrangement is arranged, in the order CD3(VH)-CD3(VL) - GPC3 (VL) - GPC3 (VH).
  • multispecific binding molecules arrangement is arranged, in the order CD3(VL)-CD3(VH) - GPC3 (VL) - GPC3 (VH).
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 11, or 27, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 12, or 28, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • multispecific binding molecules arrangement is arranged, in the order GPC3 (VH)- GPC3 (VL) - CD3 (VH) - CD3 (VL).
  • multispecific binding molecules arrangement is arranged, in the order GPC3 (VL)- GPC3(VH) - CD3 (VH) - CD3 (VL). In some embodiments, multispecific binding molecules arrangement is arranged, in the order GPC3 (VH)- GPC3 (VL) - CD3 (VL) - CD3 (VH). In some embodiments, multispecific binding molecules arrangement is arranged, in the order GPC3 (VL)- GPC3 (VH) - CD3 (VL) - CD3 (VH).
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment. In some embodiments, the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment. In some embodiments, the VH sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 11, or 27, or a fragment thereof or a variant of said amino acid sequence or fragment. In some embodiments, the VL sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 12, or 28, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • multispecific binding molecules arrangement is arranged, in the order CD3(VH)-CD3(VL) - EpCAM sdAb. In some embodiments, multispecific binding molecules arrangement is arranged, CD3(VL)-CD3(VH) - EpCAM sdAb. In some embodiments, multispecific binding molecules arrangement is EpCAM sdAb- CD3(VH)- CD3(VL). In some embodiments, multispecific binding molecules arrangement is EpCAM sdAb- CD3(VL)-CD3(VH).
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of EpCAM binding domain comprises the amino acid sequence of SEQ ID NO: 41, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • multispecific binding molecules arrangement is arranged, in the order CD3(VH)-CD3(VL) - EpCAM (VH) - EpCAM (VL). In some embodiments, multispecific binding molecules arrangement is arranged, in the order CD3(VL)-CD3(VH) - EpCAM (VH) - EpCAM (VL). In some embodiments, multispecific binding molecules arrangement is arranged, in the order CD3(VH)-CD3(VL) - EpCAM (VL) - EpCAM (VH). In some embodiments, multispecific binding molecules arrangement is arranged, in the order CD3(VL)-CD3(VH) - EpCAM (VL) - EpCAM (VH).
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment. In some embodiments, the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment. In some embodiments, the VH sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 4, or 14, or a fragment thereof or a variant of said amino acid sequence or fragment. In some embodiments, the VL sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 2, or 13, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • multispecific binding molecules arrangement is arranged, in the order EpCAM (VH)- EpCAM (VL) - CD3 (VH) - CD3 (VL). In some embodiments, multispecific binding molecules arrangement is arranged, in the order EpCAM (VL)- EpCAM (VH) - CD3 (VH) - CD3 (VL). In some embodiments, multispecific binding molecules arrangement is arranged, in the order EpCAM (VH)- EpCAM (VL) - CD3 (VL) - CD3 (VH). In some embodiments, multispecific binding molecules arrangement is arranged, in the order EpCAM (VL)- EpCAM (VH) - CD3 (VL) - CD3 (VH).
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 4, or 14, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 2, or 13, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • multispecific binding molecules arrangement is arranged, in the order CD3(VH)-CD3(VL) - EGFR sdAb. In some embodiments, multispecific binding molecules arrangement is arranged, CD3(VL)-CD3(VH) - EGFR sdAb. In some embodiments, multispecific binding molecules arrangement is EGFR sdAb- CD3(VH)-CD3(VL).
  • multispecific binding molecules arrangement is EGFR sdAb- CD3(VL)- CD3(VH).
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of EGFR binding domain comprises the amino acid sequence of SEQ ID NO: 50, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • multispecific binding molecules arrangement is arranged, in the order CD3(VH)-CD3(VL) - uPAR (ATF). In some embodiments, multispecific binding molecules arrangement is arranged, CD3(VL)-CD3(VH) - uPAR (ATF). In some embodiments, multispecific binding molecules arrangement is uPAR (ATF)- CD3(VH)-CD3(VL). In some embodiments, multispecific binding molecules arrangement is uPAR (ATF)- CD3(VL)- CD3(VH).
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of uPAR (ATF) comprises the amino acid sequence of SEQ ID NO: 17, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • multispecific binding molecules arrangement is arranged, in the order CD3(VH)-CD3(VL) - NKG2D. In some embodiments, multispecific binding molecules arrangement is arranged, CD3(VL)-CD3(VH) - NKG2D. In some embodiments, multispecific binding molecules arrangement is NKG2D - CD3(VH)-CD3(VL). In some embodiments, multispecific binding molecules arrangement is NKG2D - CD3(VL)-CD3(VH). In some embodiments, the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of NKG2D comprises the amino acid sequence of SEQ ID NO: 24, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6, and a VL region comprising the amino acid sequence of SEQ ID NO: 7, and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6, and a VL region comprising the amino acid sequence of SEQ ID NO: 7, and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 30.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15, and a VL region comprising the amino acid sequence of SEQ ID NO: 16, and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15, and a VL region comprising the amino acid sequence of SEQ ID NO: 16, and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 30.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21, and a VL region comprising the amino acid sequence of SEQ ID NO: 22, and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21, and a VL region comprising the amino acid sequence of SEQ ID NO: 22, and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 30.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 35, and a VL region comprising the amino acid sequence of SEQ ID NO: 36, and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 35, and a VL region comprising the amino acid sequence of SEQ ID NO: 36, and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 30.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 48, and a VL region comprising the amino acid sequence of SEQ ID NO: 49, and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 48, and a VL region comprising the amino acid sequence of SEQ ID NO: 49, and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 30.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6, and a VL region comprising the amino acid sequence of SEQ ID NO: 7, and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6, and a VL region comprising the amino acid sequence of SEQ ID NO: 7, and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 27, and a VL region comprising the amino acid sequence of SEQ ID NO: 28.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15, and a VL region comprising the amino acid sequence of SEQ ID NO: 16, and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15, and a VL region comprising the amino acid sequence of SEQ ID NO: 16, and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 27, and a VL region comprising the amino acid sequence of SEQ ID NO: 28.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21, and a VL region comprising the amino acid sequence of SEQ ID NO: 22, and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21, and a VL region comprising the amino acid sequence of SEQ ID NO: 22, and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 27, and a VL region comprising the amino acid sequence of SEQ ID NO: 28.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 35, and a VL region comprising the amino acid sequence of SEQ ID NO: 36, and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 35, and a VL region comprising the amino acid sequence of SEQ ID NO: 36, and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 27, and a VL region comprising the amino acid sequence of SEQ ID NO: 28.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 48, and a VL region comprising the amino acid sequence of SEQ ID NO: 49, and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 48, and a VL region comprising the amino acid sequence of SEQ ID NO: 49, and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 27, and a VL region comprising the amino acid sequence of SEQ ID NO: 28.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6, and a VL region comprising the amino acid sequence of SEQ ID NO: 7, and (ii) an EpCAM binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 41.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N- terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15, and a VL region comprising the amino acid sequence of SEQ ID NO: 16, and (ii) an EpCAM binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 41.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N- terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21, and a VL region comprising the amino acid sequence of SEQ ID NO: 22, and (ii) an EpCAM binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 41.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N- terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 35, and a VL region comprising the amino acid sequence of SEQ ID NO: 36, and (ii) an EpCAM binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 41.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N- terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 48, and a VL region comprising the amino acid sequence of SEQ ID NO: 49, and (ii) an EpCAM binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 41.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N- terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6, and a VL region comprising the amino acid sequence of SEQ ID NO: 7, and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6, and a VL region comprising the amino acid sequence of SEQ ID NO: 7, and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15, and a VL region comprising the amino acid sequence of SEQ ID NO: 16, and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15, and a VL region comprising the amino acid sequence of SEQ ID NO: 16, and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21, and a VL region comprising the amino acid sequence of SEQ ID NO: 22, and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21, and a VL region comprising the amino acid sequence of SEQ ID NO: 22, and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 35, and a VL region comprising the amino acid sequence of SEQ ID NO: 36, and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 35, and a VL region comprising the amino acid sequence of SEQ ID NO: 36, and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 48, and a VL region comprising the amino acid sequence of SEQ ID NO: 49, and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 48, and a VL region comprising the amino acid sequence of SEQ ID NO: 49, and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6, and a VL region comprising the amino acid sequence of SEQ ID NO: 7, and (ii) an EGFR binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 50.
  • the EGFR binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N- terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15, and a VL region comprising the amino acid sequence of SEQ ID NO: 16, and (ii) an EGFR binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 50.
  • the EGFR binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N- terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21, and a VL region comprising the amino acid sequence of SEQ ID NO: 22, and (ii) an EGFR binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 50.
  • the EGFR binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N- terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 35, and a VL region comprising the amino acid sequence of SEQ ID NO: 36, and (ii) an EGFR binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 50.
  • the EGFR binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N- terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 48, and a VL region comprising the amino acid sequence of SEQ ID NO: 49, and (ii) an EGFR binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 50.
  • the EGFR binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N- terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6, and a VL region comprising the amino acid sequence of SEQ ID NO: 7, and (ii) an uPAR binding domain comprising the amino acid sequence of SEQ ID NO: 17.
  • the uPAR binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15, and a VL region comprising the amino acid sequence of SEQ ID NO: 16, and (ii) an uPAR binding domain comprising the amino acid sequence of SEQ ID NO: 17.
  • the uPAR binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21, and a VL region comprising the amino acid sequence of SEQ ID NO: 22, and (ii) an uPAR binding domain comprising the amino acid sequence of SEQ ID NO: 17.
  • the uPAR binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 35, and a VL region comprising the amino acid sequence of SEQ ID NO: 36, and (ii) an uPAR binding domain comprising the amino acid sequence of SEQ ID NO: 17.
  • the uPAR binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 48, and a VL region comprising the amino acid sequence of SEQ ID NO: 49, and (ii) an uPAR binding domain comprising the amino acid sequence of SEQ ID NO: 17.
  • the uPAR binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the multispecific binding molecule provided herein comprises (i) a CD 16 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 39, and (ii) an EpCAM binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 41.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD 16 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • a long linker connects the corresponding variable heavy chain regions (VH) and the corresponding variable light chain regions (VL) to create a scFv binding domain while a short linker generally connects two scFv binding domains, or two sdAbs, or a scFv and a sdAb.
  • the linker is generally designed to provide flexibility and protease resistance, and preferably, the linker comprises glycine and/or serine amino acid residues.
  • Short peptide linkers may consist of 13 or less such as 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acids, and preferably, 5 or 6 amino acids.
  • Short peptide linkers preferably comprise the amino acid sequences SGGGGS (SEQ ID NO:5), SSGGGGS (SEQ ID NO:31), GGGGSGGG (SEQ ID NO:34), GGGGSGGGSGGG (SEQ ID NO:38), GGSGGGGS (SEQ ID NO:40), GGGGGSGGGSGGG (SEQ ID NO:42), GGGGGSGGG (SEQ ID NO:47), or GGGGS (SEQ ID NO: 115).
  • Long peptide linkers may consist of 12 or more, such as 15 to 25 or 15 to 20 or 15 to 18 amino acids.
  • Long peptide linkers preferably comprise the amino acid sequences (GGGGS)3 (SEQ ID NO:37), S(GGGGS)3 (SEQ ID NO:29) or VE(GGSGGS)2GGVD (SEQ ID NO:23). Further long peptide linkers may comprise the amino acid sequences (GGGGS)4 (SEQ ID NO:26), (GGGGS)5 (SEQ ID NO:3) or GGGGS(GGS)3GGGS (SEQ ID NO: 116).
  • the multispecific binding molecules provided herein further comprise secretion signals such as N-terminal secretion signals.
  • the signal peptide can direct the target cell to secrete the polypeptide.
  • the sequence of secretion signal is selected from the group consisting of SEQ ID NOs: 1, 43, 44, 45, 46 or 51.
  • the multispecific binding molecules provided herein further comprise C-terminal His-tags, in particular the sequence Gly - Gly - Ser - (His)6 (SEQ ID NO: 117), (His)6 (SEQ ID NOV), or VSGWRLFKKIS (SEQ ID NO:33) if present.
  • Multispecific binding molecule comprising two polypeptides
  • the multispecific binding molecule comprises two polypeptides; wherein the first polypeptide comprises a first antigen binding domain comprising a VH region binding to the first antigen; and a second antigen binding domain comprising a second VH region and a second VL region, and/or a single domain antigen binding fragment binding to the second antigen; and the second polypeptide comprises a first antigen binding domain comprising a VL region binding to the first antigen; and a third antigen binding domain comprising a third VH region and a third VL region, and/or a single domain antigen binding fragment binding to the third antigen.
  • the second binding domain and the third binding domain bind to a same antigen.
  • the second binding domain and the third binding domain bind to two different antigens. In some embodiments, the second binding domain and the third binding domain bind to two epitopes on the same antigen.
  • the immune effector cells are peripheral blood mononuclear cells (PBMCs).
  • the immune effector cells are T cells. In some embodiments, the T cells are Pan T cells. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the immune effector cells are NK cells. In some embodiments, the immune effector cells are macrophages.
  • the immune effector cells are tissue-resident macrohpages, such as Kupffer cells or peritoneal, lung, splenic, or bone marrow resident macrophages.
  • the first antigen is CD3. In some embodiments, the first antigen is CD 16. In some embodiments, the second antigen expressed on a tumor cell. In some embodiments, the second antigen is GPC3. In some embodiments, the second antigen is EpCAM. In some embodiments, the second antigen is uPAR. In some embodiments, the second antigen is NKG2DL. In some embodiments, the second antigen is EGFR. In some embodiments, the third antigen expressed on a tumor cell. In some embodiments, the third antigen is GPC3. In some embodiments, the third antigen is EpCAM. In some embodiments, the third antigen is uPAR. In some embodiments, the third antigen is NKG2DL. In some embodiments, the third antigen is EGFR.
  • the first polypeptide comprises a VH region binding to the first antigen and a Fab fragment binding to the second antigen; and the second polypeptide comprises a VL region binding to the first antigen and a Fab fragment binding to the third antigen.
  • the second binding domain and the third binding domain bind to a same antigen.
  • the second binding domain and the third binding domain bind to two different antigens.
  • the second binding domain and the third binding domain bind to two epitopes on the same antigen.
  • the first polypeptide comprises a VH region binding to the first antigen and a Fab fragment binding to the second antigen; and the second polypeptide comprises a VL region binding to the first antigen and a scFv fragment binding to the third antigen.
  • the second binding domain and the third binding domain bind to a same antigen.
  • the second binding domain and the third binding domain bind to two different antigens.
  • the second binding domain and the third binding domain bind to two epitopes on the same antigen.
  • the first polypeptide comprises a VH region binding to the first antigen and a Fab fragment binding to the second antigen; and the second polypeptide comprises a VL region binding to the first antigen and a single domain antigen binding fragment binding to the third antigen.
  • the second binding domain and the third binding domain bind to a same antigen.
  • the second binding domain and the third binding domain bind to two different antigens.
  • the second binding domain and the third binding domain bind to two epitopes on the same antigen.
  • the first polypeptide comprises a VH region binding to the first antigen and a scFv fragment binding to the second antigen; and the second polypeptide comprises a VL region binding to the first antigen and a Fab fragment binding to the third antigen.
  • the second binding domain and the third binding domain bind to a same antigen.
  • the second binding domain and the third binding domain bind to two different antigens.
  • the second binding domain and the third binding domain bind to two epitopes on the same antigen.
  • the first polypeptide comprises a VH region binding to the first antigen and a scFv fragment binding to the second antigen; and the second polypeptide comprises a VL region binding to the first antigen and a scFv fragment binding to the third antigen.
  • the second binding domain and the third binding domain bind to a same antigen.
  • the second binding domain and the third binding domain bind to two different antigens.
  • the second binding domain and the third binding domain bind to two epitopes on the same antigen.
  • the first polypeptide comprises a VH region binding to the first antigen and a scFv fragment binding to the second antigen; and the second polypeptide comprises a VL region binding to the first antigen and a single domain antigen binding fragment binding to the third antigen.
  • the second binding domain and the third binding domain bind to a same antigen.
  • the second binding domain and the third binding domain bind to two different antigens.
  • the second binding domain and the third binding domain bind to two epitopes on the same antigen.
  • the first polypeptide comprises a VH region binding to the first antigen and a single domain antigen binding fragment binding to the second antigen; and the second polypeptide comprises a VL region binding to the first antigen and a Fab fragment binding to the third antigen.
  • the second binding domain and the third binding domain bind to a same antigen.
  • the second binding domain and the third binding domain bind to two different antigens.
  • the second binding domain and the third binding domain bind to two epitopes on the same antigen.
  • the first polypeptide comprises a VH region binding to the first antigen and a single domain antigen binding fragment binding to the second antigen; and the second polypeptide comprises a VL region binding to the first antigen and a scFv fragment binding to the third antigen.
  • the second binding domain and the third binding domain bind to a same antigen.
  • the second binding domain and the third binding domain bind to two different antigens.
  • the second binding domain and the third binding domain bind to two epitopes on the same antigen.
  • the first polypeptide comprises a VH region binding to the first antigen and a single domain antigen binding fragment binding to the second antigen; and the second polypeptide comprises a VL region binding to the first antigen and a single domain antigen binding fragment binding to the third antigen.
  • the second binding domain and the third binding domain bind to a same antigen.
  • the second binding domain and the third binding domain bind to two different antigens.
  • the second binding domain and the third binding domain bind to two epitopes on the same antigen.
  • the first binding domain can be at N-terminus or C-terminus of the second binding domain.
  • the first binding domain can be at N-terminus or C-terminus of the third binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide arrangement is GPC3 sdAb-CD3 VH
  • the second polypeptide arrangement is GPC3 sdAb-CD3 VL
  • the first polypeptide arrangement is CD3 VH- GPC3 sdAb
  • the second polypeptide arrangement is CD3 VL- GPC3 sdAb.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 10 or 30, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the first polypeptide arrangement is GPC3 scFv-CD3 VH
  • the second polypeptide arrangement is GPC3 scFv -CD3 VL.
  • the scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 11, or 27, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 12, or 28, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the first polypeptide arrangement is CD3 VH- GPC3 scFv
  • the second polypeptide arrangement is CD3 VL- GPC3 scFv.
  • the scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 11, or 27, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 12, or 28, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the first polypeptide arrangement is GPC3 sdAb-CD3 VH
  • the second polypeptide arrangement is GPC3 scFv-CD3 VL
  • the first polypeptide arrangement is CD3 VH- GPC3 sdAb
  • the second polypeptide arrangement is CD3 VL- GPC3 scFv.
  • the scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 10 or 30, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 11, or 27, or a fragment thereof or a variant of said amino acid sequence or fragment. In some embodiments, the VL sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 12, or 28, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the first polypeptide arrangement is GPC3 scFv-CD3 VH
  • the second polypeptide arrangement is GPC3 sdAb -CD3 VL
  • the first polypeptide arrangement is CD3 VH- GPC3 scFv
  • the second polypeptide arrangement is CD3 VL- GPC3 sdAb.
  • the scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 10 or 30, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 11, or 27, or a fragment thereof or a variant of said amino acid sequence or fragment. In some embodiments, the VL sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 12, or 28, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the first polypeptide arrangement is EpCAM sdAb-CD3 VH
  • the second polypeptide arrangement is EpCAM sdAb-CD3 VL
  • the first polypeptide arrangement is CD3 VH- EpCAM sdAb
  • the second polypeptide arrangement is CD3 VL- EpCAM sdAb.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of EpCAM binding domain comprises amino acid sequence of SEQ ID NO: 41, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the first polypeptide arrangement is EpCAM scFv-CD3 VH
  • the second polypeptide arrangement is EpCAM scFv -CD3 VL
  • the first polypeptide arrangement is CD3 VH- EpCAM scFv
  • the second polypeptide arrangement is CD3 VL- EpCAM scFv.
  • the scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment. In some embodiments, the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment. In some embodiments, the VH sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 4, or 14, or a fragment thereof or a variant of said amino acid sequence or fragment. In some embodiments, the VL sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 2, or 13, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the first polypeptide arrangement is EpCAM sdAb-CD3 VH
  • the second polypeptide arrangement is EpCAM scFv -CD3 VL
  • the first polypeptide arrangement is CD3 VH- EpCAM sdAb
  • the second polypeptide arrangement is CD3 VL- EpCAM scFv.
  • the scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of EpCAM binding domain comprises amino acid sequence of SEQ ID NO: 41, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 4, or 14, or a fragment thereof or a variant of said amino acid sequence or fragment. In some embodiments, the VL sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 2, or 13, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the first polypeptide arrangement is EpCAM scFv-CD3 VH
  • the second polypeptide arrangement is EpCAM sdAb -CD3 VL
  • the first polypeptide arrangement is CD3 VH- EpCAM scFv
  • the second polypeptide arrangement is CD3 VL- EpCAM sdAb.
  • the scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of EpCAM binding domain comprises amino acid sequence of SEQ ID NO: 41, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 4, or 14, or a fragment thereof or a variant of said amino acid sequence or fragment. In some embodiments, the VL sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 2, or 13, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the first polypeptide arrangement is EpCAM sdAb-CD3 VH
  • the second polypeptide arrangement is GPC3 sdAb-CD3 VL
  • the first polypeptide arrangement is CD3 VH- EpCAM sdAb
  • the second polypeptide arrangement is CD3 VL- GPC3 sdAb.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 10 or 30, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of EpCAM binding domain comprises amino acid sequence of SEQ ID NO: 41, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the first polypeptide arrangement is GPC3 sdAb-CD3 VH
  • the second polypeptide arrangement is EpCAM sdAb-CD3 VL
  • the first polypeptide arrangement is CD3 VH- GPC3 sdAb
  • the second polypeptide arrangement is CD3 VL- EpCAM sdAb.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the GPC3 binding domain can be at N-terminus or C- terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 10 or 30, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of EpCAM binding domain comprises amino acid sequence of SEQ ID NO: 41, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the first polypeptide arrangement is EpCAM scFv-CD3 VH
  • the second polypeptide arrangement is GPC3 scFv -CD3 VL
  • the first polypeptide arrangement is CD3 VH- EpCAM scFv
  • the second polypeptide arrangement is CD3 VL- GPC3 scFv.
  • the scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • the EpCAM binding domain can be at N- terminus or C-terminus of the CD3 VH or VL binding domain.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 11, or 27, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 12, or 28, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 4, or 14, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 2, or 13, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the first polypeptide arrangement is GPC3 scFv-CD3 VH
  • the second polypeptide arrangement is EpCAM scFv -CD3 VL
  • the first polypeptide arrangement is CD3 VH- GPC3 scFv
  • the second polypeptide arrangement is CD3 VL- EpCAM scFv.
  • the scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • the EpCAM binding domain can be at N- terminus or C-terminus of the CD3 VH or VL binding domain.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 11, or 27, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 12, or 28, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 4, or 14, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 2, or 13, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the first polypeptide arrangement is EpCAM sdAb-CD3 VH
  • the second polypeptide arrangement is GPC3 scFv -CD3 VL
  • the first polypeptide arrangement is CD3 VH- EpCAM sdAb
  • the second polypeptide arrangement is CD3 VL- GPC3 scFv.
  • the scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • the EpCAM binding domain can be at N- terminus or C-terminus of the CD3 VH or VL binding domain.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 11, or 27, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 12, or 28, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of EpCAM binding domain comprises amino acid sequence of SEQ ID NO: 41, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the first polypeptide arrangement is GPC3 sdAb-CD3 VH
  • the second polypeptide arrangement is EpCAM scFv -CD3 VL
  • the first polypeptide arrangement is CD3 VH- GPC3 sdAb
  • the second polypeptide arrangement is CD3 VL- EpCAM scFv.
  • the scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • the EpCAM binding domain can be at N- terminus or C-terminus of the CD3 VH or VL binding domain.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 4, or 14, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 2, or 13, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 10 or 30, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the first polypeptide arrangement is EpCAM scFv-CD3 VH
  • the second polypeptide arrangement is GPC3 sdAb -CD3 VL
  • the first polypeptide arrangement is CD3 VH- EpCAM scFv
  • the second polypeptide arrangement is CD3 VL- GPC3 sdAb.
  • the scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 4, or 14, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 2, or 13, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 10 or 30, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the first polypeptide arrangement is GPC3 scFv-CD3 VH
  • the second polypeptide arrangement is EpCAM sdAb -CD3 VL
  • the first polypeptide arrangement is CD3 VH- GPC3 scFv
  • the second polypeptide arrangement is CD3 VL- EpCAM sdAb.
  • the scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • the EpCAM binding domain can be at N- terminus or C-terminus of the CD3 VH or VL binding domain.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of EpCAM binding domain comprises amino acid sequence of SEQ ID NO: 41, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 11, or 27, or a fragment thereof or a variant of said amino acid sequence or fragment. In some embodiments, the VL sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 12, or 28, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the first polypeptide arrangement is EGFR sdAb-CD3 VH
  • the second polypeptide arrangement is EGFR sdAb-CD3 VL
  • the first polypeptide arrangement is CD3 VH- EGFR sdAb
  • the second polypeptide arrangement is CD3 VL- EGFR sdAb.
  • the EGFR binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of EGFR binding domain comprises amino acid sequence of SEQ ID NO: 50, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the GPC3 binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N- terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 35 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 28, and a VL region comprising the amino acid sequence of SEQ ID NO: 29;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 36 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 28, and a VL region comprising the amino acid sequence of SEQ ID NO: 29.
  • the GPC3 binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N- terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the GPC3 binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the GPC3 binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the GPC3 binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the GPC3 binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the GPC3 binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N- terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the GPC3 binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the GPC3 binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the GPC3 binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the GPC3 binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N- terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 35 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 36 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the GPC3 binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the GPC3 binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the GPC3 binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 35 and (ii) an EGFR binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 50;
  • the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 36 and (ii) an EGFR binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 50.
  • the EGFR binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • a long linker connects the corresponding variable heavy chain regions (VH) and the corresponding variable light chain regions (VL) to create a scFv binding domain while a short linker generally connects two scFv binding domains, or two sdAbs, or a scFv and a sdAb.
  • the linker is generally designed to provide flexibility and protease resistance, and preferably, the linker comprises glycine and/or serine amino acid residues.
  • Short peptide linkers may consist of 13 or less such as 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acids, and preferably, 5 or 6 amino acids.
  • Short peptide linkers preferably comprise the amino acid sequences SGGGGS (SEQ ID NO:5), SSGGGGS (SEQ ID NO:31), GGGGSGGG (SEQ ID NO:34), GGGGSGGGSGGG (SEQ ID NO:38), GGSGGGGS (SEQ ID NO:40), GGGGGSGGGSGGG (SEQ ID NO:42), GGGGGSGGG (SEQ ID NO:47), or GGGGS (SEQ ID NO: 115).
  • Long peptide linkers may consist of 12 or more, such as 15 to 25 or 15 to 20 or 15 to 18 amino acids.
  • Long peptide linkers preferably comprise the amino acid sequences (GGGGS)3 (SEQ ID NO:37), S(GGGGS)3 (SEQ ID NO:29) or VE(GGSGGS)2GGVD (SEQ ID NO:23). Further long peptide linkers may comprise the amino acid sequences (GGGGS)4 (SEQ ID NO:26), (GGGGS)5 (SEQ ID NO:3) or GGGGS(GGS)3GGGS (SEQ ID NO: 116).
  • the multispecific binding molecule provided herein further comprises secretion signals such as N-terminal secretion signals.
  • the signal peptide can direct the target cell to secrete the polypeptide.
  • the sequence of secretion signal is selected from the group consisting of SEQ ID NOs: 1, 43, 44, 45, 46 or 51.
  • the multispecific binding molecule provided herein further comprises C - terminal His - tags, in particular the sequence Gly - Gly - Ser - (His)6 (SEQ ID NO: 117), (His)6 (SEQ ID NO:9), or VSGWRLFKKIS (SEQ ID NO:33) if present.
  • the antibodies provided herein can be humanized antibodies.
  • a humanized antibody can comprise human framework region and human constant region sequences.
  • a humanized antibody can comprise human constant region sequences.
  • a humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgGl, IgG2, IgG3 and IgG4 (e.g., variants of IgG4 and IgG4 nullbody).
  • a humanized antibody can comprise kappa or lambda light chain constant sequences.
  • Humanized antibodies can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (European Patent No. EP 239,400; International publication No. WO 91/09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (European Patent Nos. EP 592,106 and EP 519,596; Padlan, Molecular Immunology, 1991, 28(4/5):489-498; Studnicka, et al., Protein Engineering, 1994, 7(6):805-814; and Roguska, et al., Proc. Natl. Acad. Sci.
  • a humanized antibody can have one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization may be performed, for example, following the method of Jones, et al., 1986, Nature 321 :522-5; Riechmann, et al., Nature, 1988, 332:323-7; and Verhoeyen, et al., Science, 1988, 239: 1534-6, by substituting hypervariable region sequences for the corresponding sequences of a human antibody.
  • the humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the six CDRs of the parent non-human antibody (e.g., rodent) are grafted onto a human antibody framework.
  • CDR grafting in which the amino acid sequences of the six CDRs of the parent non-human antibody (e.g., rodent) are grafted onto a human antibody framework.
  • Padlan, et al. determined that only about one third of the residues in the CDRs actually contact the antigen, and termed these the “specificity determining residues,” or SDRs (Padlan, et al., FASEB J., 1995, 9: 133-9).
  • SDRs Ser, et al., FASEB J., 1995, 9: 133-9.
  • SDR grafting only the SDR residues are grafted onto the human antibody framework (see, e.g., Kashmiri, et al., Methods, 2005, 36:25-34).
  • variable domains both light and heavy
  • the sequence of the variable domain of a non-human (e.g., rodent) antibody is screened against the entire library of known human variable-domain sequences.
  • the human sequence that is closest to that of the rodent may be selected as the human framework for the humanized antibody (Sims et al., J. Immunol., 1993, 151 :2296-308; and Chothia et al., J. Mol. Biol., 1987, 196:901-17).
  • Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains.
  • the same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 1992, 89:4285-89; and Presta et al. , J. Immunol., 1993, 151 :2623-32).
  • the framework is derived from the consensus sequences of the most abundant human subclasses, VL6 subgroup I (VL6I) and VH subgroup III (VHIII).
  • VL6I VL6 subgroup I
  • VHIII VH subgroup III
  • human germline genes are used as the source of the framework regions.
  • FR homology is irrelevant.
  • the method consists of comparison of the non-human sequence with the functional human germline gene repertoire. Those genes encoding the same or closely related canonical structures to the murine sequences are then selected. Next, within the genes sharing the canonical structures with the non-human antibody, those with highest homology within the CDRs are chosen as FR donors. Finally, the non-human CDRs are grafted onto these FRs (see, e.g., Tan et al., J. Immunol., 2002, 169: 1119-25).
  • humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences.
  • Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
  • Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees, Protein Eng., 2000, 13:819-24), Modeller (Sali and Blundell, J. Mol.
  • HSC Human String Content
  • Antibody variants may be isolated from phage, ribosome, and yeast display libraries as well as by bacterial colony screening (see, e.g., Hoogenboom, Nat. Biotechnol., 2005, 23: 1105-16; Dufner, et al., Trends Biotechnol. , 2006, 24:523-9; Feldhaus, et al., Nat. Biotechnol. , 2003, 21 : 163-70; and Schlapschy et al. , Protein Eng. Des. Sei., 2004, 17:847-60).
  • residues to be substituted may include some or all of the “Vernier” residues identified as potentially contributing to CDR structure (see, e.g., Foote and Winter, J. Mol. BioL, 1992, 224:487-99), or from the more limited set of target residues identified by Baca, et al., J. Biol. Chem., 1997, 272: 10678-84.
  • FR shuffling whole FRs are combined with the non-human CDRs instead of creating combinatorial libraries of selected residue variants (see, e.g., Dall’ Acqua et al., Methods, 2005, 36:43-60).
  • the libraries may be screened for binding in a two-step process, first humanizing VL, followed by VH.
  • a one-step FR shuffling process may be used.
  • Such a process has been shown to be more efficient than the two-step screening, as the resulting antibodies exhibited improved biochemical and physicochemical properties including enhanced expression, increased affinity, and thermal stability (see, e.g., Damschroder, et al., Mol. Immunol., 2007, 44:3049-60).
  • the “humaneering” method is based on experimental identification of essential minimum specificity determinants (MSDs) and is based on sequential replacement of non-human fragments into libraries of human FRs and assessment of binding. It begins with regions of the CDR3 of non-human VH and VL chains and progressively replaces other regions of the non- human antibody into the human FRs, including the CDR1 and CDR2 of both VH and VL. This methodology typically results in epitope retention and identification of antibodies from multiple subclasses with distinct human V-segment CDRs.
  • the “human engineering” method involves altering a non-human antibody or antibody fragment, such as a mouse or chimeric antibody or antibody fragment, by making specific changes to the amino acid sequence of the antibody so as to produce a modified antibody with reduced immunogenicity in a human that nonetheless retains the desirable binding properties of the original non-human antibodies.
  • the technique involves classifying amino acid residues of a non-human (e.g., mouse) antibody as “low risk,” “moderate risk,” or “high risk” residues.
  • the classification is performed using a global risk/reward calculation that evaluates the predicted benefits of making particular substitution (e.g., for immunogenicity in humans) against the risk that the substitution will affect the resulting antibody’s folding.
  • the particular human amino acid residue to be substituted at a given position (e.g., low or moderate risk) of a non-human (e.g., mouse) antibody sequence can be selected by aligning an amino acid sequence from the non-human antibody’s variable regions with the corresponding region of a specific or consensus human antibody sequence.
  • the amino acid residues at low or moderate risk positions in the non-human sequence can be substituted for the corresponding residues in the human antibody sequence according to the alignment.
  • a composite human antibody can be generated using, for example, Composite Human AntibodyTM technology (Antitope Ltd., Cambridge, United Kingdom).
  • variable region sequences are designed from fragments of multiple human antibody variable region sequences in a manner that avoids T cell epitopes, thereby minimizing the immunogenicity of the resulting antibody.
  • Such antibodies can comprise human constant region sequences, e.g., human light chain and/or heavy chain constant regions.
  • a deimmunized antibody is an antibody in which T-cell epitopes have been removed. Methods for making deimmunized antibodies have been described (see, e.g., Jones, et al., Methods Mol Biol., 2009, 525:405-23; and De Groot, et al., Cell. Immunol., 2006, 244: 148-153).
  • Deimmunized antibodies comprise T-cell epitope-depleted variable regions and human constant regions. Briefly, VH and VL of an antibody are cloned and T-cell epitopes are subsequently identified by testing overlapping peptides derived from the VH and VL of the antibody in a T cell proliferation assay.
  • T cell epitopes are identified via in silico methods to identify peptide binding to human MHC class II. Mutations are introduced in the VH and VL to abrogate binding to human MHC class II. Mutated VH and VL are then utilized to generate the deimmunized antibody.
  • the mRNA provided herein encoding a multispecific binding molecule comprising one polypeptide; wherein the polypeptide comprises a first antigen binding domain comprising a first VH region and a first VL region, and/or a first single domain antigen binding fragment; and a second antigen binding domain comprising a second VH region and a second VL region, and/or a second single domain antigen binding; and wherein the first antigen binding domain and the second antigen binding domain bind to two different antigens.
  • the first antigen present on immune effector cell(s).
  • the immune effector cells are peripheral blood mononuclear cells (PBMCs).
  • the immune effector cells are T cells. In some embodiments, the T cells are Pan T cells. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the immune effector cells are NK cells. In some embodiments, the immune effector cells are macrophages. In some embodiments, the immune effector cells are tissue-resident macrophages, such as Kupffer cells or peritoneal, lung, splenic, or bone marrow resident macrophages.
  • the first antigen is CD3. In some embodiments, the first antigen is CD 16. In some embodiments, the second antigen expressed on a tumor cell. In some embodiments, the second antigen is GPC3. In some embodiments, the second antigen is EpCAM. In some embodiments, the second antigen is uPAR. In some embodiments, the second antigen is NKG2DL. In some embodiments, the second antigen is EGFR.
  • the polypeptide is encoded by a single mRNA.
  • RNA sequences described herein can be converted to DNA sequences by replacing the "U” (uridine) with a "T” (thymidine).
  • each of the first antigen binding domain and the second antigen binding domain is a Fab fragment, a scFv, or a single domain antigen binding fragment (sdAb).
  • the first antigen binding domain is a Fab fragment and the second antigen binding domain is a Fab fragment.
  • the first antigen binding domain is a Fab fragment and the second antigen binding domain is a scFv.
  • the first antigen binding domain is a Fab fragment and the second antigen binding domain is a single domain antigen binding fragment.
  • the first antigen binding domain is a scFv fragment and the second antigen binding domain is a Fab fragment. In some embodiments, the first antigen binding domain is a scFv fragment and the second antigen binding domain is a scFv. In some embodiments, the first antigen binding domain is a scFv fragment and the second antigen binding domain is a single domain antigen binding fragment.
  • the first antigen binding domain is a single domain antigen binding fragment and the second antigen binding domain is a Fab fragment. In some embodiments, the first antigen binding domain is a single domain antigen binding fragment and the second antigen binding domain is a scFv. In some embodiments, the first antigen binding domain is a single domain antigen binding fragment and the second antigen binding domain is a single domain antigen binding fragment.
  • the first binding domain can be at N-terminus or C-terminus of the second binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising one polypeptide, wherein the multispecific binding molecules arrangement is CD3 sdAb-GPC3 sdAb.
  • the multispecific binding molecules arrangement is GPC3 sdAb -CD3 sdAb.
  • the multispecific binding molecules arrangement is CD3 scFv-GPC3 sdAb.
  • the multispecific binding molecules arrangement is GPC3 sdAb-CD3 scFv.
  • multispecific binding molecules arrangement is CD3 sdAb-GPC3 scFv.
  • multispecific binding molecules arrangement is GPC3 scFv- CD3 sdAb.
  • the multispecific binding molecules arrangement is CD3 scFv-GPC3 scFv. In some embodiments, the multispecific binding molecules arrangement is GPC3 scFv-CD3 scFv. In some embodiments, the multispecific binding molecules arrangement is CD3 sdAb- EpCAM sdAb. In some embodiments, the multispecific binding molecules arrangement is EpCAM sdAb -CD3 sdAb. In some embodiments, the multispecific binding molecules arrangement is CD3 scFv- EpCAM sdAb. In some embodiments, the multispecific binding molecules arrangement is EpCAM sdAb-CD3 scFv. In some embodiments, multispecific binding molecules arrangement is CD3 sdAb- EpCAM scFv. In some embodiments, multispecific binding molecules arrangement is CD3 sdAb- EpCAM scFv. In some embodiments, multispecific binding molecules arrangement is CD3 sdAb- EpCAM scFv.
  • multispecific binding molecules arrangement is EpCAM scFv- CD3 sdAb. In some embodiments, the multispecific binding molecules arrangement is EpCAM scFv-CD3 scFv. In some embodiments, the multispecific binding molecules arrangement is CD3 scFv- EpCAM scFv. In some embodiments, the multispecific binding molecules arrangement is CD3 sdAb- EGFR sdAb. In some embodiments, the multispecific binding molecules arrangement is EGFR sdAb -CD3 sdAb. In some embodiments, the multispecific binding molecules arrangement is CD3 scFv- EGFR sdAb.
  • the multispecific binding molecules arrangement is EGFR sdAb-CD3 scFv. In some embodiments, multispecific binding molecules arrangement is CD3 sdAb- EGFR scFv. In some embodiments, multispecific binding molecules arrangement is EGFR scFv- CD3 sdAb. In some embodiments, the multispecific binding molecules arrangement is EGFR scFv-CD3 scFv. In some embodiments, the multispecific binding molecules arrangement is CD3 scFv- EGFR scFv. In some embodiments, the multispecific binding molecules arrangement is CD3 sdAb - uPAR (ATF).
  • the multispecific binding molecules arrangement is uPAR (ATF) - CD3 sdAb. In some embodiments, the multispecific binding molecules arrangement is uPAR (ATF) - CD3 scFv. In some embodiments, the multispecific binding molecules arrangement is CD3 scFv - uPAR (ATF). In some embodiments, the multispecific binding molecules arrangement is CD3 sdAb - NKG2D. In some embodiments, the multispecific binding molecules arrangement is NKG2D - CD3 sdAb. In some embodiments, the multispecific binding molecules arrangement is NKG2D - CD3 scFv. In some embodiments, the multispecific binding molecules arrangement is CD3 scFv - NKG2D. The scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising one polypeptide, wherein the multispecific binding molecules arrangement is CD16 sdAb-GPC3 sdAb.
  • the multispecific binding molecules arrangement is GPC3 sdAb - CD16 sdAb.
  • the multispecific binding molecules arrangement is CD16 scFv-GPC3 sdAb.
  • the multispecific binding molecules arrangement is GPC3 sdAb- CD 16 scFv.
  • multispecific binding molecules arrangement is CD 16 sdAb-GPC3 scFv.
  • multispecific binding molecules arrangement is GPC3 scFv- CD16 sdAb.
  • the multispecific binding molecules arrangement is CD 16 scFv-GPC3 scFv. In some embodiments, the multispecific binding molecules arrangement is GPC3 scFv- CD16 scFv. In some embodiments, the multispecific binding molecules arrangement is CD16 sdAb- EpCAM sdAb. In some embodiments, the multispecific binding molecules arrangement is EpCAM sdAb - CD16 sdAb. In some embodiments, the multispecific binding molecules arrangement is CD16 scFv- EpCAM sdAb. In some embodiments, the multispecific binding molecules arrangement is EpCAM sdAb- CD 16 scFv. In some embodiments, multispecific binding molecules arrangement is CD 16 sdAb- EpCAM scFv. In some embodiments, multispecific binding molecules arrangement is CD 16 sdAb- EpCAM scFv.
  • multispecific binding molecules arrangement is EpCAM scFv- CD16 sdAb. In some embodiments, the multispecific binding molecules arrangement is EpCAM scFv- CD 16 scFv. In some embodiments, the multispecific binding molecules arrangement is CD 16 scFv- EpCAM scFv. In some embodiments, the multispecific binding molecules arrangement is CD16 sdAb- EGFR sdAb. In some embodiments, the multispecific binding molecules arrangement is EGFR sdAb - CD16 sdAb. In some embodiments, the multispecific binding molecules arrangement is CD 16 scFv- EGFR sdAb. In some embodiments, the multispecific binding molecules arrangement is EGFR sdAb CD 16 scFv. In some embodiments, the multispecific binding molecules arrangement is EGFR sdAb CD 16 scFv. In some embodiments, the multispecific binding molecules arrangement is EGFR sdAb CD 16 scFv.
  • multispecific binding molecules arrangement is CD 16 sdAb- EGFR scFv. In some embodiments, multispecific binding molecules arrangement is EGFR scFv- CD16 sdAb. In some embodiments, the multispecific binding molecules arrangement is EGFR scFv- CD 16 scFv. In some embodiments, the multispecific binding molecules arrangement is CD 16 scFv- EGFR scFv. In some embodiments, the multispecific binding molecules arrangement is CD 16 sdAb - uPAR (ATF). In some embodiments, the multispecific binding molecules arrangement is uPAR (ATF) - CD16 sdAb.
  • the multispecific binding molecules arrangement is uPAR (ATF) - CD 16 scFv. In some embodiments, the multispecific binding molecules arrangement is CD 16 scFv - uPAR (ATF). In some embodiments, the multispecific binding molecules arrangement is CD 16 sdAb - NKG2D. In some embodiments, the multispecific binding molecules arrangement is NKG2D - CD16 sdAb. In some embodiments, the multispecific binding molecules arrangement is NKG2D - CD 16 scFv. In some embodiments, the multispecific binding molecules arrangement is CD 16 scFv - NKG2D. The scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising one polypeptide, wherein the multispecific binding molecules arrangement is arranged, in the order CD3(VH)-CD3(VL) - GPC3 sdAb. In some embodiments, the multispecific binding molecules arrangement is arranged, CD3(VL)-CD3(VH) - GPC3 sdAb. In some embodiments, the multispecific binding molecules arrangement is GPC3 sdAb- CD3(VH)- CD3(VL). In some embodiments, the multispecific binding molecules arrangement is GPC3 sdAb- CD3(VL)-CD3(VH).
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 10 or 30, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • mRNAs provided herein encode multispecific binding molecules comprising one polypeptide, wherein the multispecific binding molecules arrangement is arranged, in the order CD3(VH)-CD3(VL) - GPC3 (VH) - GPC3 (VL). In some embodiments, the multispecific binding molecules arrangement is arranged, in the order CD3(VL)-CD3(VH) - GPC3 (VH) - GPC3 (VL). In some embodiments, the multispecific binding molecules arrangement is arranged, in the order CD3(VH)-CD3(VL) - GPC3 (VL) - GPC3 (VH).
  • the multispecific binding molecules arrangement is arranged, in the order CD3(VL)-CD3(VH) - GPC3 (VL) - GPC3 (VH).
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 11, or 27, or a fragment thereof or a variant of said amino acid sequence or fragment. In some embodiments, the VL sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 12, or 28, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • mRNAs provided herein encode multispecific binding molecules comprising one polypeptide, wherein the multispecific binding molecules arrangement is arranged, in the order GPC3 (VH)- GPC3 (VL) - CD3 (VH) - CD3 (VL). In some embodiments, the multispecific binding molecules arrangement is arranged, in the order GPC3 (VL)- GPC3(VH) - CD3 (VH) - CD3 (VL). In some embodiments, the multispecific binding molecules arrangement is arranged, in the order GPC3 (VH)- GPC3 (VL) - CD3 (VL) - CD3 (VH).
  • the multispecific binding molecules arrangement is arranged, in the order GPC3 (VL)- GPC3 (VH) - CD3 (VL) - CD3 (VH).
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 11, or 27, or a fragment thereof or a variant of said amino acid sequence or fragment. In some embodiments, the VL sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 12, or 28, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • mRNAs provided herein encode multispecific binding molecules comprising one polypeptide, wherein the multispecific binding molecules arrangement is arranged, in the order CD3(VH)-CD3(VL) - EpCAM sdAb. In some embodiments, the multispecific binding molecules arrangement is arranged, CD3(VL)-CD3(VH) - EpCAM sdAb. In some embodiments, the multispecific binding molecules arrangement is EpCAM sdAb- CD3(VH)-CD3(VL). In some embodiments, the multispecific binding molecules arrangement is EpCAM sdAb- CD3(VL)-CD3(VH).
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of EpCAM binding domain comprising amino acid sequence of SEQ ID NO: 41, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • mRNAs provided herein encode multispecific binding molecules comprising one polypeptide, wherein the multispecific binding molecules arrangement is arranged, in the order CD3(VH)-CD3(VL) - EpCAM (VH) - EpCAM (VL). In some embodiments, the multispecific binding molecules arrangement is arranged, in the order CD3(VL)-CD3(VH) - EpCAM (VH) - EpCAM (VL). In some embodiments, the multispecific binding molecules arrangement is arranged, in the order CD3(VH)-CD3(VL) - EpCAM (VL) - EpCAM (VH).
  • the multispecific binding molecules arrangement is arranged, in the order CD3(VL)-CD3(VH) - EpCAM (VL) - EpCAM (VH).
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 4, or 14, or a fragment thereof or a variant of said amino acid sequence or fragment. In some embodiments, the VL sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 2, or 13, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • mRNAs provided herein encode multispecific binding molecules comprising one polypeptide, wherein the multispecific binding molecules arrangement is arranged, in the order EpCAM (VH)- EpCAM (VL) - CD3 (VH) - CD3 (VL). In some embodiments, the multispecific binding molecules arrangement is arranged, in the order EpCAM (VL)- EpCAM (VH) - CD3 (VH) - CD3 (VL). In some embodiments, the multispecific binding molecules arrangement is arranged, in the order EpCAM (VH)- EpCAM (VL) - CD3 (VL) - CD3 (VH).
  • the multispecific binding molecules arrangement is arranged, in the order EpCAM (VL)- EpCAM (VH) - CD3 (VL) - CD3 (VH).
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 4, or 14, or a fragment thereof or a variant of said amino acid sequence or fragment. In some embodiments, the VL sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 2, or 13, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • mRNAs provided herein encode multispecific binding molecules comprising one polypeptide, wherein the multispecific binding molecules arrangement is arranged, in the order CD3(VH)-CD3(VL) - EGFR sdAb. In some embodiments, the multispecific binding molecules arrangement is arranged, CD3(VL)-CD3(VH) - EGFR sdAb. In some embodiments, the multispecific binding molecules arrangement is EGFR sdAb- CD3(VH)- CD3(VL). In some embodiments, the multispecific binding molecules arrangement is EGFR sdAb- CD3(VL)-CD3(VH).
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of EGFR binding domain comprises amino acid sequence of SEQ ID NO: 50, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • mRNAs provided herein encode multispecific binding molecules comprising one polypeptide, wherein the multispecific binding molecules arrangement is arranged, in the order CD3(VH)-CD3(VL) - uPAR (ATF). In some embodiments, the multispecific binding molecules arrangement is arranged, CD3(VL)-CD3(VH) - uPAR (ATF). In some embodiments, the multispecific binding molecules arrangement is uPAR (ATF)- CD3(VH)-CD3(VL). In some embodiments, the multispecific binding molecules arrangement is uPAR (ATF)- CD3(VL)-CD3(VH).
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of uPAR (ATF) comprises amino acid sequence of SEQ ID NO: 17, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • mRNAs provided herein encode multispecific binding molecules comprising one polypeptide, wherein the multispecific binding molecules arrangement is arranged, in the order CD3(VH)-CD3(VL) - NKG2D. In some embodiments, the multispecific binding molecules arrangement is arranged, CD3(VL)-CD3(VH) - NKG2D. In some embodiments, the multispecific binding molecules arrangement is NKG2D - CD3(VH)- CD3(VL). In some embodiments, the multispecific binding molecules arrangement is NKG2D - CD3(VL)-CD3(VH).
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of NKG2D comprises amino acid sequence of SEQ ID NO: 24, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6, and a VL region comprising the amino acid sequence of SEQ ID NO: 7, and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6, and a VL region comprising the amino acid sequence of SEQ ID NO: 7, and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 30.
  • the GPC3 binding domain can be at N- terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15, and a VL region comprising the amino acid sequence of SEQ ID NO: 16, and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15, and a VL region comprising the amino acid sequence of SEQ ID NO: 16, and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 30.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21, and a VL region comprising the amino acid sequence of SEQ ID NO: 22, and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21, and a VL region comprising the amino acid sequence of SEQ ID NO: 22, and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 30.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 35, and a VL region comprising the amino acid sequence of SEQ ID NO: 36, and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 35, and a VL region comprising the amino acid sequence of SEQ ID NO: 36, and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 30.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 48, and a VL region comprising the amino acid sequence of SEQ ID NO: 49, and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 48, and a VL region comprising the amino acid sequence of SEQ ID NO: 49, and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 30.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6, and a VL region comprising the amino acid sequence of SEQ ID NO: 7, and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6, and a VL region comprising the amino acid sequence of SEQ ID NO: 7, and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 27, and a VL region comprising the amino acid sequence of SEQ ID NO: 28.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15, and a VL region comprising the amino acid sequence of SEQ ID NO: 16, and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15, and a VL region comprising the amino acid sequence of SEQ ID NO: 16, and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 27, and a VL region comprising the amino acid sequence of SEQ ID NO: 28.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21, and a VL region comprising the amino acid sequence of SEQ ID NO: 22, and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21, and a VL region comprising the amino acid sequence of SEQ ID NO: 22, and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 27, and a VL region comprising the amino acid sequence of SEQ ID NO: 28.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 35, and a VL region comprising the amino acid sequence of SEQ ID NO: 36, and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 35, and a VL region comprising the amino acid sequence of SEQ ID NO: 36, and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 27, and a VL region comprising the amino acid sequence of SEQ ID NO: 28.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 48, and a VL region comprising the amino acid sequence of SEQ ID NO: 49, and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 48, and a VL region comprising the amino acid sequence of SEQ ID NO: 49, and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 27, and a VL region comprising the amino acid sequence of SEQ ID NO: 28.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6, and a VL region comprising the amino acid sequence of SEQ ID NO: 7, and (ii) an EpCAM binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 41.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N- terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15, and a VL region comprising the amino acid sequence of SEQ ID NO: 16, and (ii) an EpCAM binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 41.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N- terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21, and a VL region comprising the amino acid sequence of SEQ ID NO: 22, and (ii) an EpCAM binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 41.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N- terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 35, and a VL region comprising the amino acid sequence of SEQ ID NO: 36, and (ii) an EpCAM binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 41.
  • the EpCAM binding domain can be atN-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N- terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 48, and a VL region comprising the amino acid sequence of SEQ ID NO: 49, and (ii) an EpCAM binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 41.
  • the EpCAM binding domain can be atN-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N- terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6, and a VL region comprising the amino acid sequence of SEQ ID NO: 7, and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6, and a VL region comprising the amino acid sequence of SEQ ID NO: 7, and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15, and a VL region comprising the amino acid sequence of SEQ ID NO: 16, and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15, and a VL region comprising the amino acid sequence of SEQ ID NO: 16, and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21, and a VL region comprising the amino acid sequence of SEQ ID NO: 22, and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21, and a VL region comprising the amino acid sequence of SEQ ID NO: 22, and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 35, and a VL region comprising the amino acid sequence of SEQ ID NO: 36, and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 35, and a VL region comprising the amino acid sequence of SEQ ID NO: 36, and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 48, and a VL region comprising the amino acid sequence of SEQ ID NO: 49, and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 48, and a VL region comprising the amino acid sequence of SEQ ID NO: 49, and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6, and a VL region comprising the amino acid sequence of SEQ ID NO: 7, and (ii) an EGFR binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 50.
  • the EGFR binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C- terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15, and a VL region comprising the amino acid sequence of SEQ ID NO: 16, and (ii) an EGFR binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 50.
  • the EGFR binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N- terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21, and a VL region comprising the amino acid sequence of SEQ ID NO: 22, and (ii) an EGFR binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 50.
  • the EGFR binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N- terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 35, and a VL region comprising the amino acid sequence of SEQ ID NO: 36, and (ii) an EGFR binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 50.
  • the EGFR binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N- terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 48, and a VL region comprising the amino acid sequence of SEQ ID NO: 49, and (ii) an EGFR binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 50.
  • the EGFR binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N- terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6, and a VL region comprising the amino acid sequence of SEQ ID NO: 7, and (ii) an uPAR binding domain comprising the amino acid sequence of SEQ ID NO: 17.
  • the uPAR binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15, and a VL region comprising the amino acid sequence of SEQ ID NO: 16, and (ii) an uPAR binding domain comprising the amino acid sequence of SEQ ID NO: 17.
  • the uPAR binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide
  • the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21, and a VL region comprising the amino acid sequence of SEQ ID NO: 22, and (ii) an uPAR binding domain comprising the amino acid sequence of SEQ ID NO: 17.
  • the uPAR binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 35, and a VL region comprising the amino acid sequence of SEQ ID NO: 36, and (ii) an uPAR binding domain comprising the amino acid sequence of SEQ ID NO: 17.
  • the uPAR binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule provided herein comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 48, and a VL region comprising the amino acid sequence of SEQ ID NO: 49, and (ii) an uPAR binding domain comprising the amino acid sequence of SEQ ID NO: 17.
  • the uPAR binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNA provided herein encodes a multispecific binding molecule comprising one polypeptide, wherein the multispecific binding molecule comprises (i) a CD 16 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 39, and (ii) an EpCAM binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 41.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD 16 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • a long linker connects the corresponding variable heavy chain regions (VH) and the corresponding variable light chain regions (VL) to create a scFv binding domain, while a short linker generally connects two scFv binding domains, or two sdAbs, or a scFv and a sdAb.
  • the linker is generally designed to provide flexibility and protease resistance, and preferably, the linker comprises glycine and/or serine amino acid residues.
  • Short peptide linkers may consist of 12 or less such as 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acids, and preferably, 5 or 6 amino acids.
  • Short peptide linkers preferably comprise the amino acid sequences SGGGGS (SEQ ID NO:5), SSGGGGS (SEQ ID NO:31), GGGGSGGG (SEQ ID NO:34), GGGGSGGGSGGG (SEQ ID NO:38), GGSGGGGS (SEQ ID NO:40), GGGGGSGGGSGGG (SEQ ID NO:42), GGGGGSGGG (SEQ ID NO:47), or GGGGS (SEQ ID NO: 115).
  • Long peptide linkers may consist of 12 or more, such as 15 to 25 or 15 to 20 or 15 to 18 amino acids.
  • Long peptide linkers preferably comprise the amino acid sequences (GGGGS)3 (SEQ ID NO:37), S(GGGGS)3 (SEQ ID NO:29) or VE(GGSGGS)2GGVD (SEQ ID NO:23). Further long peptide linkers may comprise the amino acid sequences (GGGGS)4 (SEQ ID NO:26), (GGGGS)5 (SEQ ID NO:3) or GGGGS(GGS)3GGGS (SEQ ID NO: 116).
  • the multispecific binding molecules encoded by mRNAs provided herein further comprise secretion signals such as N-terminal secretion signals.
  • the signal peptide can direct the target cell to secrete the polypeptide.
  • the sequence of secretion signal is selected from the group consisting of SEQ ID NOs: 1, 43, 44, 45, 46 or 51.
  • the multispecific binding molecules encoded by mRNAs provided herein further comprise C-terminal His-tags, in particular the sequence Gly - Gly - Ser - (His)6 (SEQ ID NO: 117), (His)6 (SEQ ID NO:9), or VSGWRLFKKIS (SEQ ID NO:33) if present.
  • the mRNA(s) provided herein encoding multispecific binding molecules comprising two polypeptides; wherein the first polypeptide comprises a first antigen binding domain comprising a VH region binding to the first antigen; and a second antigen binding domain comprising a second VH region and a second VL region, and/or a single domain antigen binding fragment binding to the second antigen; and the second polypeptide comprises a first antigen binding domain comprising a VL region binding to the first antigen; and a third antigen binding domain comprising a third VH region and a third VL region, and/or a single domain antigen binding fragment binding to the third antigen.
  • the second binding domain and the third binding domain bind to a same antigen. In some embodiments, the second binding domain and the third binding domain bind to two different antigens. In some embodiments, the second binding domain and the third binding domain bind to two epitopes on the same antigen. In some embodiments, the first antigen present on immune effector cell(s). In some embodiments, the immune effector cells are peripheral blood mononuclear cells (PBMCs). In some embodiments, the immune effector cells are T cells. In some embodiments, the T cells are Pan T cells. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the immune effector cells are NK cells. In some embodiments, the immune effector cells are macrophages.
  • PBMCs peripheral blood mononuclear cells
  • the immune effector cells are T cells. In some embodiments, the T cells are Pan T cells. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the immune effector cells are NK cells. In some
  • the immune effector cells are tissue-resident macrohpages, such as Kupffer cells or peritoneal, lung, splenic, or bone marrow resident macrophages.
  • the first antigen is CD3.
  • the first antigen is CD 16.
  • the second antigen is GPC3.
  • the second antigen is EpCAM.
  • the second antigen is uPAR.
  • the second antigen is NKG2DL.
  • the second antigen is EGFR.
  • the third antigen is EpCAM. In some embodiments, the third antigen is uPAR. In some embodiments, the third antigen is NKG2DL. In some embodiments, the third antigen is EGFR. [00438] In some embodiments, the two polypeptides are encoded by a single mRNA. In some embodiments, the two polypeptides are separated by a 2A cleavable linker, such as a T2A, P2A, E2A, or F2A linker. In some embodiments, the two polypeptides are separated by an internal ribosome entry site (IRES).
  • IRS internal ribosome entry site
  • the two polypeptides are encoded by separate mRNAs.
  • the mRNAs encoding the two polypeptides are administered together.
  • the mRNAs encoding the two polypeptides are administered separately.
  • the mRNAs encoding the two polypeptides may be delivered via a single delivery vehicle.
  • the mRNAs encoding the two polypeptides may be delivered via separate delivery vehicles.
  • the mRNAs encoding the two polypeptides may be packaged separately but delivered simultaneously. Alternatively, the mRNAs encoding the two polypeptides may be packaged separately and delivered sequentially.
  • RNA sequences described herein can be converted to DNA sequences by replacing the "U” (uridine) with a "T” (thymidine).
  • the first polypeptide comprises a VH region binding to the first antigen and a Fab fragment binding to the second antigen; and the second polypeptide comprises a VL region binding to the first antigen and a Fab fragment binding to the third antigen.
  • the second binding domain and the third binding domain bind to a same antigen.
  • the second binding domain and the third binding domain bind to two different antigens.
  • the second binding domain and the third binding domain bind to two epitopes on the same antigen.
  • the first polypeptide comprises a VH region binding to the first antigen and a Fab fragment binding to the second antigen; and the second polypeptide comprises a VL region binding to the first antigen and a scFv fragment binding to the third antigen.
  • the second binding domain and the third binding domain bind to a same antigen.
  • the second binding domain and the third binding domain bind to two different antigens.
  • the second binding domain and the third binding domain bind to two epitopes on the same antigen.
  • the first polypeptide comprises a VH region binding to the first antigen and a Fab fragment binding to the second antigen; and the second polypeptide comprises a VL region binding to the first antigen and a single domain antigen binding fragment binding to the third antigen.
  • the second binding domain and the third binding domain bind to a same antigen.
  • the second binding domain and the third binding domain bind to two different antigens.
  • the second binding domain and the third binding domain bind to two epitopes on the same antigen.
  • the first polypeptide comprises a VH region binding to the first antigen and a scFv fragment binding to the second antigen; and the second polypeptide comprises a VL region binding to the first antigen and a Fab fragment binding to the third antigen.
  • the second binding domain and the third binding domain bind to a same antigen.
  • the second binding domain and the third binding domain bind to two different antigens.
  • the second binding domain and the third binding domain bind to two epitopes on the same antigen.
  • the first polypeptide comprises a VH region binding to the first antigen and a scFv fragment binding to the second antigen; and the second polypeptide comprises a VL region binding to the first antigen and a scFv fragment binding to the third antigen.
  • the second binding domain and the third binding domain bind to a same antigen.
  • the second binding domain and the third binding domain bind to two different antigens.
  • the second binding domain and the third binding domain bind to two epitopes on the same antigen.
  • the first polypeptide comprises a VH region binding to the first antigen and a scFv fragment binding to the second antigen; and the second polypeptide comprises a VL region binding to the first antigen and a single domain antigen binding fragment binding to the third antigen.
  • the second binding domain and the third binding domain bind to a same antigen.
  • the second binding domain and the third binding domain bind to two different antigens.
  • the second binding domain and the third binding domain bind to two epitopes on the same antigen.
  • the first polypeptide comprises a VH region binding to the first antigen and a single domain antigen binding fragment binding to the second antigen; and the second polypeptide comprises a VL region binding to the first antigen and a Fab fragment binding to the third antigen.
  • the second binding domain and the third binding domain bind to a same antigen.
  • the second binding domain and the third binding domain bind to two different antigens.
  • the second binding domain and the third binding domain bind to two epitopes on the same antigen.
  • the first polypeptide comprises a VH region binding to the first antigen and a single domain antigen binding fragment binding to the second antigen; and the second polypeptide comprises a VL region binding to the first antigen and a scFv fragment binding to the third antigen.
  • the second binding domain and the third binding domain bind to a same antigen.
  • the second binding domain and the third binding domain bind to two different antigens.
  • the second binding domain and the third binding domain bind to two epitopes on the same antigen.
  • the first polypeptide comprises a VH region binding to the first antigen and a single domain antigen binding fragment binding to the second antigen; and the second polypeptide comprises a VL region binding to the first antigen and a single domain antigen binding fragment binding to the third antigen.
  • the second binding domain and the third binding domain bind to a same antigen.
  • the second binding domain and the third binding domain bind to two different antigens.
  • the second binding domain and the third binding domain bind to two epitopes on the same antigen.
  • the first binding domain can be at N-terminus or C-terminus of the second binding domain.
  • the first binding domain can be at N-terminus or C-terminus of the third binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide arrangement is GPC3 sdAb-CD3 VH, and the second polypeptide arrangement is GPC3 sdAb-CD3 VL.
  • the first polypeptide arrangement is CD3 VH- GPC3 sdAb
  • the second polypeptide arrangement is CD3 VL- GPC3 sdAb.
  • the GPC3 binding domain can be at N- terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 10 or 30, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide arrangement is GPC3 scFv-CD3 VH, and the second polypeptide arrangement is GPC3 scFv -CD3 VL.
  • the first polypeptide arrangement is CD3 VH- GPC3 scFv
  • the second polypeptide arrangement is CD3 VL- GPC3 scFv.
  • the scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 11, or 27, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 12, or 28, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide arrangement is GPC3 sdAb-CD3 VH, and the second polypeptide arrangement is GPC3 scFv-CD3 VL.
  • the first polypeptide arrangement is CD3 VH- GPC3 sdAb
  • the second polypeptide arrangement is CD3 VL- GPC3 scFv.
  • the scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 10 or 30, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 11, or 27, or a fragment thereof or a variant of said amino acid sequence or fragment. In some embodiments, the VL sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 12, or 28, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide arrangement is GPC3 scFv-CD3 VH, and the second polypeptide arrangement is GPC3 sdAb -CD3 VL.
  • the first polypeptide arrangement is CD3 VH- GPC3 scFv
  • the second polypeptide arrangement is CD3 VL- GPC3 sdAb.
  • the scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 10 or 30, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 11, or 27, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 12, or 28, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide arrangement is EpCAM sdAb-CD3 VH, and the second polypeptide arrangement is EpCAM sdAb-CD3 VL.
  • the first polypeptide arrangement is CD3 VH- EpCAM sdAb
  • the second polypeptide arrangement is CD3 VL- EpCAM sdAb.
  • the EpCAM binding domain can be at N- terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of EpCAM binding domain comprises amino acid sequence of SEQ ID NO: 41, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide arrangement is EpCAM scFv-CD3 VH, and the second polypeptide arrangement is EpCAM scFv -CD3 VL.
  • the first polypeptide arrangement is CD3 VH- EpCAM scFv
  • the second polypeptide arrangement is CD3 VL- EpCAM scFv.
  • the scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 4, or 14, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 2, or 13, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide arrangement is EpCAM sdAb-CD3 VH, and the second polypeptide arrangement is EpCAM scFv -CD3 VL.
  • the first polypeptide arrangement is CD3 VH- EpCAM sdAb
  • the second polypeptide arrangement is CD3 VL- EpCAM scFv.
  • the scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of EpCAM binding domain comprises amino acid sequence of SEQ ID NO: 41, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 4, or 14, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 2, or 13, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide arrangement is EpCAM scFv-CD3 VH, and the second polypeptide arrangement is EpCAM sdAb -CD3 VL.
  • the first polypeptide arrangement is CD3 VH- EpCAM scFv
  • the second polypeptide arrangement is CD3 VL- EpCAM sdAb.
  • the scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of EpCAM binding domain comprises amino acid sequence of SEQ ID NO: 41, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 4, or 14, or a fragment thereof or a variant of said amino acid sequence or fragment. In some embodiments, the VL sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 2, or 13, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide arrangement is EpCAM sdAb-CD3 VH, and the second polypeptide arrangement is GPC3 sdAb-CD3 VL.
  • the first polypeptide arrangement is CD3 VH- EpCAM sdAb
  • the second polypeptide arrangement is CD3 VL- GPC3 sdAb.
  • the EpCAM binding domain can be at N- terminus or C-terminus of the CD3 VH or VL binding domain.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs:
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 10 or 30, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of EpCAM binding domain comprises amino acid sequence of SEQ ID NO: 41, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide arrangement is GPC3 sdAb-CD3 VH, and the second polypeptide arrangement is EpCAM sdAb-CD3 VL.
  • the first polypeptide arrangement is CD3 VH- GPC3 sdAb
  • the second polypeptide arrangement is CD3 VL- EpCAM sdAb.
  • the EpCAM binding domain can be at N- terminus or C-terminus of the CD3 VH or VL binding domain.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs:
  • sequence of single domain antibody region (such as a VHH region) of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 10 or 30, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • sequence of single domain antibody region (such as a VHH region) of EpCAM binding domain comprises amino acid sequence of SEQ ID NO: 41, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide arrangement is EpCAM scFv-CD3 VH, and the second polypeptide arrangement is GPC3 scFv -CD3 VL.
  • the first polypeptide arrangement is CD3 VH- EpCAM scFv
  • the second polypeptide arrangement is CD3 VL- GPC3 scFv.
  • the scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 11, or 27, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 12, or 28, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 4, or 14, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 2, or 13, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide arrangement is GPC3 scFv-CD3 VH, and the second polypeptide arrangement is EpCAM scFv -CD3 VL.
  • the first polypeptide arrangement is CD3 VH- GPC3 scFv
  • the second polypeptide arrangement is CD3 VL- EpCAM scFv.
  • the scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 11, or 27, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 12, or 28, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 4, or 14, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 2, or 13, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide arrangement is EpCAM sdAb-CD3 VH, and the second polypeptide arrangement is GPC3 scFv -CD3 VL.
  • the first polypeptide arrangement is CD3 VH- EpCAM sdAb
  • the second polypeptide arrangement is CD3 VL- GPC3 scFv.
  • the scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 11, or 27, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 12, or 28, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of EpCAM binding domain comprises amino acid sequence of SEQ ID NO: 41, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide arrangement is GPC3 sdAb-CD3 VH, and the second polypeptide arrangement is EpCAM scFv -CD3 VL.
  • the first polypeptide arrangement is CD3 VH- GPC3 sdAb
  • the second polypeptide arrangement is CD3 VL- EpCAM scFv.
  • the scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 4, or 14, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 2, or 13, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 10 or 30, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide arrangement is EpCAM scFv-CD3 VH, and the second polypeptide arrangement is GPC3 sdAb -CD3 VL.
  • the first polypeptide arrangement is CD3 VH- EpCAM scFv
  • the second polypeptide arrangement is CD3 VL- GPC3 sdAb.
  • the scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 4, or 14, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of EpCAM binding domain is selected from the group consisting of SEQ ID NOs: 2, or 13, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 10 or 30, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide arrangement is GPC3 scFv-CD3 VH, and the second polypeptide arrangement is EpCAM sdAb -CD3 VL.
  • the first polypeptide arrangement is CD3 VH- GPC3 scFv
  • the second polypeptide arrangement is CD3 VL- EpCAM sdAb.
  • the scFv comprises a VH region and a VL region, the VH region can be at N-terminus or C-terminus of the VL region.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of EpCAM binding domain comprises amino acid sequence of SEQ ID NO: 41, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VH sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 11, or 27, or a fragment thereof or a variant of said amino acid sequence or fragment. In some embodiments, the VL sequence of GPC3 binding domain is selected from the group consisting of SEQ ID NOs: 12, or 28, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide arrangement is EGFR sdAb-CD3 VH, and the second polypeptide arrangement is EGFR sdAb-CD3 VL.
  • the first polypeptide arrangement is CD3 VH- EGFR sdAb
  • the second polypeptide arrangement is CD3 VL- EGFR sdAb.
  • the EGFR binding domain can be at N- terminus or C-terminus of the CD3 VH or VL binding domain.
  • the VH sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 6, 15, 21, 35, or 48, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the VL sequence of CD3 binding domain is selected from the group consisting of SEQ ID NOs: 7, 16, 22, 36, or 49, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • the sequence of single domain antibody region (such as a VHH region) of EGFR binding domain comprises amino acid sequence of SEQ ID NO: 50, or a fragment thereof or a variant of said amino acid sequence or fragment.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the GPC3 binding domain can be at N- terminus or C-terminus of the CD3 binding domain.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the GPC3 binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N- terminus or C-terminus of
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 35 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 28, and a VL region comprising the amino acid sequence of SEQ ID NO: 29; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 36 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 28, and a VL region comprising the amino acid sequence of SEQ ID NO:
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 35 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 36 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C- terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the GPC3 binding domain can be atN-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C- terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the GPC3 binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the GPC3 binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 6 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 7 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the GPC3 binding domain can be at N- terminus or C-terminus of the CD3 binding domain.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the GPC3 binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N- terminus or C-terminus of
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C- terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the GPC3 binding domain can be atN-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C- terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the GPC3 binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the GPC3 binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 16 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the GPC3 binding domain can be at N- terminus or C-terminus of the CD3 binding domain.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the GPC3 binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N- terminus or C-terminus of
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C- terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the GPC3 binding domain can be atN-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) a GPC3 binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 10.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C- terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2.
  • the GPC3 binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 4, and a VL region comprising the amino acid sequence of SEQ ID NO: 2; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13.
  • the GPC3 binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C- terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 21 and (ii) an EpCAM binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 14, and a VL region comprising the amino acid sequence of SEQ ID NO: 13; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 22 and (ii) a GPC3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 11, and a VL region comprising the amino acid sequence of SEQ ID NO: 12.
  • the GPC3 binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the EpCAM binding domain can be at N-terminus or C-terminus of the CD3 binding domain.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • the VH region can be at N-terminus or C-terminus of the VL region.
  • mRNAs provided herein encode multispecific binding molecules comprising two polypeptides, wherein the first polypeptide comprises (i) a CD3 binding domain comprising a VH region comprising the amino acid sequence of SEQ ID NO: 35 and (ii) an EGFR binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 50; the second polypeptide comprises (i) a CD3 binding domain comprising a VL region comprising the amino acid sequence of SEQ ID NO: 36 and (ii) an EGFR binding domain comprising a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 50.
  • the EGFR binding domain can be at N- terminus or C-terminus of the CD3 binding domain.
  • a long linker connects the corresponding variable heavy chain regions (VH) and the corresponding variable light chain regions (VL) to create a scFv binding domain while a short linker generally connects two scFv binding domains, or two sdAbs, or a scFv and a sdAb.
  • the linker is generally designed to provide flexibility and protease resistance, and preferably, the linker comprises glycine and/or serine amino acid residues.
  • Short peptide linkers may consist of 13 or less such as 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acids, and preferably, 5 or 6 amino acids.
  • Short peptide linkers preferably comprise the amino acid sequences SGGGGS (SEQ ID NO:5), SSGGGGS (SEQ ID NO:31), GGGGSGGG (SEQ ID NO:34), GGGGSGGGSGGG (SEQ ID NO:38), GGSGGGGS (SEQ ID NO:40), GGGGGSGGGSGGG (SEQ ID NO:42), GGGGGSGGG (SEQ ID NO:47), or GGGGS (SEQ ID NO: 115).
  • Long peptide linkers may consist of 12 or more, such as 15 to 25 or 15 to 20 or 15 to 18 amino acids.
  • Long peptide linkers preferably comprise the amino acid sequences (GGGGS)3 (SEQ ID NO:37), S(GGGGS)3 (SEQ ID NO:29) or VE(GGSGGS)2GGVD (SEQ ID NO:23). Further long peptide linkers may comprise the amino acid sequences (GGGGS)4 (SEQ ID NO:26), (GGGGS)5 (SEQ ID NO:3) or GGGGS(GGS)3GGGS (SEQ ID NO: 116).
  • the multispecific binding molecules encoded by mRNAs provided herein further comprise secretion signals such as N-terminal secretion signals.
  • the signal peptide can direct the target cell to secrete the polypeptide.
  • the sequence of secretion signal is selected from the group consisting of SEQ ID NOs: 1, 43, 44, 45, 46 or 51.
  • the multispecific binding molecules encoded by mRNAs provided herein further comprise C-terminal His-tags, in particular the sequence Gly - Gly - Ser - (His)6 (SEQ ID NO: 117), (His)6 (SEQ ID NO:9), or VSGWRLFKKIS (SEQ ID NO:33) if present.
  • the mRNAs encoding a multispecific binding molecule when administered to a patient may be present in naked form or in a suitable delivery vehicle such as in the form of lipid nanoparticles, liposomes or viral particles, or within a host cell.
  • the nucleic acid provided can produce the multispecific binding molecules over extended time periods in a sustained manner mitigating the instability at least partially observed for therapeutic antibodies, in particular bispecific antibodies.
  • the delivery vehicle is a lipid vehicle.
  • the delivery vehicle is a lipid nanoparticle.
  • the lipid nanoparticle comprises a cationic or ionizable lipid.
  • the lipid nanoparticle comprises an ionizable lipid and one or more non-cationic lipids.
  • said noncationic lipids comprise a phospholipid and cholesterol.
  • said delivery vehicle comprises a polyethylene glycol conjugate such as DMG-PEG.
  • said delivery vehicle is administered intravenously.
  • said delivery vehicle preferentially transfects hepatocytes.
  • the mRNAs encoding multispecific binding molecules provided herein are delivered to a target cell by a lipid nanoparticle with at least one targeting moiety which leads to selective transfection of the target cell.
  • the targeting moiety is cholesterol.
  • the target cell is a liver cancer cell. 5.3.4 Synthesis of mRNA
  • mRNAs according to the present invention may be synthesized according to any of a variety of known methods.
  • mRNAs according to the present invention may be synthesized via in vitro transcription (IVT).
  • IVT in vitro transcription
  • IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and/or RNAse inhibitor.
  • RNA polymerase e.g., T3, T7 or SP6 RNA polymerase
  • a DNA template is transcribed in vitro.
  • a suitable DNA template typically has a promoter, for example a T3, T7 or SP6 promoter, for in vitro transcription, followed by desired nucleotide sequence for desired antibody encoding (e.g., heavy chain or light chain encoding) mRNA and a termination signal.
  • mRNA according to the present invention may be synthesized as unmodified or modified mRNA. In order to increase expression and/or stability of the RNA used according to the present invention, it may be modified, preferably without altering the sequence of the expressed peptide or protein.
  • modification in the context of RNA as used according to the present invention includes any modification of RNA which is not naturally present in said RNA.
  • the RNA used according to the present disclosure does not have uncapped 5 '-triphosphates. Removal of such uncapped 5 '-triphosphates can be achieved by treating RNA with a phosphatase.
  • RNA according to the present disclosure may have modified naturally occurring or synthetic ribonucleotides in order to increase its stability and/or decrease cytotoxicity.
  • uridine is substituted, partially or completely, preferably completely, with one of the nucleotides selected from the group consisting of 5-methoxy uridine, pseudouridine, 5- methyl uridine, N1 -methyl pseudouridine, N1 -ethyl pseudouridine, 5-carboxy methyl ester uridine, 2-thio uridine, 5-hydroxymethyluridine, N1 -propylpseudouridine, 5 -hydroxyuridine, or N1 -methoxymethylpseudouridine.
  • cytidine is substituted, partially or completely, preferably completely, with one of the nucleotides selected from the group consisting of 5-methyl cytidine, 5 -hydroxymethyl cytidine, or 5-methoxy cytidine.
  • both uridine and cytidine are substituted, partially or completely, preferably completely, with one or more of the modified ribonucleotides described herein.
  • uridine is substituted, partially or completely, preferably completely, with two or more of the modified ribonucleotides described herein.
  • cytidine is substituted, partially or completely, preferably completely, with two or more of the modified ribonucleotides described herein.
  • the term “modification” relates to providing an RNA with a 5'- cap or 5 '-cap analog.
  • the term “5 '-cap” refers to a cap structure found on the 5 '-end of an mRNA molecule and generally consists of a guanosine nucleotide connected to the mRNA via an unusual 5' to 5' triphosphate linkage. In one embodiment, this guanosine is methylated at the 7- position.
  • the term “conventional 5 '-cap” refers to a naturally occurring RNA 5 '-cap, preferably to the 7-methylguanosine cap (m7G).
  • the term “5 '-cap” includes a 5 '-cap analog that resembles the RNA cap structure and is modified to possess the ability to stabilize RNA if attached thereto, preferably in vivo and/or in a cell.
  • RNA with a 5 '-cap or 5 '-cap analog may be achieved by in vitro transcription of a DNA template in the presence of said 5 '-cap or 5 '-cap analog, wherein said 5'- cap is co-transcriptionally incorporated into the generated RNA strand, or the RNA may be generated, for example, by in vitro transcription, and the 5 '-cap may be attached to the RNA post-transcriptionally using capping enzymes, for example, capping enzymes of vaccinia virus.
  • the RNA may comprise further modifications.
  • RNA used in the present invention may be an extension or truncation of the naturally occurring poly(A) tail or an alteration of the 5'- or 3 '-untranslated regions (UTR) such as introduction of a UTR which is not related to the coding region of said RNA, for example, the insertion of one or more, preferably two copies of a 3'-UTR derived from a globin gene, such as alpha2-globin, alpha 1 -globin, beta-globin, preferably beta-globin, more preferably human betaglobin.
  • UTR 5'- or 3 '-untranslated regions
  • the RNA used according to the present invention may be modified so as to be present in conjunction with a poly-A sequence, preferably having a length of 10 to 500, more preferably 30 to 300, even more preferably 65 to 200 and especially 70 to 150 adenosine residues.
  • the poly-A sequence has a length of approximately 80 adenosine residues.
  • the poly-A sequence has a length of approximately 120 adenosine residues.
  • UTR 3 '-non translated regions
  • IVT mRNA expression can also be carried out according to standard procedures well known by one skilled in the art. Briefly, the DNA of the constructs are synthesized by GenScript and cloned into the IVT template plasmid (Bratkowski et al. 2020. Cell Reports, 32(5), 107999). Cloning is conducted using the Kpnl and Notl restriction enzyme sites. The 5’ cap of mRNA (m7G(5’)ppp(5’)(2’OMeA)pG) is added co-transcriptionally with CleanCap reagent AG (TriLink Biotechnologies, #N7113). The 80 polyA tail is added using a PCR-based method.
  • Linearized DNA templates are subjected to in vitro transcription using MEGAscript T7 Kit according to the manufacturer's guidelines.
  • Purification of IVT -mRNA is carried out with the MEGAclear Kit according to the manufacturer's guidelines. Concentration and quality of the IVT-RNA are assessed by spectrophotometry using the NanoDrop system. All mRNA is generated with adenine, thymine, cytosine and a modified nucleotide selected from the group consisting of Pseudouridine, N1 -Methylpseudouridine, or 5-Methoxyuridine (TriLink Biotechnologies). mRNA purity is analyzed via Agilent Bioanalyzer High Sensitivity RNA analysis using the manufacturer’s standard protocol.
  • the target cells are tumor cells.
  • mRNAs coding multispecific binding molecules described herein may be delivered, with or without delivery vehicles, to a subject in need of delivery such that fully assembled desired multispecific binding molecules are expressed in vivo.
  • desired multispecific binding molecules are as described in Section 5.2.
  • a method of treating a disease or disorder in a subject comprising administering to a target tissue of the subject mRNA encoding a multispecific binding molecule, wherein the multispecific binding molecule comprises one or more binding domains with affinity for a target cell in the target tissue, and one or more binding domains with affinity for an immune cell.
  • the multispecific binding molecule induces the binding of the target cell and the immune cell, and induces target cell elimination.
  • the immune cell is a peripheral blood mononuclear cell (PBMC).
  • the immune cell is a T cell.
  • the T cell is a Pan T cell.
  • the T cell is a CD8+ T cell.
  • the immune cell is an NK cell. In some embodiments, the immune cell is a macrophage. In some embodiments, the immune cell is a tissue-resident macrophage, such as a Kupffer cell or a peritoneal, lung, splenic, or bone marrow resident macrophage. In some embodiments, the target cell is a tumor cell. In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is lung cancer. In some emodiments, the cancer is skin cancer.
  • the composition comprises one or more mRNA(s) encoding multispecific binding molecule(s) with binding affinity for one or more target cells and one or more immune effector cells as described in Section 5.2 and 5.3, to achieve the selective, immune-cell-mediated elimination of target cells.
  • the immune effector cells are peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • the immune effector cells are T cells.
  • the T cells are Pan T cells.
  • the T cells are CD8+ T cells.
  • the immune effector cells are NK cells.
  • the immune effector cells are macrophages.
  • the immune effector cells are tissueresident macrophages, such as a Kupffer cells or a peritoneal, lung, splenic, or bone marrow resident macrophages.
  • the target cells are tumor cells.
  • the cancer is liver cancer.
  • the cancer is lung cancer.
  • the cancer is skin cancer.
  • a tumor therapeutic for delivery to a target cell comprising mRNA encoding a multispecific binding molecule, wherein the multispecific binding molecule comprises (1) a first binding domain capable of binding to a first antigen present on an immune cell, and (2) a second binding domain capable of binding to a second antigen present on the target cell, wherein the multispecific binding molecule is expressed and secreted by the target target cell, wherein the multispecific binding molecule binds to the second antigen on the target cell in an autocrine-like fashion, and wherein the multispecific binding molecule binds to the first antigen on an immune cell, inducing the immune cell to kill the target cell.
  • the method provided herein could achieve target cell elimination via autocrine-like secretion and binding of multispecific immune cell engagers (multispecific binding molecule with binding affinity for one or more target cells and one or more immune effector cells as described in Section 5.2 and 5.3).
  • the immune cell engagers are expressed at a higher level in the target tissue compared to a non-target tissue of said subject.
  • the method provided herein could reduce or eliminate shed antigen inhibition.
  • the method provided herein could reduce or eliminate on-target off-tumor toxicity.
  • the method provided herein could achieve high degree of cell killing compared with equivalent amounts of antibody treatment.
  • the method provided herein could achieve high local cell surface concentrations of immune engagers.
  • the method provided herein could reduce barriers to tumor penetration compared with traditional antibody therapies.
  • Autocrine-like secretion and binding refers to the secretion by a target cell of a multispecific immune cell engager with a binding domain capable of binding to an antigen present on the surface of the target cell.
  • the immune cell is a peripheral blood mononuclear cell (PBMC).
  • PBMC peripheral blood mononuclear cell
  • the immune cell is a T cell.
  • the T cell is a Pan T cell.
  • the T cell is a CD8+ T cell.
  • the immune cell is an NK cell.
  • the immune cell is a macrophage.
  • the immune cell is a tissue-resident macrophage, such as a Kupffer cell or a peritoneal, lung, splenic, or bone marrow resident macrophage.
  • the target cell is a tumor cell.
  • mRNA-induced autocrine-like secretion and binding of immune engagers eliminates the substantial barriers to tumor penetration which traditional antibody therapies suffer from.
  • Traditional antibody-based therapeutics when delivered as proteins (e.g., intravenously or subcutaneously), often have poor tumor penetrance which can limit efficacy in solid tumors.
  • Antibodies are excluded from tumors in part because of high hydrostatic pressure in tumors which reduces convection (see Thurber et al., 2008, Adv Drug Deliv Rev, 60(12)1421- 1434 ).
  • Antibodies may also be excluded from the center of tumors due to rapid antigen binding and internalization at the outer surface of the tumor (i.e., the binding site barrier effect; see Singh et al., 2020, AAPS J, 22(2), 28 ). These effects prevent entry of traditional antibodies into deeper portions of tumors and can hinder efficacy.
  • mRNA may be delivered to target cells by numerous means which are not hindered by the same physical barriers as traditional antibodies, including a delivery vehicle (e.g., lipid nanoparticle), tissue-targeted delivery vehicle, target-antigen targeted delivery vehicle, and/or direct (e.g., intratumoral) injection.
  • a delivery vehicle e.g., lipid nanoparticle
  • tissue-targeted delivery vehicle e.g., lipid nanoparticle
  • target-antigen targeted delivery vehicle e.g., intratumoral
  • the immune cell is a peripheral blood mononuclear cell (PBMC).
  • PBMC peripheral blood mononuclear cell
  • the immune cell is a T cell.
  • the T cell is a Pan T cell.
  • the T cell is a CD8+ T cell.
  • the immune cell is an NK cell.
  • the immune cell is a macrophage.
  • the immune cell is a tissue-resident macrophage, such as a Kupffer cell or a peritoneal, lung, splenic, or bone marrow resident macrophage.
  • the target cell is a tumor cell.
  • the multispecific binding molecule provided herein includes multispecific binding molecule described in section 5.2.
  • the mRNA encoding multispecific binding molecule provided herein includes mRNA described in section 5.3.
  • Target cells e.g., cancer cells
  • other cells may secrete or “shed” target antigen molecules and/or other molecules with similar structure or antigen epitopes.
  • Target antigen molecules may be shed due to the activity of, for example, matrix melatoproteasis in the tumor microenvironment (as may be the case in uPAR or NKG2DL shedding).
  • Target antigen molecules may be shed due to an inherent function of the target antigen (as may be the case in uPAR or GPC3 shedding).
  • This soluble antigen is capable of binding to immunotherapy constructs such as CAR-T cells, systemically administered bispecific antibodies, or other systemically administered antibodies, antibody-drug conjugates, antibody-based immunotoxins, etc.
  • target binding domains of an immunotherapy are bound to shed antigen, binding to the target antigen on the target cell surface is inhibited, thus decreasing or eliminating target cell killing.
  • the methods disclosed herein using mRNA-encoded multispecific binding molecules are demonstrated to exhibit reduced or negligible inhibition when in the presence of shed antigen.
  • the immune cell is a peripheral blood mononuclear cell (PBMC).
  • PBMC peripheral blood mononuclear cell
  • the immune cell is a T cell.
  • the T cell is a Pan T cell.
  • the T cell is a CD8+ T cell.
  • the immune cell is an NK cell.
  • the immune cell is a macrophage.
  • the immune cell is a tissue-resident macrophage, such as a Kupffer cell or a peritoneal, lung, splenic, or bone marrow resident macrophage.
  • the target cell is a tumor cell.
  • the multispecific binding molecule provided herein includes multispecific binding molecule described in section 5.2.
  • the mRNA encoding multispecific binding molecule provided herein includes mRNA described in section 5.3.
  • target cell killing By transfecting a target tissue with mRNA encoding multispecific binding molecules, an increased level of target cell killing with a decreased level of total systemic antibody exposure may be achieved.
  • the high local concentrations and low distant concentrations of multispecific antibody attainable by the methods disclosed herein are illustrated in FIG. 17.
  • target cells e.g., cancer cells
  • target cell killing is achieved with significantly lower concentrations of protein than with exogenously produced protein.
  • the result is target cell elimination with lower overall systemic immune cell engager concentrations (as compared to traditional antibody therapies), thus improving upon both potency and toxicity.
  • the immune cell is a peripheral blood mononuclear cell (PBMC).
  • PBMC peripheral blood mononuclear cell
  • the immune cell is a T cell.
  • the T cell is a Pan T cell.
  • the T cell is a CD8+ T cell.
  • the immune cell is an NK cell.
  • the immune cell is a macrophage.
  • the immune cell is a tissue-resident macrophage, such as a Kupffer cell or a peritoneal, lung, splenic, or bone marrow resident macrophage.
  • the target cell is a tumor cell.
  • the multispecific binding molecule provided herein includes multispecific binding molecule described in section 5.2.
  • the mRNA encoding multispecific binding molecule provided herein includes mRNA described in section 5.3. [00551]
  • the method provided herein could achieve high local cell surface concentrations of immune engagers.
  • the concentration of secreted molecules at the surface of the target cell is believed to be high relative to the concentration throughout the system (e.g., cell culture well or animal), and in some cases the antibody may in fact be saturating available binding sites.
  • the concentration at the cell surface, and thus the potential number of binding events, is believed to be greatly increased via the method provided herein when compared to the delivery of equivalent amounts of antibody into a system (e.g., cell culture media or animal).
  • the method provided herein is believed to lead to greatly increased cell surface concentrations and thus increased target cell killing, when compared to an equivalent among of systemically administered recombinant antibody.
  • the method of mRNA encoding a multispecific antibody provided herein may reduce total systemic exposure while retaining or increasing the level of target cell killing.
  • the method provided herein could reduce barriers to tumor penetration compared with traditional antibody therapies.
  • mRNA-induced autocrine-like secretion and binding of immune engagers reduces and/or eliminates the substantial barriers to tumor penetration which traditional antibody therapies suffer from.
  • Traditional antibody-based therapeutics when delivered as proteins (e.g., intravenously or subcutaneously), often have poor tumor penetrance which can limit efficacy in solid tumors.
  • Antibodies are excluded from tumors in part because of high hydrostatic pressure in tumors which reduces convection. Antibodies may also be excluded from the center of tumors due to rapid antigen binding and internalization at the outer surface of the tumor (i.e., the binding site barrier effect).
  • mRNA may be delivered to target cells by numerous means which are not hindered by the same physical barriers as traditional antibodies, including a delivery vehicle (e.g., lipid nanoparticle), tissue- targeted delivery vehicle, target-antigen targeted delivery vehicle, and/or direct (e.g., intratumoral) injection.
  • a delivery vehicle e.g., lipid nanoparticle
  • tissue- targeted delivery vehicle e.g., lipid nanoparticle
  • target-antigen targeted delivery vehicle e.g., intratumoral
  • the expression of the multispecific binding molecule is greater in the target tissue compared to non-target tissue of the subject.
  • the target cell is transfected with mRNA encoding the multispecific binding molecule, and the target cell expresses and secretes the multispecific binding molecule, and the multispecific binding molecule binds to the target cell and the immune cell.
  • a nontarget cell within the target tissue is transfected with mRNA encoding the multispecific binding molecule, and the non-target cell expresses and secretes the multispecific binding molecule, and the multispecific binding molecule binds to the target cell and the immune cell.
  • the expression of the multispecific binding molecule at least 5 times greater in the target tissue compared to non-target tissue. In some embodiments, the expression of the multispecific binding molecule at least 10 times greater in the target tissue compared to non-target tissue. In some embodiments, the expression of the multispecific binding molecule at least 100 times greater in the target tissue compared to non-target tissue.
  • target cell refers to a cell or tissue to which antibody encoding mRNA(s) is to be directed or targeted.
  • the hepatocyte represents the target cell.
  • mRNAs encoding multispecific binding molecule described herein may be delivered to a variety of target cells or tissues.
  • Target cells include, but not limited to cancer cell, senescent cell, activated fibroblast, tumor-associated fibroblast, tumor-associated macrophage, pro-tumorigenic cell, tumormicroenvironment cell or pro-inflammatory cell.
  • Target tissues include, but not limited to liver, lung, or spleen.
  • Delivery of mRNAs to target cells and tissues may be accomplished by both passive and active targeting means.
  • passive targeting exploits the natural distributions patterns of a transfer vehicle in vivo without relying upon the use of additional excipients or means to enhance recognition of the transfer vehicle by target cells.
  • transfer vehicles which are subject to phagocytosis by the cells of the reticulo-endothelial system are likely to accumulate in the liver or spleen, and accordingly may provide means to passively direct the delivery of the compositions to such target cells.
  • delivery of mRNAs to target cells and tissues may be accomplished by active targeting, which involves the use of additional excipients, referred to herein as “targeting ligands” that may be bound (either covalently or non-covalently) to the transfer vehicle to encourage localization of such transfer vehicle at certain target cells or target tissues.
  • targeting may be mediated by the inclusion of one or more endogenous targeting ligands (e.g., apolipoprotein E) in or on the transfer vehicle to encourage distribution to the target cells or tissues.
  • endogenous targeting ligands e.g., apolipoprotein E
  • the composition can comprise a ligand capable of enhancing affinity of the composition to the target cell.
  • Targeting ligands may be linked to the outer bilayer of the lipid particle during formulation or post-formulation.
  • compositions of the present invention demonstrate improved transfection efficacies, and/or demonstrate enhanced selectivity towards target cells or tissues of interest.
  • compositions which comprise one or more ligands (e.g., peptides, aptamers, oligonucleotides, a vitamin or other molecules) that are capable of enhancing the affinity of the compositions and their nucleic acid contents for the target cells or tissues.
  • ligands may optionally be bound or linked to the surface of the transfer vehicle.
  • the targeting ligand may span the surface of a transfer vehicle or be encapsulated within the transfer vehicle.
  • Suitable ligands and are selected based upon their physical, chemical or biological properties (e.g., selective affinity and/or recognition of target cell surface markers or features) Cell-specific target sites and their corresponding targeting ligand can vary widely.
  • compositions of the invention may include surface markers (e.g., apolipoprotein-B or apolipoprotein-E) that selectively enhance recognition of, or affinity to hepatocytes (e.g., by receptor-mediated recognition of and binding to such surface markers).
  • surface markers e.g., apolipoprotein-B or apolipoprotein-E
  • the use of galactose as a targeting ligand would be expected to direct the compositions of the present invention to parenchymal hepatocytes, or alternatively the use of mannose containing sugar residues as a targeting ligand would be expected to direct the compositions of the present invention to liver endothelial cells (e.g., mannose containing sugar residues that may bind preferentially to the asialoglycoprotein receptor or mannose receptor present in hepatocytes).
  • liver endothelial cells e.g., mannose containing sugar residues that may bind preferentially to the asialoglycoprotein receptor or mannose receptor present in hepatocytes.
  • targeting ligands that have been conjugated to moieties present in the transfer vehicle (e.g., a lipid nanoparticle) therefore facilitate recognition and uptake of the compositions of the present invention in target cells and tissues.
  • suitable targeting ligands include one or more peptides, proteins, aptamers, vitamins and oligonucleotides.
  • the term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, to which the mRNAs and compositions of the present invention are administered.
  • the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.
  • mRNAs e.g., multispecific binding molecule encoding mRNAs
  • delivery vehicles can be formulated in combination with one or more additional nucleic acids, carriers, targeting ligands, targeting moieties, or stabilizing reagents, or in pharmacological compositions where it is mixed with suitable excipients.
  • Multispecific binding molecule encoding mRNAs and compositions containing the same may be administered and dosed in accordance with current medical practice, taking into account the clinical condition of the subject, the site and method of administration, the scheduling of administration, the subject's age, sex, body weight and other factors relevant to clinicians of ordinary skill in the art.
  • the "effective amount" for the purposes herein may be determined by such relevant considerations as are known to those of ordinary skill in experimental clinical research, pharmacological, clinical and medical arts.
  • the amount administered is effective to achieve at least some stabilization, improvement or elimination of symptoms and other indicators as are selected as appropriate measures of disease progress, regression or improvement by those of skill in the art.
  • a suitable amount and dosing regimen is one that causes at least transient antibody production.
  • Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal, pulmonary including intratracheal or inhaled, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • mRNAs and compositions of the invention may be administered in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a targeted tissue, preferably in a sustained release formulation. Local delivery can be affected in various ways, depending on the tissue to be targeted.
  • compositions of the present invention can be inhaled (for nasal, tracheal, or bronchial delivery); compositions of the present invention can be injected into the site of injury, disease manifestation, or pain, for example; compositions can be provided in lozenges for oral, tracheal, or esophageal application; can be supplied in liquid, tablet or capsule form for administration to the stomach or intestines, can be supplied in suppository form for rectal or vaginal application; or can even be delivered to the eye by use of creams, drops, or even injection.
  • Formulations containing compositions of the present invention complexed with therapeutic molecules or ligands can even be surgically administered, for example in association with a polymer or other structure or substance that can allow the compositions to diffuse from the site of implantation to surrounding cells. Alternatively, they can be applied surgically without the use of polymers or supports.
  • the compositions of the invention are formulated such that they are suitable for extended-release of the mRNA contained therein.
  • Such extended-release compositions may be conveniently administered to a subject at extended dosing intervals.
  • the compositions of the present invention are administered to a subject twice day, daily or every other day.
  • the compositions of the present invention are administered to a subject twice a week, once a week, every ten days, every two weeks, every three weeks, or more preferably every four weeks, once a month, every six weeks, every eight weeks, every other month, every three months, every four months, every six months, every eight months, every nine months or annually.
  • compositions and liposomal vehicles which are formulated for depot administration (e.g., intramuscularly, subcutaneously, intravitreally) to either deliver or release a mRNA over extended periods of time.
  • depot administration e.g., intramuscularly, subcutaneously, intravitreally
  • the extended-release means employed are combined with modifications made to the mRNA to enhance stability.
  • Embodiment 1 A method of treating a disease or disorder in a subject comprising administering to a target tissue of said subject one or more mRNA(s) encoding a multispecific binding molecule, wherein the multispecific binding molecule comprises (1) a first binding domain capable of binding to a first antigen present on an immune cell, and (2) a second binding domain capable of binding to a second antigen present on a target cell in said target tissue.
  • Embodiment 2 The method of embodiment 1, wherein the first binding domain comprises a VH and a VL region, and/or a single domain antigen binding fragment.
  • Embodiment 3 The method of embodiment 1, wherein the second binding domain comprises a VH and a VL region, and/or a single domain antigen binding fragment.
  • Embodiment 4 The method of any one of embodiments 1 to 3, wherein the multispecific binding molecule comprises one polypeptide, wherein the polypeptide comprises the first binding domain and the second binding domain.
  • Embodiment 5 The method of embodiment 4, wherein the polypeptide further comprises a secretion signal, wherein optionally the secretion signal is N-terminal secretion signal.
  • Embodiment 6 The method of embodiment 4, wherein the first binding domain and the second binding domain are connected via a linker.
  • Embodiment 7 The method of any one of embodiments 4-6, wherein the polypeptide is encoded by a single mRNA.
  • Embodiment 8 The method of any one of embodiments 1-6, wherein the first antigen is CD3.
  • Embodiment 9 The method of embodiment 8, wherein the first binding domain comprises: (i) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 6, and a VL comprising the amino acid sequence of SEQ ID NO: 7, (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and a VL comprising the amino acid sequence of SEQ ID NO: 16, (iii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 21, and a VL comprising the amino acid sequence of SEQ ID NO: 22, (iv) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 35, and a VL comprising the amino acid sequence of SEQ ID NO: 36, or (v) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 48, and a VL comprising the amino acid sequence of SEQ ID NO: 49.
  • VH heavy chain variable
  • Embodiment 10 The method of any one of embodiments 1-6, wherein the first antigen is CD16.
  • Embodiment 11 The method of embodiment 10, wherein the first binding domain is a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 39.
  • Embodiment 12 The method of any one of embodiments 1-11, wherein the second antigen is GPC3.
  • Embodiment 13 The method of embodiment 12, wherein the second binding domain is a single domain antigen binding fragment comprising (i) the amino acid sequence of SEQ ID NO: 10 or (ii) the amino acid sequence of SEQ ID NO: 30.
  • Embodiment 14 The method of embodiment 12, wherein the second binding domain comprises: (i) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 11, and a VL comprising the amino acid sequence of SEQ ID NO: 12 or (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 27, and a VL comprising the amino acid sequence of SEQ ID NO: 28.
  • VH heavy chain variable region
  • Embodiment 15 The method of any one of embodiments 1-11, wherein the second antigen is EpCAM.
  • Embodiment 16 The method of embodiment 15, wherein the second binding domain is a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 41.
  • Embodiment 17 The method of embodiment 15, wherein the second binding domain comprises: (i) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 4, and a VL comprising the amino acid sequence of SEQ ID NO: 2, or (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 14, and a VL comprising the amino acid sequence of SEQ ID NO: 13.
  • VH heavy chain variable region
  • VL comprising the amino acid sequence of SEQ ID NO: 2
  • VH heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14
  • VL comprising the amino acid sequence of SEQ ID NO: 13
  • Embodiment 18 The method of any one of embodiments 1-11, wherein the second antigen is uPAR.
  • Embodiment 19 The method of embodiment 18, wherein the wherein the second binding domain is derived from the amino terminal fragment of uPA.
  • Embodiment 20 The method of any one of embodiments 1-11, wherein the second antigen is an NKG2D ligand.
  • Embodiment 21 The method of embodiment 20, wherein the NKG2D ligand is selected from MICA, MICB, RAET1E, RAET1G, RAET1L/ULBP6, ULBP1, ULBP2, ULBP3 or ULBP4.
  • Embodiment 22 The method of embodiment 21, wherein the second binding domain is derived from the extracellular domain of NKG2D.
  • Embodiment 23 The method of any one of embodiments 1-11, wherein the second antigen is EGFR.
  • Embodiment 24 The method of embodiment 23, wherein the second binding domain is a single domain antigen binding fragment comprising the amino acid sequence of SEQ ID NO: 50.
  • Embodiment 25 The method of embodiment 6, wherein the sequence of secretion signal is selected from the group consisting of SEQ ID NOs: 1, 43, 44, 45, 46 or 51.
  • Embodiment 26 The method of embodiment 4, wherein the multispecific binding molecule comprises one polypeptide; wherein the polypeptide comprises (1) the first binding domain binding to the first antigen present on an immune cell, and (2) the second binding domain binding to the second antigen present on a target cell; wherein the first binding domain is a scFv comprising a VH and a VL region, and the second binding domain is a scFv comprising a VH and a VL region.
  • Embodiment 27 The method of embodiment 26, wherein the first antigen is CD3.
  • Embodiment 28 The method of embodiment 4, wherein the multispecific binding molecule comprises one polypeptide; wherein the polypeptide comprises (1) the first binding domain binding to the first antigen present on an immune cell, and (2) the second binding domain binding to the second antigen present on a target cell; wherein the first binding domain is a scFv comprising a VH and a VL region, and the second binding domain is a single domain antigen binding fragment.
  • Embodiment 29 The method of embodiment 28, wherein the first antigen is CD3.
  • Embodiment 30 The method of any one of embodiments 1-29, wherein the multispecific binding molecule(s) bind said immune cell and said target cell inducing said target cell elimination.
  • Embodiment 31 The method of any one of embodiments 1-29, wherein the multispecific binding molecule is expressed at a higher level in the target tissue compared to a non-target tissue of said subject.
  • Embodiment 32 The method of any one of embodiments 1-29, wherein the target cell is transfected with the mRNA(s) encoding the multispecific binding molecule, the target cell expresses and secretes the multispecific binding molecule, and the multispecific binding molecule binds to the target cell.
  • Embodiment 33 The method of any one of embodiments 1-29, wherein a non-target cell is transfected with the mRNA(s) encoding the multispecific binding molecule, the non-target cell expresses and secretes the multispecific binding molecule, and the multispecific binding molecule binds to the target cell.
  • Embodiment 34 The method of any one of embodiments 1-29, comprises (1) delivery of the mRNA to the target cell, and (2) the multispecific binding molecule is expressed and secreted by the target cell, and (3) the multispecific binding molecule binds to the second antigen on the target cell in an autocrine-like fashion, and (4) the multispecific binding molecule binds to the first antigen on an immune cell, inducing the immune cell to kill the target cell.
  • Embodiment 35 The method of any one of embodiments 1-34, wherein the mRNA is administered to the subject via a delivery vehicle, wherein optionally the delivery vehicle is a lipid nanoparticle.
  • Embodiment 36 The method of any one of embodiments 1-34, wherein the mRNAs and/or compositions comprising the mRNAs are administered via intravenous injection.
  • Embodiment 37 The method of any one of embodiments 1-34, wherein the mRNAs and/or compositions comprising the mRNAs are administered via direct injection.
  • Embodiment 38 The method of any one of embodiments 1-37, wherein the target tissue is liver.
  • Embodiment 39 The method of any one of embodiments 1-37, wherein the target tissue is lung.
  • Embodiment 40 The method of any one of embodiments 1-37, wherein the target tissue is spleen.
  • Embodiment 41 The method of any one of embodiments 1-40, wherein the target cell is a cancer cell.
  • Embodiment 42 The method of embodiment 41, wherein the cancer cell is a liver cancer cell.
  • Embodiment 43 The method of any one of embodiments 1-40, wherein the target cell is a senescent cell.
  • Embodiment 44 The method of any one of embodiments 1-40, wherein the target cell is an activated fibroblast.
  • Embodiment 45 The method of any one of embodiments 1-40, wherein the target cell is a tumor-associated fibroblast.
  • Embodiment 46 The method of any one of embodiments 1-40, wherein the target cell is a tumor-associated macrophage.
  • Embodiment 47 The method of any one of embodiments 1-40, wherein the target cell is a pro-tumorigenic cell.
  • Embodiment 48 The method of any one of embodiments 1-40, wherein the target cell is a tumor-microenvironment cell.
  • Embodiment 49 The method of any one of embodiments 1-40, wherein the target cell is a pro-inflammatory cell.
  • Embodiment 50 The method of any one of embodiments 1-49, wherein the method reduces or eliminates antigen shed inhibition.
  • Embodiment 51 The method of any one of embodiments 1-49, wherein the method reduces or eliminates systemic antibody exposure .
  • Embodiment 52 The method of any one of embodiments 1-49, wherein the method reduces or eliminates on-target off-tumor toxicity.
  • Embodiment 53 The method of any one of embodiments 1-49, wherein the method achieves increased target cell killing compared with equivalent amounts of antibody treatment.
  • Embodiment 54 The method of any one of embodiments 1-49, wherein the method achieves high local cell surface concentrations of the multispecific binding molecule encoded by the mRNA.
  • Embodiment 55 The method of any one of embodiments 1-49, wherein the method reduces or eliminates barriers to tumor penetration.
  • Embodiment 56 A tumor therapeutic for delivery to a target cell, comprising mRNA encoding a multispecific binding molecule, wherein the multispecific binding molecule comprises (1) a first binding domain capable of binding to a first antigen present on an immune cell, and (2) a second binding domain capable of binding to a second antigen present on the target cell, wherein the multispecific binding molecule is expressed and secreted by the target target cell, wherein the multispecific binding molecule binds to the second antigen on the target cell in an autocrine-like fashion, and wherein the multispecific binding molecule binds to the first antigen on an immune cell, inducing the immune cell to kill the target cell.
  • the multispecific binding molecule comprises (1) a first binding domain capable of binding to a first antigen present on an immune cell, and (2) a second binding domain capable of binding to a second antigen present on the target cell, wherein the multispecific binding molecule is expressed and secreted by the target target cell, wherein the multispecific binding molecule binds to the second anti
  • the invention is generally disclosed herein using affirmative language to describe the numerous embodiments.
  • the invention also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, procedures, assays or analysis.
  • the invention is generally not expressed herein in terms of what the invention does not include, aspects that are not expressly included in the invention are nevertheless disclosed herein.
  • mRNA constructs encoding multispecific binding molecules were prepared.
  • the binding domain of the multispecific binding molecules arrangement were selected from, for example, the group summarized in Table below.
  • T-Cell Dependent Cellular Cytotoxicity (TDCC) assay was used to assess the ability of mRNA encoding constructs that bind to GPC3 and CD3 to generate specific T-cell mediated target cell killing.
  • HepG2 is a human hepatoma cell line that is known to express GPC3.
  • GPC3 is a fetal protein and tumor associated antigen frequently overexpressed in liver cancers.
  • HepG2 cells were plated in a 96 well plate at 10,000 cells per well. Cells were then transfected using MessengerMax with mRNA encoding one of four bispecific antibodies (Construct 31, 32, 53, or 54) or GFP (control).
  • Each bispecific antibody has a CD3 binding domain and one of two different GPC3 binding domains (a single domain antibody (sdAb) or a single-chain variable fragments (scFv)).
  • the binding domain arrangement of the multispecific binding molecules encoded by the 4 mRNA are shown in the table above.
  • two relative orientations of the GPC3 and CD3 binding domains were tested.
  • CD8+ cytotoxic T cells were added at an effectortarget ratio of 10: 1. Cells were co-incubated for 48 hours, then washed to remove T cells.
  • the ATP-dependent luciferase-based cell viability assay Cell Titer Gio (Promega) was then performed to determine the number of live target cells per well. Complete lysis was achieved in control wells with Triton X-100 at 2% v/v final concentration per well.
  • FIG. 5 shows the results of a TDCC assay performed as described, utilizing the GPC3 negative fibroblast cell line HFF1.
  • the binding domain with affinity for an immune cell is “split” across two multispecific antibodies, such that immune cell binding can only occur when these two or more antibodies are in close proximity.
  • VH and VL domains of an anti-CD3 (“a-CD3”) single chain variable fragment (scFv ) are encoded in separate mRNA constructs which generate separate multispecific antibodies, such as Construct 57 and 58 as listed in the table above (Construct 57 comprises the VH domain of CD3, Construct 58 comprises the VL domain of CD3).
  • Construct 57 comprises the VH domain of CD3
  • Construct 58 comprises the VL domain of CD3
  • FIG. 2 illustrates such a design.
  • FIG. 3 further illustrates such a design, wherein the target cell surface antigens (marked 4 and 5) may be the same antigen or different antigens.
  • FIG. 6 shows the results of a TDCC assay performed, utilizing mRNA encoding bispecific antibodies with intact (construct 54) or split (constructs 57 and 58) CD3 binding domains.
  • the binding domain arrangement of the multispecific binding molecules encoded by the Construct 54, 57, and 58 are shown in the table above.
  • FIG. 8 shows the results of a TDCC assay utilizing mRNA encoding anti-EpCAM x anti-CD3 bispecific antibodies.
  • EpCAM is frequently overexpressed in liver cancers and other cancers, and is known to be overexpressed in HepG2 cells.
  • FIG. 9 shows the results of a TDCC assay performed, utilizing mRNA encoding anti- EpCAM x anti-GPC3 x anti-CD3 Trispecific Binding Molecule(s) with split CD3 binding domains (Construct 59 and 58), each encoding target cell-binding scFvs with affinity for different target antigens (EpCAM and GPC3, respectively).
  • FIG. 10 demonstrates the absence of inhibition of target cell killing in the presence of shed target antigen when target cells express bispecific immune engaging protein via mRNA transfection.
  • a TDCC assay performed as described in previous examples with the following modifications: 20 uL of assay media was substituted for an equal volume of a PBS solution containing recombinant human GPC3 (rGPC3) at a concentration of 0 or 100 ug/mL, yielding a final concentration of 0 and 20 ug/mL, respectively. Samples were harvested at 24 hours. As previously observed, mRNA encoding bispecific antibody constructs (Construct 53, 55+56, 54, 57+58, 58+59) lead to a decrease in viable cell count. Across conditions, minimal differences in specific lysis were observed in the groups in presence or absence of shed target antigen.
  • FIG. 11 is a diagram representing the inhibition of immunotherapies via shed antigens and hypothesis for the observed reduced or eliminated inhibition in the context of mRNA delivery to target cells.
  • a second set of 96 well plates was seeded with HepG2 target cells, and following attachment overnight, the media was removed and replaced with conditioned media from producer cells (12.4).
  • mRNA was administered to wells containing conditioned media without antibody, while lipid vehicle only was added to wells containing antibody conditioned media (12.5).
  • CD8+ cytotoxic T cells were added to all wells (12.6). After a 48 hour incubation, a Cell Titer Gio assay was performed as described in Fig 2, and the degree of cytotoxicity was determined (12.7).
  • FIG. 13 shows the results of the assay described in FIG. 12 for anti-GPC3 bispecific constructs 53 and 54. Cytotoxicity normalized to vehicle control (HepG2 + Lipid Vehicle Only; 100% viability) is shown. A greater degree of target cell lysis is observed in the mRNA transfected target cells as compared to the target cells incubated with antibody containing supernatant.
  • FIG. 14 shows the results of the assay described in Figure 12 for anti-EpCAM bispecific constructs 23 and 63.
  • FIG. 15 shows the results of the assay described in FIG. 12 for anti-GPC3 bispecific constructs with split CD3 binding domains 55+56 (derived from construct 53) and 58+57 (derived from construct 54). A greater degree of target cell lysis is observed in the mRNA transfected target cells as compared to the target cells incubated with antibody containing supernatant.
  • FIG. 16 shows the results of the assay described in FIG.12 for anti-EpCAM bispecific constructs with split CD3 binding domains 59+46 (derived from construct 23), 62+61 (derived from construct 63), and 57+59 (anti-GPC3 split CD3 construct derived from construct 54 + anti- EpCAM split CD3 construct derived from construct 23).
  • a greater degree of target cell lysis is observed in the mRNA transfected target cells as compared to the target cells incubated with antibody containing supernatant for constructs 59+46 and 62+61.
  • Example 8 - TDCC Assay to Assess mRNA(s) Encoding anti-EpCAM x anti- CD3 and anti-uPAR x anti-CD3 Bispecific Binding Molecule (s).
  • FIG. 18 shows the results of a TDCC assay utilizing mRNA encoding anti-EpCAM x anti-CD3 and anti-uPAR x anti-CD3 bispecific antibodies.
  • EpCAM is frequently overexpressed in ovarian cancers and other cancers, and is known to be overexpressed in OV90 ovarian cancer cells.
  • This assay included anti-EpCAM Construct 23.
  • uPAR urokinase plasminogen activator surface receptor, or CD87
  • CD87 urokinase plasminogen activator surface receptor
  • This assay included anti-uP AR Construct 27, which comprises an anti-CD3 scFv linked to the amino terminal fragment (ATF) of uPA, a natural ligand for uPAR which binds with high affinity.
  • FIG. 19 shows the results of a TDCC assay utilizing mRNA encoding anti-mouse CD3 x anti-mouse uPAR [mouse uPA ATF] and anti-mouse CD3 x anti-mouse NKG2D Ligand (L) [mouse NKG2D] bispecific antibodies.
  • uPAR and NKG2D ligands are frequently overexpressed in colorectal cancers and other cancers, and are known to be overexpressed in the mouse colorectal cancer cell line MC38.
  • Constructs 21 and 22 utilize anti-mouse CD3 binding domains.
  • Construct 21 utilizes the murine uPA ATF (corresponding to the murine homolog of the human uPA ATF employed in construct 27).
  • Construct 22 utilizes the extracellular domain of mouse NKG2D, which has affinity for multiple ligands (e.g., the murine homologs of the human ligands MICA, MICB, and others) which are upregulated in response to cellular stress such as genomic instability, and often overexpressed in cancer and senescence.
  • Example 10 - TDCC Assay to Assess mRNA(s) Encoding anti-EpCAM scFv x anti-CD3 scFv and anti-CD16 sdAb x anti-EpCAM sdAb Bispecific Binding Molecule (s).
  • A431 cells stably expressing luciferase were plated 3.75 hours before transfection with mRNA encoding GFP, C103 (anti-EpCAM scFvXanti-CD3 scFv), or C190 (anti-CD16 sdAbxanti-EpCAM sdAb).
  • a dilution series of mRNA complexed with MessengerMAX transfection reagent (ThermoFisher, per manufacturer’s protocol) was prepared.
  • PBMCs isolated from a healthy donor were plated 2.75 hours after mRNA transfection. Luciferase signal intensity was measured at 72 hours post transfection. Viability was normalized to GFP transfected wells for each mRNA concentration. Results are shown in FIG. 20. Data are from two replicates per concentration. Standard error of the mean is displayed. [00653]
  • the data in FIG. 20 demonstrate activity of mRNA-encoded immune cell engager targeting CD3 and, separately, mRNA-encoded immune cell engager targeting CD 16. Maximum cytotoxicity of 94% and 61% were observed for anti-CD3 x anti-EpCAM and anti-CD16 x anti- EpCAM bispecific immune cell engagers, respectively.
  • Hep3B cells stably expressing luciferase were plated 1 day before transfection with mRNA encoding GFP or experimental constructs.
  • a dilution series of mRNA complexed with MessengerMAX transfection reagent (ThermoFisher, per manufacturer’s protocol) was prepared.
  • Pan T cells isolated from a healthy donor were plated 2-4 hours after mRNA transfection. Luciferase signal intensity was measured at 48 hours post transfection. Viability was normalized to GFP transfected wells for each mRNA concentration. Results are shown in FIG. 21. Data are from two replicates per concentration. Standard error of the mean is displayed.
  • FIG. 21 show that mRNA(s) encoding anti-CD3 x anti- GPC3 bispecific binding molecules with a range of secretion signals and orientations are active.
  • HepG2 and A431 cells stably expressing luciferase were plated 1 day before transfection with mRNA encoding experimental construct.
  • a dilution series of mRNA complexed with MessengerMAX transfection reagent (ThermoFisher, per manufacturer’s protocol) was prepared.
  • wells were sampled to determine the concentration of protein produced via HiBit assay (Promega, per manufacturer’s protocol, using C terminus HiBit tag on experimental constructs). Luciferase signal intensity was measured at 48 hours post transfection. Results are shown in FIG. 22 (HepG2 transfection) and FIG. 23 (A431 transfection).
  • A431 cells stably expressing luciferase were plated 1 day before transfection with mRNA encoding experimental construct.
  • a dilution series of mRNA complexed with MessengerMAX transfection reagent (ThermoFisher, per manufacturer’s protocol) was prepared.
  • mRNA encoding GFP or Cl 03 (anti-EpCAM) was used.
  • Pan T cells isolated from a healthy donor were plated 2-4 hours after mRNA transfection. At 48 hours, wells were sampled to determine the concentration of protein produced via HiBit assay (Promega, per manufacturer’s protocol, using C terminus HiBit tag [“HB”] on experimental constructs). Luciferase signal intensity was measured at 48 hours post transfection.
  • Expi 293 (Thermo Fisher) was transfected with plasmid DNA encoding experimental constructs. Supernatant was collected and protein was quantified by HiBit assay as previously described. A dilution series of supernatant was prepared for experimental constructs and concentration-mated mock control (no experimental construct). A TDCC assay was performed in parallel with mRNA conditions as described above.
  • Results are shown in FIG. 24. Data are from two replicates per concentration. Raw luminescence counts are shown. Standard error of the mean is displayed.
  • FIG. 25 Further data in FIG. 25 show a high degree of linearity for experimental construct protein concentration (as measured by HiBit) relative to mRNA transfection in A431 cells. 7.14 Example 14 - TDCC Assay Using Different Subsets of Immune Effector Cells to Assess mRNAs Encoding anti-CD3 x anti-GPC3 and anti-CD3 x anti-EpCAM Bispecific Binding Molecules.
  • HepG2 cells stably expressing luciferase were plated 1 day before transfection with mRNA encoding GFP or experimental constructs.
  • a dilution series of mRNA complexed with MessengerMAX transfection reagent (ThermoFisher, per manufacturer’s protocol) was prepared.
  • mRNA encoding GFP or immune cell engager C073 (anti-CD3 x anti-GPC3) or C023 A (anti- CD3 x anti-EpCAM) were used.
  • Activated and Expanded Pan T cells, Pan T Cells, CD8+ T cells, or PBMCs isolated from the same healthy donor (“Dl l”) were plated 2-4 hours after mRNA transfection. Luciferase signal intensity was measured at 48 hours post transfection. Raw luminescence counts are displayed. Results are shown in FIGs. 26A-E. Data are from two replicates per concentration. Standard error of the mean is displayed.
  • A431 stably transfected with luciferase and GPC3 were plated 1 day before transfection with mRNA encoding GFP or experimental constructs.
  • a dilution series of mRNA complexed with MessengerMAX transfection reagent (ThermoFisher, per manufacturer’s protocol) was prepared.
  • Pan T cells isolated from a healthy donor were plated 2-4 hours after mRNA transfection. Luciferase signal intensity was measured at 48 hours post transfection. Viability was normalized to GFP transfected wells for each mRNA concentration. Results are shown in FIGs. 27A-B. Data are from two replicates per concentration. Standard error of the mean is displayed.
  • HepG2 cells stably transfected with luciferase were plated 1 day before transfection with mRNA encoding GFP or experimental constructs.
  • a dilution series of mRNA complexed with MessengerMAX transfection reagent (ThermoFisher, per manufacturer’s protocol) was prepared.
  • Pan T cells isolated from a healthy donor were plated 2-4 hours after mRNA transfection. Luciferase signal intensity was measured at 48 hours post transfection. Viability was normalized to GFP transfected wells for each mRNA concentration. Results are shown in FIGs. 28A-B. Data are from two replicates per concentration. Standard error of the mean is displayed.
  • Constructs include anti-EpCAM Cl 03 and anti-GPC3 C109, C183-184, C212-215, and C223-228. Luciferase signal intensity was measured at 48 hours post transfection. Viability was normalized to GFP transfected wells for each mRNA concentration. Results are shown in FIG. 29. Data are from two replicates per concentration. Standard error of the mean is displayed.
  • Lipid nanoparticles were formulated using the Precision Nanosystems Ignite (per manufacturer’s protocol) for all in vivo studies. Lipid nanoparticles were prepared using a commercially available formulation (Cayman Chemical Lipid Nanoparticle Exploration Kit LNP-0315) comprising: ionizable lipid (((4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2- hexyldecanoate), molar ratio 46.3), cholesterol (molar ratio 42.7), peg lipid (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, molar ratio 1.6), and phospholipid (1, 2-distearoyl-sn- glycero-3 -phosphocholine (DSPC), molar ratio 9.4); unless otherwise noted, this exemplary formulation was used for all LNP
  • mRNA Lipid nanoparticles encapsulating firefly luciferase mRNA were formulated.
  • NCG mice 6-8 week old male and female immunodeficient NCG mice (Charles River) were used. HepG2 human liver cancer cells (ATCC) were stably transfected with luciferase (Luc) to enable non-invasive bioluminescence (BLI) imaging. At Week -1, Mice were engrafted with 5xlOE6 HepG2 Luc+ cells in the left lobe of the liver, with a final volume of 20 uL consisting of 10 uL cell solution and 10 uL Cultrex Reduced Growth Factor Basement Membrane Extract (Cat# 3433-001-01). At Week 0, one week after tumor engraftment, mice were assessed via BLI for baseline tumor size. Mice with established tumors were pair-match randomized to treatment or control groups.
  • mice were engrafted with lxlOE7 human PBMCs (STEMCELL Technologies) intraperitoneally. [00679] Mice then received 3x weekly injections (M, W, F) of 0.6 mg/kg (encapsulated mRNA / body weight) of MRNA encoding either active bispecific antibody construct C080 (anti-CD3, anti-GPC3 immune cell engager) or control GFP mRNA. Results are shown in FIG. 33.
  • NCG mice 6-8 week old male and female immunodeficient NCG mice (Charles River) were used. HepG2 human liver cancer cells (ATCC) were stably transfected with luciferase (Luc) to enable non-invasive bioluminescence (BLI) imaging. At Day -4, Mice were engrafted with lxlOE6 HepG2 Luc+ cells in the left lobe of the liver, with a final volume of 20 uL consisting of 10 uL cell solution and 10 uL Cultrex Reduced Growth Factor Basement Membrane Extract (Cat# 3433-001-01). At Day 0, mice were assessed via BLI for baseline tumor size. Mice with established tumors were randomized to treatment or control groups. Mice were engrafted with lxlOE7 Pan T cells intraperitoneally.
  • mice then received 3x weekly injections (M, W, F) of 0.6 mg/kg (encapsulated mRNA / body weight) of MRNA encoding either active bi specific antibody construct C073 or control GFP mRNA. Results are shown in FIG. 34.
  • NCG mice 6-8 week old male and female immunodeficient NCG mice (Charles River) were used. HepG2 human liver cancer cells (ATCC) were stably transfected with luciferase (Luc) to enable non-invasive bioluminescence (BLI) imaging. At Day -4, Mice were engrafted with lxlOE6 HepG2 Luc+ cells in the left lobe of the liver, with a final volume of 20 uL consisting of 10 uL cell solution and 10 uL Cultrex Reduced Growth Factor Basement Membrane Extract (Cat# 3433-001-01). At Day 0, mice were assessed via BLI for baseline tumor size. Mice with established tumors were randomized to treatment or control groups.
  • mice were engrafted with lxlOE7 Pan T cells intraperitoneally. [00685] Mice then received 3x weekly injections (M, W, F) of 0.6 mg/kg (encapsulated mRNA / body weight) of MRNA encoding either active bispecific antibody construct C023A (anti-CD3, anti-EpCAM immune cell engager) or control GFP mRNA. Results are shown in FIGs. 35-36.
  • Bioluminescence data in FIG. 35 demonstrate that by Day 44, all tumors were eliminated from treated animals, with large tumors present in all control mRNA animals. Qualification of the luminescence data is shown in FIG. 36.
  • Cells in Condition 2 were transfected with a dilution series of mRNA encoding experimental construct C271; control plate cells were transfected with mRNA encoding GFP; other conditions were untransfected.
  • HTS Transwell 96 well inserts (07200742, Corning) were coated with 30 uL of 50% Cultrex Reduced Growth Factor Basement Membrane Extract (Cat# 3433-001-01); Cultrex served as a model for the ECM of a tumor, acting as a rate limiter for diffusion of circulating multispecific antibodies.
  • Transwells (“peripheral” compartment) in Condition 1 received a dilution series of Expi293 supernatant containing experimental construct C271 protein in culture media.
  • condition 2 The data demonstrate that locally-produced protein via mRNA transfection of the tumor (condition 2) induces cytotoxicity with significantly lower systemic concentrations of immune engaging protein as compared to systemically delivered protein (condition 1).
  • condition 1 The data reflect the increased potency of locally expressed immune cell engagers relative to systematically administered immune cell engagers.
  • Lipid nanoparticles were formulated using the Precision Nanosystems Ignite (per manufacturer’s protocol). mRNA Lipid nanoparticles encapsulating firefly luciferase mRNA (TriLink Biotechnologies) were formulated.
  • Lipid nanoparticles were administered intratumorally at 5 ug mRNA per injection. After 24 hours, bioluminescence imaging was performed using the IVIS spectrum imaging system (PerkinElmer) to assess the level of protein production and injection site specificity. Whole mouse bioluminescent imaging as shown in FIG. 39 illustrates the high level of localized protein production following intratumoral injection.
  • NCG mice 6-8 week old male and female immunodeficient NCG mice (Charles River) were used.
  • Hep3B human liver cancer cells ATCC
  • Luc luciferase
  • BLI bioluminescence
  • 20 uL of Geltrex was mixed with 30 uL containing 3E6 Hep3B-Luc liver cancer cells and engrafted subcutaneously in the rear flank of mice. Xenografts were allowed to grow for 31 days prior to treatment.
  • Lipid nanoparticles were formulated using the Precision Nanosystems Ignite (per manufacturer’s protocol). mRNA Lipid nanoparticles encapsulating control (GFP) or C080 mRNA encoding an exemplary immune cell engager.
  • GFP Precision Nanosystems Ignite
  • C080 C080 mRNA encoding an exemplary immune cell engager.
  • mice with established tumors were randomized to treatment or control groups and blioluminescence imaging was performed to established baseline tumor size (“Day 0”). Lipid nanoparticles were administered intratumorally at 5 ug mRNA per injection.
  • bioluminescence imaging was again performed to establish change in tumor size.
  • the bioluminescence data in FIG. 40 demonstrate that one week following treatment, tumor shrinkage was observed in 2 of 3 treated mice (-50% and -63% respectively) compared with 0 of 3 control mice. 7.25 Example 25 - Transfection of local and metastatic HCC cells via intravenous nanoparticle administration in mice
  • HepG2 and Hep3B cells are characterized as positive for ApoE receptors such as LDLR.
  • HepG2 and Hep3B cells are stably transfected with a conditional reporter plasmid to generate conditional reporter cells.
  • the conditional reporter plasmid contains a LoxP flanked stop cassette prior to one or both of the reporter genes TdTomato and Luciferase.
  • Cre-mediated recombination excises the stop cassette and induces expression of the reporter genes.
  • Conditional reporter cells are engrafted into immunocompromised mice (NCG mice, Charles River) orthotopically (onto the left lobe of the liver) and heterotopically (subcutaneous xenograft).
  • LNPs lipid nanoparticles
  • LNPs are administered intravenously to animals with orthotopic and heterotopic xenografts. Expression of luciferase is monitored via bioluminescence imaging (BLI). BLI data show conditional expression in tumors of mice that received Cre-encoding mRNA; these data demonstrate that LNPs are transfecting HCC cells in orthotopic and heterotopic locations.

Abstract

L'invention concerne une méthode de traitement d'une maladie ou d'une affection chez un sujet, comprenant l'administration à un tissu cible dudit sujet d'un ou de plusieurs ARNm codant une molécule de liaison multispécifique ainsi que la sécrétion et la liaison de type autocrine des anticorps multispécifiques recrutant des cellules immunitaires (molécule de liaison multispécifique présentant une affinité de liaison pour une ou plusieurs cellules cibles et une ou plusieurs cellules effectrices immunitaires) pour obtenir l'élimination sélective, médiée par des cellules immunitaires, de cellules cibles.
PCT/US2022/079055 2021-11-02 2022-11-01 Matériels et méthodes de traitement avec un arnm codant des molécules de liaison multispécifiques WO2023081644A1 (fr)

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WO2021138600A1 (fr) * 2019-12-31 2021-07-08 Fred Hutchinson Cancer Research Center Systèmes de nanoparticules pour stimuler et maintenir la réactivité d'un système immunitaire au niveau de sites de traitement

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Publication number Priority date Publication date Assignee Title
WO2021138600A1 (fr) * 2019-12-31 2021-07-08 Fred Hutchinson Cancer Research Center Systèmes de nanoparticules pour stimuler et maintenir la réactivité d'un système immunitaire au niveau de sites de traitement

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