WO2012162418A1 - Procédés et compositions pour ligands de ciblage hétérodimères - Google Patents

Procédés et compositions pour ligands de ciblage hétérodimères Download PDF

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Publication number
WO2012162418A1
WO2012162418A1 PCT/US2012/039187 US2012039187W WO2012162418A1 WO 2012162418 A1 WO2012162418 A1 WO 2012162418A1 US 2012039187 W US2012039187 W US 2012039187W WO 2012162418 A1 WO2012162418 A1 WO 2012162418A1
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cancer
molecule
heterodimeric
binding
domain
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PCT/US2012/039187
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English (en)
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Rihe Liu
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The University Of North Carolina At Chapel Hill
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0032—Methine dyes, e.g. cyanine dyes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0058—Antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • C07K2318/00—Antibody mimetics or scaffolds
    • C07K2318/20—Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics

Definitions

  • the present invention is directed to targeting ligands that bind to cell surface and ⁇ r intracellular molecules with high affinity and specificity, for use as therapeutic agents, imaging and/or diagnostic agents and/or as agents to deliver therapeutic agents to target cells.
  • Targeting ligands with avidity have major advantages, for example, in cancer cell targeting.
  • An important parameter for satisfactory cell targeting is the valency of a targeting ligand. This is particularly important when the concentration of a targeting ligand is low, such as in the circulatory system. It is believed that monovalent targeting ligands, even those with very high binding affinities, tend to have fast dissociation rates and provide only modest retention time on target antigen in a nonequilibrium environment of, e.g., tumor tissues.
  • One solution to address the problem is to develop targeting ligands that bind two different regions on a target protein.
  • the present invention overcomes previous shortcomings in the art by providing heterodimeric targeting ligands that bind to cell surface and/or intracellular biomarkers with exceptionally high affinity and specificity, for use as therapeutic agents and/or for targeted delivery of imaging and/or therapeutic agents.
  • the present invention provides a heterodimeric targeting ligand, comprising: a) a first target binding domain specific for a first site on an extracellular or intracellular domain of a target molecule; b) a second target binding domain specific for a second site on the extracellular or intracellular domain of the target molecule of (a), wherein the first site and the second site are nonoverlapping on the target molecule; and c) a linker peptide linking the first target binding domain of (a) and the second target binding domain of (b).
  • the above-described linker can be, but is not limited to, a) a linker comprising the amino acid sequence of SEQ ID NO: 1 ((GGGGS) n , wherein n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.; e.g., (GGGGS) 3 ); b) a linker comprising the amino acid sequence of SEQ ID NO:2 ((GGGS) terme, wherein n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.); c) a linker comprising the amino acid sequence of SEQ ID NO:3 ((GSGSGS) n , wherein n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.); d) a linker comprising the amino acid sequence of SEQ ID NO:4 ((TPPTPSP) n , wherein n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.); e) a linker comprising the amino acid sequence of SEQ ID NO:5 ((PQPQ
  • the target molecule to which the heterodimeric targeting ligand of this invention binds can comprise, consist essentially of or consist of an extracellular domain of a cell surface receptor, which can be, e.g., an epidermal growth factor receptor family member (e.g., EGFR, HER2, HER3, etc.), c-MET, VEGFR, insulin receptor, insulinlike growth factor receptor, prostate specific membrane antigen, mesothelin, hepsin, an integrin, mucin, MUC16, a cell surface cluster of differentiation (CD) molecule (e.g., CD20, CD22, CD30, CD33, CD44, CD56, etc.) and any combination thereof.
  • a cell surface receptor which can be, e.g., an epidermal growth factor receptor family member (e.g., EGFR, HER2, HER3, etc.), c-MET, VEGFR, insulin receptor, insulinlike growth factor receptor, prostate specific membrane antigen, mesothelin, heps
  • the target molecule of this invention can comprise, consist essentially of or consist of a catalytic domain, regulatory domain and/or a binding partner- interacting region of an intracellular, secreted and/or membrane bound protein.
  • an intracellular, secreted and/or membrane-bound protein of this invention include a growth factor, a cytokine, VEGF, bFGF, EGF, IGF, PDGF, TGF, TNF, bFGF receptor, TGF receptor, TNF receptor, IgE, IgE receptor, a tyrosine kinase, PI3K, AKT, MEK, a tyrosine kinase receptor, EGFR, HER2, VEGFR, PDGFR, c-MET, insulin-like growth factor receptor, BRAF, a phosphatase, PTP1B, Cdc25, PTEN, SHP2, a protease, DPP-IV, caspase-3, cathepsin D,
  • the first target binding domain of (a) and/or the second target binding domain of (b) can comprise, consist essentially of or consist of an antibody fragment, a single domain antibody fragment, a single chain polypeptide of a VH or VL domain of an antibody, a peptide or protein derived from a binding and/or framework region of an antibody, a single domain antibody mimic based on a non- immunoglobulin scaffold, a Z domain, a FN3 domain, a designed ankyrin repeat protein (DARPIN), an epidermal growth factor receptor (EGFR)-binding domain, a HER2 -binding domain, a PSMA-binding domain, an v P3-binding domain, a target-binding peptide containing natural and/or unnatural amino acids, a molecule or binding portion thereof which specifically binds to the extracellular domain of a cell surface receptor or to the binding partner- interacting surface of an intracellular protein, and any combination thereof.
  • DARPIN ankyrin repeat protein
  • the present invention provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a heterodimeric targeting ligand of this invention and/or a nucleic acid molecule encoding a heterodimeric targeting ligand of this invention, wherein the first and second target binding domains are specific for a target molecule on a cancer cell in the subject and the heterodimeric targeting ligand binds the target molecule on cancer cells in the subject, thereby treating the cancer in the subject.
  • a method of detecting and/or localizing cancer cells in a subject comprising administering to the subject an effective amount of a heterodimeric targeting ligand of this invention and/or a nucleic acid encoding a heterodimeric targeting ligand of this invention, wherein the first and second targeting domains are specific for a target molecule on a cancer cell in the subject and the heterodimeric targeting ligand further comprises an imaging molecule and/or detectable molecule, whereby the heterodimeric targeting ligand binds the target molecule on cancer cells in the subject and the imaging molecule is visualized and/or the detectable molecule is detected at its binding location on cancer cells in the subject, thereby detecting and/or localizing cancer cells in the subject.
  • the present invention also provides a method of diagnosing cancer in a subject, comprising administering to the subject an effective amount of a heterodimeric targeting ligand of this invention or a nucleic acid molecule encoding a heterodimeric targeting ligand of this invention, wherein the first and second targeting domains are specific for a target molecule on a cancer cell in the subject and the heterodimeric targeting ligand further comprises an imaging molecule and/or detectable molecule, whereby the heterodimeric targeting ligand binds the target molecule on cancer cells in the subject and the imaging molecule is visualized and/or the detectable molecule is detected on cancer cells in the subject, thereby diagnosing cancer in the subject.
  • a kit comprising a heterodimeric targeting ligand of this invention and/or a nucleic acid molecule of this invention and/or a vector of this invention and/or a cell of this invention and instructions for their use in the treatment of cancer in a subject and/or detection and/or localization of cancer cells and/or other diseased cells in a subject and/or diagnosis of cancer and/or other disorders in a subject.
  • a further embodiment of this invention includes a composition comprising, consisting essentially of or consisting of a heterodimeric targeting ligand of this invention, a nucleic acid molecule of this invention, a vector of this invention and/or a cell of this invention, as individual entities or in any combination, in a pharmaceutically acceptable carrier.
  • FIG. 1 Top: ELISA analysis of the competitive binding to immobilized EGFR between Erbitux and monomeric (A or B) or heterodimeric (A-B) targeting ligand.
  • Figure 3 Cell-based ELISA analysis of the inhibition of EGF-induced phosphorylation of EGFR by monomeric (A or B) or heterodimeric (A-B) targeting ligand using H292 cells. The phosphorylation of Y1068 of EGFR was determined by using PathScan pEGFR ELISA kit.
  • Figure 4. Xenogen imaging of A. nude mice bearing human A431 tumor (tumor size: 300 mg in al, 50 mg in a2), B. orthotopic AsPC-1 pancreatic cancer (bl: Luc signal in tumor, b2: NIR signal in whole body), both using 0.5 nmol IRDye-BiEGFR by i.v. injection.
  • C Biodistribution of IRDye-BiEGFR for A.
  • D. Preblocking of the binding of IRDye-BiEGFR with tumor by unlabeled BiEGFR (left) or buffer (right).
  • FIG. 7 SPR analysis of the competitive binding to immobilized EGFR between two different targeting ligands.
  • the binding of a monomeric (A or B) or heterodimeric (A-B) targeting ligand to EGFR immobilized on the surface of CM5 chip is demonstrated by the initial injection.
  • the binding saturation is demonstrated by the second injection of the same targeting ligand.
  • the competition binding of the first injected ligand to EGFR with another targeting ligand is examined by a third (and fourth in d) injection of an equal amount mixture of two targeting ligands to be compared.
  • FIG. 8 Binding of heterodimer to EGFR on CM5 chip is demonstrated by initial injection of heterodimer. Both A and B units on heterodimer are used for binding as demonstrated by no further binding of subsequently injected EGFR in the flow phase.
  • FIG. 9 Comparison of anti-tumor efficacy of the monomeric and heterodimeric ligands using EGFR-expressing H292 xenografts.
  • the PEGylated monomeric or heterodimeric ligands in PBS were i.p. injected every three days at a dose of 50 mg/kg.
  • the mean tumor sizes were obtained from groups of 3 mice.
  • a can mean one or more than one, depending on the context in which it is used.
  • a cell can mean one cell or multiple cells.
  • the term "about,” as used herein when referring to a measurable value such as an amount of a compound or agent of this invention, dose, time, temperature, and the like, is meant to encompass variations of ⁇ 20%, ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5%, or even ⁇ 0.1% of the specified amount.
  • the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.
  • any feature or combination of features set forth herein can be excluded or omitted.
  • the present invention is based on the unexpected discovery of a heterodimeric targeting ligand that binds a target molecule with high affinity and specificity, for use as a therapeutic agent, imaging and/or diagnostic agent and/or as an agent to deliver therapeutic agents to target cells.
  • the present invention provides a heterodimeric targeting ligand, comprising: a) a first target binding domain specific for a first site on an extracellular and/or intracellular domain of a target molecule; b) a second target binding domain specific for a second site on the extracellular and/or intracellular domain of the target molecule of (a), wherein the first site and the second site are nonoverlapping on the target molecule; and c) a linker peptide operably linking the first target binding domain of (a) and the second target binding domain of (b).
  • FIG. 1A An illustration of an exemplary heterodimeric targeting ligand of this invention is shown in Figure 1A, wherein the ligand has a first targeting binding domain labeled A and a second targeting binding domain labeled B and a linker peptide (black line) linking the two domains.
  • both A and B can be within the native ligand binding site or one of the domains A or B can be within the native ligand binding site and the other domain can be outside of the native ligand binding site as long as the functional effect of the binding of the heterodimeric targeting ligand is to modulate (e.g., disrupt or alter) binding of the native ligand to the receptor or modulate (e.g., disrupt or alter) some other interaction of the native ligand with the receptor due to the steric hindrance effect of the presence of the heterodimeric targeting ligand.
  • the first target binding domain and/or the second target binding domain can be based on or derived from protein domains identified and isolated from combinatorial libraries of single domain antibody or non-immunoglobuin antibody mimics with high diversity, as are known in the art.
  • Nonlimiting examples of such libraries include immunoglobulin antibody libraries with highly diversified complementarity determining (CDR) regions, non-immunoglobuin antibody mimic libraries such as Z domain, FN3 domain, or DARPI libraries with the interacting surface residues or loop residues partially or totally randomized.
  • the linker peptide of the heterodimeric targeting ligand of this invention can comprise, consist essentially of or consist of the following nonlimiting examples: a) a linker comprising of the amino acid sequence of SEQ ID NO: 1 ((GGGGS) n , wherein n can be any number, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.; e.g., (GGGGS) 3 ); b) a linker comprising the amino acid sequence of SEQ ID NO:2
  • ((GGGS) n wherein n can be any number, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.); c) a linker comprising the amino acid sequence of SEQ ID NO:3 ((GSGSGS) n , wherein n can be any number, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.); d) a linker comprising the amino acid sequence of SEQ ID NO:4 ((TPPTPSP) n , wherein n can be any number, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.); e) a linker comprising the amino acid sequence of SEQ ID NO:5
  • ((PQPQPK) n wherein n can be any number such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.); f) a linker comprising the amino acid sequence of SEQ ID NO:6 ((PQPQPE) n , wherein n can be any number such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.); g) a linker comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NO:7 (GPQPQP PQPK); h) a linker comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NO:8 (TPPTPSPSTPPTPSP; human IgAl heavy chain); i) a linker comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NO:9
  • PQPQPQPKPQPKPEPE camel IgG
  • k a linker comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NO: l 1 (PEPEPQPQGG); and 1) any combination of (a)-(k) above.
  • the linker peptide of this invention can also be a peptide of about 5 to about 50 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 56, 47, 48, 49, or 50) having an amino acid composition that provides for the functional feature of having the appropriate length and flexibility to facilitate the positioning of the first target binding domain and the second target binding domain of the heterodimeric targeting ligand for binding at their respective sites on the target molecule.
  • amino acids e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 56, 47, 48, 49, or 50
  • the length of the linker peptide can be determined and optimized based on the positioning of the target of the first targeting domain and the target of the second targeting domain relative to one another such that the heterodimeric targeting ligand binds the respective targets as described herein.
  • the target molecule to which the amino acid sequence of the invention is selected from the group consisting of: N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-N-(2-aminoethyl)-2-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • heterodimeric targeting ligand of this invention binds can comprise, consist essentially of or consist of an extracellular domain of a cell surface receptor such as, for example, a member of the epidermal growth factor receptor family (e.g., EGFR, HER1, HER2, HER3, HER4, etc., as are well known in the art), c-MET, vascular endothelial growth factor receptor (e.g., VEGFRl, VEGFR2, VEGFR3), insulin receptor, insulin-like growth factor receptor (IGFR), prostate specific membrane antigen (PSMA), mesothelin, hepsin, an integrin, mucin (e.g., MUC16), a cell surface cluster of differentiation (CD) molecule (nonlimiting examples of which include CD20, CD22, CD30, CD33, CD44, CD56, etc.), and any combination thereof.
  • a cell surface receptor such as, for example, a member of the epidermal growth factor receptor family (e.g., EGFR,
  • the target molecule to which the amino acid sequence of the invention is selected from the group consisting of: N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-N-(2-aminoethyl)-2-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • heterodimeric targeting ligand of this invention binds can comprise, consist essentially of or consist of a catalytic domain, regulatory domain and/or binding partner-interacting region of an intracellular, secreted, and/or membrane-bound protein such as a growth factor, a cytokine, a secreted protein (e.g., VEGF, bFGF, EGF, IGF, PDGF, TGF, TNF, IgE and their respective receptors, a kinase (e.g., PI3 , AKT, MEK, BRAF), a tyrosine kinase receptor (e.g., EGFR, HER2, VEGFR, PDGFR, c-MET, IGFR, etc.), a phosphatase (e.g., PTP1B, Cdc25, PTEN, SHP2, etc.), a protease (e.g., DPP-IV, caspase-3, cathepsin D, matript
  • methyltransferase a histone demethylase, etc.
  • the first targeting domain and/or the second target binding domain of the heterodimeric targeting ligand can comprise, consist essentially of or consist of an antibody fragment, a single domain antibody fragment, a single chain polypeptide of a VH or VL domain of an antibody, a peptide or protein derived from a binding and/or framework region of an antibody, a single domain antibody mimic based on non- immunoglobulin scaffolds (such as Z domains, FN3 domains, DARPIN, etc.), an epidermal growth factor receptor (EGFR)-binding, a HER2 -binding, an ot v p 3 -binding Z domain or FN3 domain or DARPIN, a short target-binding peptide containing natural and/or unnatural amino acids, a molecule or binding portion thereof which specifically binds to the extracellular domain of a cell surface receptor or to the binding partner-interacting region of an intracellular protein, and any combination thereof.
  • EGFR epidermal growth factor receptor
  • Nonlimiting examples of target binding domains of this invention include the following:
  • VDNKFNKEMWAAWEEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKLNDAQ APK (Friedman et al. Journal of Molecular Biology 376: 1388-1402 (2008))
  • BiEGFR-ZP (Ramamurthy et al. Structure 20(2):259-269 (2012) and Emanuel et al. PCT Publication No. WO2010060095) BiEGFR-ZP:
  • VDNKFNKEMWAAWEEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKLNDAQ APK (Friedman et al. Journal of Molecular Biology 376: 1388-1402 (2008))
  • VDNKFNKEMWAAWEEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKLNDAQ APK (Friedman et al. Journal of Molecular Biology 376: 1388-1402 (2008))
  • the peptides and polypeptides of this invention may also contain conservative substitutions where a naturally occurring amino acid is replaced by one having similar properties and which does not alter the function of the polypeptide or peptide. Such conservative substitutions are well known in the art.
  • modifications and changes may be made in the nucleic acid and/or amino acid sequence of the peptides and polypeptides of the present invention and still obtain a peptide or polypeptide having like or otherwise desirable characteristics. Such changes may occur in natural isolates or may be synthetically introduced using site-specific mutagenesis, the procedures for which, such as mis-match polymerase chain reaction (PCR), are well known in the art.
  • polypeptides and nucleic acids that contain modified amino acids and nucleotides, respectively (e.g., to increase the half-life and/or the therapeutic efficacy of the molecule), can be used in the methods of the invention.
  • the heterodimeric targeting ligand of this invention can further comprise a diagnostic molecule, a therapeutic molecule, an imaging molecule, or any combination thereof.
  • the heterodimeric targeting ligand can further comprise an N- and/or C- terminal cysteine for site-specific conjugation with other molecules such as a therapeutic molecule, a diagnostic molecule and/or an imaging molecule.
  • the heterodimeric targeting ligand of this invention can have a binding strength (affinity) for a target molecule that is increased about 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 500 fold, 1000 fold, 2000 fold, 3000 fold, 4000 fold, 5000 fold or 10,000 fold as compared with a monomer control (e.g., a monomer of the first target binding domain or a monomer of the second target binding domain).
  • a monomer control e.g., a monomer of the first target binding domain or a monomer of the second target binding domain.
  • the binding strength is increased from about 100 fold to about 1000 fold relative to a monomer control.
  • the heterodimeric targeting ligand of this invention has a target binding domain (e.g., the first target binding domain or the second target binding domain) that binds the extracellular domain of the target molecule at or near a binding site of a native ligand of the target molecule (e.g., EGF binding site of EGF receptor, etc.).
  • a target binding domain e.g., the first target binding domain or the second target binding domain
  • a native ligand of the target molecule e.g., EGF binding site of EGF receptor, etc.
  • the other target binding domain binds at a different location at or near the binding site of the native ligand of the target molecule such that binding of the heterodimeric ligand modulates (e.g., disrupts, prevents, alters) the biological interaction between the native ligand and the target molecule (e.g., modulates the binding of EGF to the EGF receptor, etc.).
  • the heterodimeric targeting ligand of this invention has a target binding domain (e.g., the first target binding domain or the second target binding domain) that binds a catalytic domain, regulatory domain and/or binding partner-interacting region of an intracellular, secreted, and/or membrane-bound target molecule at or near a binding site of the cofactor, substrate, or binding partner (e.g., ATP- or substrate-binding region of a kinase).
  • a target binding domain e.g., the first target binding domain or the second target binding domain
  • a catalytic domain, regulatory domain and/or binding partner-interacting region of an intracellular, secreted, and/or membrane-bound target molecule at or near a binding site of the cofactor, substrate, or binding partner (e.g., ATP- or substrate-binding region of a kinase).
  • the other target binding domain binds at a different location at or near the binding site of the cofactor, substrate, or binding partner such that binding of the heterodimeric ligand modulates (e.g., disrupts, prevents, alters) the biological interaction between the cofactor, substrate, or binding partner and the target molecule (e.g., the binding of a substrate to a kinase, etc.).
  • the present invention also provides an isolated nucleic acid molecule encoding the heterodimeric targeting ligand of this invention, a vector comprising the nucleic acid molecule of this invention, a cell (e.g., an isolated cell and/or transformed cell) comprising the nucleic acid molecule of this invention and a cell (e.g., an isolated cell and/or transformed cell) comprising the vector of this invention.
  • a vector comprising the nucleic acid molecule of this invention, a cell (e.g., an isolated cell and/or transformed cell) comprising the nucleic acid molecule of this invention and a cell (e.g., an isolated cell and/or transformed cell) comprising the vector of this invention.
  • Nucleic acid refers to single- or double-stranded molecules which may be DNA, comprised of the nucleotide bases A, T, C and G, or RNA, comprised of the bases A, U (substitutes for T), C, and G.
  • the nucleic acid may represent a coding strand or its complement.
  • Nucleic acids may be identical in sequence to the sequence, which is naturally occurring or may include alternative codons, which encode the same amino acid as that which is found in the naturally occurring sequence.
  • nucleic acids may include codons, which represent conservative substitutions of amino acids as are well known in the art.
  • the nucleic acids of this invention can also comprise any nucleotide analogs and/or derivatives as are well known in the art.
  • isolated nucleic acid means a nucleic acid separated or substantially free from at least some of the other components of the naturally occurring organism, for example, the cell structural components commonly found associated with nucleic acids in a cellular environment and/or other nucleic acids.
  • the isolation of nucleic acids can therefore be accomplished by well-known techniques such as cell lysis followed by phenol plus chloroform extraction, followed by ethanol precipitation of the nucleic acids.
  • the nucleic acids of this invention can be isolated from cells according to methods well known in the art for isolating nucleic acids.
  • the nucleic acids of the present invention can be synthesized according to standard protocols well described in the literature for synthesizing nucleic acids. Modifications to the nucleic acids of the invention are also contemplated, provided that the essential structure and function of the peptide or polypeptide encoded by the nucleic acid are maintained.
  • the nucleic acid encoding the peptide or polypeptide of this invention can be part of a recombinant nucleic acid construct comprising any combination of restriction sites and/or functional elements as are well known in the art that facilitate molecular cloning and other recombinant DNA manipulations.
  • the present invention further provides a recombinant nucleic acid construct comprising a nucleic acid encoding a peptide and/or polypeptide of this invention.
  • the present invention further provides a vector comprising a nucleic acid encoding a peptide and/or polypeptide of this invention.
  • the vector can be any expression vector (e.g., prokaryotic or eukaryotic) which contains all of the genetic components required for expression of the nucleic acid in cells into which the vector has been introduced, as are well known in the art.
  • the expression vector can be a commercial expression vector or it can be constructed in the laboratory according to standard molecular biology protocols.
  • the expression vector can comprise, for example, viral nucleic acid including, but not limited to, vaccinia virus, adenovirus, retrovirus, alphavirus and/or adeno-associated virus nucleic acid.
  • the nucleic acid or vector of this invention can also be in a liposome or a delivery vehicle, which can be taken up by a cell via receptor-mediated or other type of endocytosis.
  • compositions comprising the heterodimeric targeting ligand of this invention, the nucleic acid molecule of this invention, the vector of this invention and/or the cell of this invention, as individual components or in any combination, in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is meant a carrier that is compatible with other ingredients in the pharmaceutical composition and that is not harmful or deleterious to the subject.
  • the carrier can be a solid or a liquid, or both, and is preferably formulated with the composition of this invention as a unit-dose formulation, for example, a tablet, which may contain from about 0.01 or 0.5% to about 95% or 99% by weight of the composition.
  • the pharmaceutical compositions are prepared by any of the well-known techniques of pharmacy including, but not limited to, admixing the components, optionally including one or more accessory ingredients.
  • compositions of this invention can be used, for example, in the production of a medicament for the use in treatment of a disease and/or disorder as described herein.
  • compositions of this invention include those suitable for oral, rectal, topical, inhalation (e.g., via an aerosol) buccal (e.g., sub-lingual), vaginal, parenteral (e.g., subcutaneous, intramuscular, intradermal, intraarticular, intrapleural, intraperitoneal, intracerebral, intraarterial, intraocular or intravenous), topical (i.e., both skin and mucosal surfaces, including airway surfaces) and transdermal administration, although the most suitable route and dosage intervals in any given case will depend, as is well known in the art, on such factors as the species, age, gender and overall condition of the subject, the nature and severity of the condition being treated and/or on the nature of the particular composition (i.e., dosage, formulation, mode of administration) that is being administered.
  • the composition of this invention can be administered to a subject as an eye drop solution and/or via injection into the eye.
  • nucleic acids of this invention can be achieved by any one of numerous, well-known approaches, for example, but not limited to, direct transfer of the nucleic acids, in a plasmid or viral vector, or via transfer in cells or in combination with carriers such as cationic liposomes. Such methods are well known in the art and readily adaptable for use in the methods described herein.
  • a subject of this invention can be any animal that is susceptible to any of the disorders (e.g., cancer, diabetes, asthma, etc.) and can be treated and/or diagnosed and/or detected according to the methods described herein.
  • a subject of this invention include a mammal, a reptile, an avian or an amphibian (e.g., mouse, bird, dog, cat, cow, horse, fish).
  • the subject is a mammalian subject and in particular embodiments, the subject is a human.
  • a subject "in need thereof is a subject who is susceptible to having, is at increased risk of having, has been diagnosed as having, or is suspected of having a disorder (e.g., cancer, diabetes, asthma) of this invention
  • Effective amount refers to an amount of a protein, fragment, nucleic acid molecule, vector and/or composition of this invention that is sufficient to produce a desired effect, which can be a therapeutic effect and/or an improvement.
  • a “treatment effective” or “effective” amount is an amount that will provide some alleviation, mitigation, decrease or stabilization in at least one clinical symptom/sign in the subject.
  • An “effective amount” of a compound of this invention also refers to a nontoxic but sufficient amount to provide a desired therapeutic effect. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.
  • the effective amount or treatment effective amount will vary with the age, general condition of the subject, the severity of the condition being treated, the particular compound, agent, substance or composition administered, the duration of the treatment, the nature of any concurrent treatment, the pharmaceutically acceptable carrier used if any, and like factors within the knowledge and expertise of those skilled in the art.
  • an "effective amount” or “treatment effective amount” in any individual case can be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and/or by using routine pharmacological procedures. (Remington, The Science And Practice of Pharmacy, latest edition).
  • compositions of the invention are believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the subject. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides or other antagonists, and can generally be estimated based on EC50S found to be effective in in vitro and in vivo animal models.
  • a nonlimiting example of a dosage range for administration of a heterodimeric targeting ligand protein of this invention to a subject includes about 10 mg/kg to about 1000 mg/kg (e.g., about 1.0 mg/kg, about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, about 50 mg/kg, about 75 mg/kg, about 100 mg/kg, about 200 mg/kg, about 300 mg/kg, about 500 mg/kg, about 700 mg/kg, about 1000 mg/kg, about 2000 mg kg, etc.).
  • the protein may be given once or more daily, every other day, every three days, weekly, monthly or yearly, including over the lifetime of the subject
  • a nonlimiting example of a dosage range for administration of a nucleic acid molecule encoding the heterodimeric targeting ligand protein of this invention to a subject includes about 50 ⁇ g to about 10 mg per dose (e.g., about 10 ⁇ g, about 20 ⁇ g, about 50 ⁇ g, about 100 ⁇ 3 ⁇ 4 about 200 ⁇ g, about 500 ⁇ g, about 700 ⁇ g, about 1 mg, about 2 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg per dose, etc.).
  • the nucleic acid may be given once or more daily, every other day, every three days, weekly, monthly or yearly, including over the lifetime of the subject
  • Treat” or “treating” as used herein refers to any type of action or implementation that imparts a benefit to a subject that is diagnosed with, at risk of having, suspected to have and/or likely to have a disease or disorder that can be responsive in a positive way to a compound or composition of this invention.
  • a benefit can include an improvement in the condition of the subject (e.g., in one or more symptoms), delay and/or reversal in the progression of the condition, prevention or delay of the onset of the disease or disorder, etc.
  • the present invention provides various methods employing the heterodimeric targeting ligands of this invention.
  • the present invention provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of the heterodimeric targeting ligand of this invention and/or the nucleic acid molecule of this invention and/or the vector of this invention and/or the cell of this invention, wherein the first target binding domain and the second target binding domain are specific for a target molecule on a cancer cell in the subject and the heterodimeric targeting ligand binds the target molecule on cancer cells in the subject, thereby treating the cancer in the subject.
  • the heterodimeric targeting ligand can further comprise a cytotoxic moiety that kills the cancer cell subsequent to binding of the heterodimeric targeting ligand to cancer cells in the subject.
  • the cytotoxic moiety can be but is not limited to a small molecule, isotope, drug-containing nanoparticle, protein toxin, nucleic acid-based therapeutic agents (e.g., siRNA, miRNA, antisense, anti-gene oligonucleotide, etc.), or any combination thereof.
  • Nonlimiting examples of cytotoxic small molecules include auristatin E, maytansinoids, SN-38, calicheamicin, taxoids, epothilones, vinblastine, breflate, depsipeptide, and jasplakinolide or their derivatives as are known in the art.
  • Nonlimiting examples of radioisotopes include copper-67, yttrium-90, and indium- 1 1 1.
  • Nonlimiting examples of cytotoxic protein toxins include ricin, diphtheria toxin, colicin la, exotoxin A, abrin, and gelonin.
  • Nonlimiting examples of nanoparticles include gold nanoparticles, magnetite nanoparticles, PLGA-based nanoparticles, and liposome nanoparticles.
  • Nonlimiting examples of nucleic acid-based agents include siRNA, miRNA, antisense oligonucleotides, anti-gene oligonucleotides, etc., as are well known in the art.
  • the present invention further provides a method of detecting and/or localizing cancer cells in a subject, comprising administering to the subject an effective amount of the heterodimeric targeting ligand of this invention and/or the nucleic acid molecule of this invention and/or the vector of this invention and/or the cell of this invention, wherein the first targeting domain and the second targeting domain of the heterodimeric targeting ligand are specific for a target molecule on a cancer cell in the subject and the heterodimeric targeting ligand further comprises an imaging molecule and/or detectable molecule, whereby the heterodimeric targeting ligand binds the target molecule on cancer cells in the subject and the imaging molecule is visualized and/or the detectable molecule is detected at its binding location on cancer cells in the subject, thereby detecting and/Or localizing cancer cells in the subject.
  • the heterodimeric targeting ligand can also be simultaneously acting as a therapeutic agent to treat the cancer in the subject
  • the present invention provides a method of diagnosing cancer in a subject, comprising administering to the subject an effective amount of the heterodimeric targeting ligand of this invention and/or the nucleic acid molecule of this invention and/or the vector of this invention and/or the cell of this invention, wherein the first and second targeting domains are specific for a target molecule on a cancer cell in the subject and the heterodimeric targeting ligand further comprises an imaging molecule and/or detectable molecule, whereby the heterodimeric targeting ligand binds the target molecule on cancer cells in the subject and the imaging molecule is visualized and/or the detectable molecule is detected on cancer cells in the subject, thereby diagnosing cancer in the subject.
  • the imaging molecule can be but is not limited to an MRI contrast agent, a radioisotope for PET and nuclear medicine (e.g., 64 Cu-ATSM, 18 F- FDG, fluoride, FLT, FMISO, gallium, technetium-99m, etc.), a near-IR fluorescence molecule, a nanoparticle-containing imaging agent or any combination thereof.
  • MRI contrast agent e.g., 64 Cu-ATSM, 18 F- FDG, fluoride, FLT, FMISO, gallium, technetium-99m, etc.
  • a near-IR fluorescence molecule e.g., a near-IR fluorescence molecule, a nanoparticle-containing imaging agent or any combination thereof.
  • any suitable imaging modality can be used to view and/or image the imaging molecule in the body of a subject.
  • imaging modalities/systems include, but are not limited to, MRI Scanners, Ultrasound systems, X-ray systems including Computed Tomography (CT) Scanners, Combined Positron Emission Tomography and Computed Tomography (PET/CT) Scanners, Multispectral fluorescence camera systems, external and intraoperative fiber optic camera or detection systems, and implantable or catheter based medical sensors or detectors and the like as are well known in the art.
  • CT Computed Tomography
  • PET/CT Computed Tomography
  • Multispectral fluorescence camera systems Multispectral fluorescence camera systems
  • external and intraoperative fiber optic camera or detection systems and implantable or catheter based medical sensors or detectors and the like as are well known in the art.
  • Nonlimiting examples of a cancer antigen include, HER2/neu and BRCA1 antigens for breast cancer, MART-l/MelanA, gplOO, tyrosinase, TRP-1, TRP-2, NY-ESO-1, CDK-4, ⁇ -catenin, MUM-1, Caspase-8, KIAA0205, HPVE7, SART-1, PRAME, and pl5 antigens, members of the MAGE family, the BAGE family (such as BAGE-1), the DAGE/PRAME family (such as DAGE-1), the GAGE family, the RAGE family (such as RAGE-1), the SMAGE family, NAG, TAG-72, CA125, mutated proto-oncogenes such as p21ras, mutated tumor suppressor genes such as p53, tumor associated viral antigens (e.g., HPV16 E
  • MAGE-1, MAGE-2, MAGE-3, MAGE-4 and MAGE-11 Members of the GAGE family include, but are not limited to, GAGE-1, GAGE-6. See, e.g., review by Van den Eynde and van der Bruggen (1997) in Curr. Opin. Immunol. 9: 684-693, Sahin et al. (1997) in Curr. Opin. Immunol. 9: 709-716, and Shawler et al. (1997), the entire contents of which are incorporated by reference herein for their teachings of cancer antigens.
  • the cancer antigen/target molecule can also be, but is not limited to, human epithelial cell mucin (Muc-1; a 20 amino acid core repeat for Muc-1 glycoprotein, present on breast cancer cells and pancreatic cancer cells), MUC-2, MUC-3, MUC-18, the Ha-ras oncogene product, carcino-embryonic antigen (CEA), the raf oncogene product, CA-125, GD2, GD3, GM2, TF, sTn, gp75, EBV-LMP 1 & 2, HPV-F4, 6, 7, prostatic serum antigen (PSA), prostate-specific membrane antigen (PSMA), alpha-fetoprotein (AFP), C017-1A, GA733, gp72, p53, the ras oncogene product, ⁇ -HCG, gp43, HSP-70 , pl7 mel, HSP-70, gp43, HMW, HOJ-1, melanoma gan
  • the cancer antigen/target molecule of this invention can also be an antibody produced by a B cell tumor (e.g., B cell lymphoma; B cell leukemia; myeloma; hairy cell leukemia), a fragment of such an antibody, which contains an epitope of the idiotype of the antibody, a malignant B cell antigen receptor, a malignant B cell immunoglobulin idiotype, a variable region of an immunoglobulin, a hypervariable region or complementarity determining region (CDR) of a variable region of an immunoglobulin, a malignant T cell receptor (TCR), a variable region of a TCR and/or a hypervariable region of a TCR.
  • the cancer antigen of this invention can be a single chain antibody (scFv), comprising linked VH, and VL domains, which retains the conformation and specific binding activity of the native idiotype of the antibody.
  • the present invention is in no way limited to the cancer antigens listed herein.
  • Other cancer antigens be identified, isolated and cloned by methods known in the art such as those disclosed in U.S. Pat. No. 4,514,506, the entire contents of which are incorporated by reference herein.
  • Nonlimiting examples of a cancer of this invention include (in any combination) B cell lymphoma, T cell lymphoma, myeloma, leukemia, hematopoietic neoplasias, thymoma, lymphoma, sarcoma, lung cancer, , non-Hodgkins lymphoma, Hodgkins lymphoma, uterine cancer, adenocarcinoma, breast cancer, pancreatic cancer, colon cancer, colorectal cancer, anal cancer, lung cancer, renal cancer, bladder cancer, liver cancer, prostate cancer, ovarian cancer, skin cancer, primary or metastatic melanoma, squamous cell carcinoma, basal cell carcinoma, brain cancer, angiosarcoma, hemangiosarcoma, head and neck carcinoma, thyroid carcinoma, soft tissue sarcoma, bone cancer, bone sarcoma, testicular cancer, uterine cancer, cervical cancer, gastrointestinal cancer, stomach cancer, esophageal cancer
  • the target molecule can comprise, consist essentially of or consist of an extracellular domain of a cell surface receptor (such as epidermal growth factor receptor family members (EGFR, HER2, HER3, HER4, etc.), c-MET, VEGFR, insulin receptor, insulin-like growth factor receptor, prostate specific membrane antigen, mesothelin, hepsin, an integrin, mucin (e.g., MUC16, etc.), a cell surface cluster of differentiation (CD) molecule, (e.g., CD20, CD22, CD30, CD33, CD44, CD56, etc.), and any combination thereof.
  • a cell surface receptor such as epidermal growth factor receptor family members (EGFR, HER2, HER3, HER4, etc.
  • c-MET VEGFR
  • insulin receptor insulin-like growth factor receptor
  • prostate specific membrane antigen mesothelin
  • mesothelin hepsin
  • mucin e.g., MUC16, etc.
  • CD cell surface
  • the target molecule can comprise, consist essentially of or consist of acatalytic domain, regulatory domain and/or binding partner- interacting region of an intracellular, secreted, and/or membrane-bound protein [e.g., a growth factor, a cytokine, a secreted protein (e.g., VEGF, bFGF, EGF, IGF, PDGF, TGF, TNF, IgE and their respective receptors)], a kinase, a tyrosine kinase receptor (e.g., PI3K, AKT, MEK, EGFR, HER2, VEGFR, PDGFR, c-MET, insulin-like growth factor receptor, BRAF, etc.), a phosphatase (e.g., PTP1B, Cdc25, PTEN, SHP2), a protease (e.g., DPP-TV, caspase-3, cathepsin D, ma
  • a growth factor e.g
  • the present invention also provides a kit comprising a heterodimeric targeting ligand of this invention and/or a nucleic acid molecule of this invention and/or a vector of this invention and/or a cell of this invention and instructions, reagents, buffers, diluents, etc., for their use in the treatment of cancer in a subject and/or detection and/or localization of cancer cells and/or other diseased cells in a subject and/or diagnosis of cancer and/or other disorders (e.g., diabetes, asthma) in a subject.
  • a kit comprising a heterodimeric targeting ligand of this invention and/or a nucleic acid molecule of this invention and/or a vector of this invention and/or a cell of this invention and instructions, reagents, buffers, diluents, etc., for their use in the treatment of cancer in a subject and/or detection and/or localization of cancer cells and/or other diseased cells in a subject and/or diagnosis of cancer and/or
  • Targeting ligands that bind to the same target such as EGFR at two different sites could have synergistic avidity effect and therefore possess significantly increased target- binding strength (Fig. 1).
  • the targeting ligand could effectively compete with EGF to disrupt the biologically important interaction between EGF and its receptor.
  • Such targeting ligands have the potential to serve as the next generation targeted anti-cancer biopharmaceuticals, while being used as an excellent targeting ligand for targeted delivery of various therapeutic and/or imaging agents. Since the target-binding protein domains can be isolated from a library with very high diversity, sequences that bind at different regions on the target can be isolated. With the availability of such target-binding protein domains, fusion proteins can be generated from two monomeric targeting ligands, each binding at a unique region on the ECD of the EGFR. This can be done by competitive-binding experiments using surface plasma resonance (SPR), flow cytometry, and confocal microscopy.
  • SPR surface plasma resonance
  • a flexible linker as a recombinant fusion protein (A-B heterodimer w/linker), as shown in Fig. 1.
  • a flexible linker such as (GGGGS) n is used, and the linker length is tuned and optimized to achieve the best effect.
  • An EGFR-binding heterodimeric targeting ligand (“A-B” or "BiEGFR”) has been generated from two monomeric single domain antibody mimics (Z domain- and FN3 domain- based EGFR-binding ligands) with each binding to a different region on the EGFR.
  • the target-binding properties of the heterodimeric BiEGFR were compared with the respective monomers and with commercially available monoclonal antibodies using the methods described herein. As shown in Fig. 1 (right), both monomers bound to EGFR with affinities around 20 nM.
  • the A-B heterodimer (BiEGFR) bound to EGFR much more tightly, with a binding affinity around 30 pM, about 600-800 times higher than that of the monomers.
  • monomer A nor monomer B blocked the binding of Erbitux to EGFR.
  • the binding of Erbitux to EGFR was completely blocked by heterodimeric BiEGFR as shown in Fig. 2 (left), indicating that BiEGFR binds to EGFR much more strongly than Erbitux.
  • Fig. 2 shows the binding is highly specific to EGFR-positive AsPC-1 cells, but not to normal HPNE pancreas cells.
  • heterodimeric targeting ligand inhibits phosphorylation of specific tyrosine residues in the cytoplasmic tail of EGFR
  • positive or control cells are incubated with different concentrations of monomeric or heterodimeric targeting ligands, followed by stimulation with EGF.
  • the cells are lysed and used to determine the phosphorylation of critical residues at the cytoplasmic tail using a PathScan pEGFR kit (Cell Signaling) for phosphorylated tyrosine residues.
  • PathScan pEGFR kit Cell Signaling
  • heterodimeric targeting ligands In vivo cancer targeting using heterodimeric targeting ligands. Experiments were conducted to investigate whether such heterodimeric targeting ligands are effective in targeting tumor cells in vivo.
  • the A-B heterodimer (BiEGFR) was conjugated with a near- infrared (700 nm-900 nm) fluorescence (NIRF) probe such as IRDye 800 (from Li-COR) that is safe to use and able to penetrate into living tissues to depths of several centimeters with a high transmission and low autofluorescence background.
  • NIRF near- infrared fluorescence
  • a recombinant BiEGFR was generated and purified containing the only Cys at the C-terminus, which allowed for site-specific conjugation with IRDye 800CW-maleimide (Li-COR).
  • the IRDye- BiEGFR was injected via the tail vein into mice bearing EGFR-expressing A431, H292 or AsPC-1 tumor xenografts.
  • whole-body fluorescence imaging of A431 bearing mice demonstrated the strongest tumor accumulation with no or minimal accumulation in the heart, lungs, or spleen (Figs. 4A and 4C). There was modest accumulation in the liver and kidneys presumably due to the elimination of the protein.
  • orthotopic pancreatic cancer was detected very clearly, consistent with the tumor signal from the luciferase activity in the primary tumor and metastases. It should be noted that such targeted imaging reagents can effectively block the tumor-inducing EGF/EGFR pathway, and therefore possess the unique advantage of conferring therapeutic effect during the imaging process, a desirable feature for the next generation of theranostics.
  • EGFR-positive A431, AsPC-1, H292, HER2-positive SKOV3, PSMA-positive LNCaP cells and the corresponding negative control cells were obtained from the UNC Tissue Culture Facility or ATCC. All cell lines were maintained by serial passage at 37°C in 5% C0 2 in an appropriate medium supplemented with 10% fetal bovine serum, 100 units/mL of penicillin, and 0.1 mg/mL streptomycin.
  • the codon-optimized DNA sequences that code for various monomeric and heterodimeric targeting ligands were codon-optimized and custom-synthesized by GenScript (Piscataway, NJ). After PCR amplification, the gene products were digested with Nco I and Xho I. The digested fragments were cloned into the corresponding sites (Nco I and Xho I) of pET28b (Novagen, Darmstadt, Germany). The cloned plasmids were confirmed by sequencing prior to use in protein expression.
  • Each expression vector was transformed into E. coli BL21 (DE3) Rosseta cells (Novagen, Darmstadt, Germany). The positive clones were selected on LB plates containing kanamycin (50 ⁇ g/mL) and chloramphenicol (34 ⁇ g/mL). A single colony was selected and grown in 10 mL of LB medium overnight at 37°C. The resulting culture was added to a flask with 1 L of LB media containing kanamycin (50 ⁇ g/mL) and chloramphenicol (34 ⁇ g/mL). The cells were grown at 37°C until the optical density (at 600 nm) reached 0.6 to 1.0.
  • IPTG IPTG with a final concentration of 0.5 to 1 mM was then added to the cell cultures, followed by incubation at 30°C for 16 h. After induction, the cells were spun down at 3,000 g for 10 min at 4°C, and the pellet was stored at -20°C until use. To purify the monomeric and heterodimeric ligands, the cell pellet was resuspended in buffer A (25mM HEPES pH 7.4 and 300mM NaCl) and sonicated for 1 min for a total of 5 times. The soluble fraction was recovered by centrifugation at 12,000 g for 10 min at 4°C.
  • buffer A 25mM HEPES pH 7.4 and 300mM NaCl
  • the resulting fraction was loaded onto a TALON metal affinity column (Clontech, Mountainview, CA) pre-equilibrated with buffer A. After washing (-20 column volumes) with buffer B (buffer A with 20mM imidazole), the protein of interest was eluted with buffer C (buffer A with 200mM imidazole). The quality of the purified proteins was examined by SDS-PAGE.
  • each monomeric and heterodimeric molecule was reacted with fluorescein isothiocyanate (FITC) (ACROS organics) or IRDye 800CW NHS ester (LI-COR) in 50 mM borate buffer (pH 8.5). Briefly, 1 mg of each protein was reacted with a 25 molar excess of FITC or IRDye 800CW NHS ester in the reaction buffer and incubated at room temperature for 2 h. The resulting mixture was quenched by the addition of 100 mM Tris-HCl (pH 8.8) at room temperature for 1 h.
  • FITC fluorescein isothiocyanate
  • IRDye 800CW NHS ester LI-COR
  • each monomeric or heterodimeric molecule containing a C-terminal cysteine residue was reacted with IRDye 800CW maleimide (LI-COR) in a buffer containing 20 mM sodium phosphate and 150 mM NaCl, pH 7.2. Briefly, 1 mg of each protein was reacted with a 25 molar excess of IRDye 800CW maleimide in the reaction buffer and incubated at room temperature for 2 h.
  • Cell-based ELISA was performed by incubating EGFR-expressing H292 cells in 24- well plates with serial dilutions of different targeting ligands followed by stimulation with 100 ng/ml of EGF. Cells were then lysed and the phosphorylation of Y1068 of EGFR was determined by using PathScan Phospho-EGFR Sandwich ELISA kit from Cell Signaling. BIAcore analysis
  • the BIAcore 2000 (BIAcore AB, Uppsala, Sweden) was used for surface plasmon resonance analysis. 1 ⁇ g of purified extracellular domain of recombinant human EGFR ECD- Fc or HER2 ECD-Fc (R&D System, Minneapolis, MN) was diluted in a buffer containing 10 mM sodium acetate pH 5.0 and immobilized on CM5 sensor chip (GE healthcare) by amine coupling according to the manufacturer's instruction (about 4,000 resonance units).
  • the competition binding of the first injected ligand to EGFR with another targeting ligand was performed by a third injection of an equal amount mixture of two targeting ligands to be compared.
  • the binding of heterodimer to EGFR immobilized on CM5 chip was first achieved by the initial injection of heterodimer, followed by a second injection of Erbitux.
  • the extracellular domain of EGFR immobilized on CM5 chip was first saturated with heterodimer, followed by a subsequent injection of EGFR in the flow phase.
  • the dissociation equilibrium constant (K D ), the association rate (K a ), and the dissociation rate (3 ⁇ 4) were calculated using BIAevaluation software (BIAcore) by fitting the data on a one to one Langmuir binding model.
  • Purified monomeric or heterodimeric targeting ligand containing a C-terminal cysteine residue was first treated with 10 mM DTT followed by exchanging to PBS buffer using a NAP- 10 column. PEG(5 kDa or 30 kDa)-maleimide was then added to 5 to 10 molar excess and incubated at room temperature for 2 to 4 hours. The PEGylated targeting ligands were then purified by Ni-NTA column and characterized by SDSA-PAGE. The resulting targeting ligands were passed through an endotoxin-removal column prior to use in animals. In vivo imaging of tumor in xenograft animal models.
  • Immunodeficient SCID nude mice purchased at 5-6 weeks of age, either male or female, were used for in vivo tumor targeting studies. The mice were first subcutaneously implanted with EGFR-expressing cancer cells, including A431, H292, and AsPc-1 cells, to establish a tumor with appropriate size. An AsPC-1 orthotopic animal model was also used for in vivo imaging. 0.5 to 2.0 nmol of IRDye 800CW-labeled monomeric or heterodimeric targeting ligand was injected through the tail vein into mice bearing EGFR-expressing A431, H292 or AsPC-1 tumor xenografts. Whole-body fluorescence imaging of the mice was performed at multiple time points using an IVIS Imaging System.
  • heterodimeric ligand was first injected to block EGFR on the surface of cancer cells prior to the injection of IRDye-labeled heterodimer.
  • EGFR-expressing H292 cells were first subcutaneously implanted in the flank of the nude mice (3-8 mice per group) to establish tumor sizes of approximately 100-1 0 mm 3 in 3 weeks.
  • the PEGylated monomeric or A-B heterodimer targeting ligands were highly purified and prepared in PBS for intraperitoneal (i.p.) injection at a dose of 50 mg/kg every three days.
  • the mean tumor sizes were obtained from groups of 3 to 8 mice.
  • This invention is directed to the generation of a class of novel heterodimeric targeting ligands that tightly and specifically bind to a drug target of interest at two nonoverlapping epitopes with synergistic avidity effect.
  • Such heterodimeric targeting ligands can bind to the target of interest much more tightly than the corresponding monomers with significantly reduced off rate and therefore retain on target for a much longer time.
  • the heterodimeric ligand could effectively inhibit the biologically important interaction between the natural ligand and its receptor, and therefore block the signaling pathways mediated by this receptor.
  • the target can be any intracellular or extracellular protein or any protein with post-translational modifications, including but not limited to numerous extracellular domains of many membrane-bound receptors. Since many drug targets are membrane-bound receptors, as nonlimiting examples, this technology was demonstrated by developing a heterodimeric targeting ligand (A-B) from two different single domain targeting ligands with each binding to a unique region on the extracellular domain of EGFR as illustrated in Figure 5. The target binding and biochemical properties of this heterodimeric targeting ligand against EGFR were analyzed.
  • A-B heterodimeric targeting ligand
  • SPR surface plasmon resonance
  • the 0ff of the optimal heterodimer is more than 10,000 times slower than that of the monomers.
  • FIG. 7A shows that monomer A and monomer B do not compete with each other and therefore bind at different sites on the EGFR.
  • Figures 7B and 7C show that A-B heterodimer can still bind to EGFR that is saturated by either monomer A or monomer B, respectively.
  • Figure 7D shows that when EGFR is saturated with A-B heterodimer, neither monomer A nor monomer B can compete with it. Further studies were done to investigate whether both the A and the B units of A-B heterodimer were used in interaction with EGFR.
  • heterodimeric targeting ligand can more effectively inhibit the EGF-induced activation of EGFR signaling pathways.
  • EGFR-positive H292 lung cancer cells were incubated with serial dilutions of a monomeric or heterodimeric targeting ligand to block EGFR, followed by stimulation of cells with EGF.
  • the IC50 for the inhibition of EGF-induced phosphorylation of EGFR is at least one magnitude lower than that of the monomers, suggesting the A-B heterodimer is not only a much better targeting ligand for the extracellular domain of EGFR, but also more effectively blocks the EGF- induced activation of EGFR, presumably by competitively blocking the binding of endogenous EGF to EGFR.
  • This unique feature makes it possible to use such heterodimeric targeting ligands as theranostic agents, simultaneously serving as an imaging agent and a therapeutic agent.
  • the in vivo targeting efficacy of the EGFR-binding A-B heterodimer was investigated using xenograft and orthotopic animal models.
  • a recombinant A-B heterodimer containing the only cysteine residue at the C-terminus was generated and purified, which allowed for site-specific conjugation with an imaging probe.
  • the A-B heterodimer was conjugated with a near-infrared (700 nm-900 nm) fluorescence (NIRF) probe such as IRDye 800CW maleimide (from Li-COR) that is safe to use and able to penetrate into living tissues to depths of several centimeters with a high transmission and low autofluorescence background.
  • NIRF near-infrared fluorescence
  • the purified IRDye-labeled A-B heterodimer was injected via the tail vein to mice bearing EGFR- expressing A431, H292 or AsPC-1 tumor xenografts.
  • whole-body fluorescence imaging of A431 bearing mice demonstrated the strongest tumor accumulation with no or minimal accumulation in the heart, lungs, or spleen (Figs.
  • mice xenografted with EGFR-expressing tumor were used.
  • the mice were first subcutaneously implanted with EGFR-expressing H292 cells.
  • the purified PEGylated monomeric or A-B heterodimer targeting ligands in PBS were i.p. injected at a dose of 50 mg'kg every three days.
  • Figure 9 shows that compared to the monomeric targeting ligand, the A-B heterodimer has significantly improved anti-tumor activity, with the tumor sizes remaining almost unchanged during the period of treatment.

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Abstract

L'invention concerne un ligand de ciblage hétérodimère comprenant : a) un premier domaine de liaison à une cible spécifique pour un premier site sur un domaine extracellulaire ou intracellulaire d'une molécule cible ; b) un second domaine de liaison à une cible spécifique pour un second site sur le domaine extracellulaire ou intracellulaire de la molécule cible de (a), le premier site et le second site ne se chevauchant pas sur la molécule cible ; et c) un peptide de liaison qui lie le premier domaine de liaison à une cible de (a) et le second domaine de liaison à une cible de (b). L'invention concerne également des procédés d'utilisation dudit ligand de ciblage hétérodimère afin de traiter et/ou de diagnostiquer des troubles.
PCT/US2012/039187 2011-05-23 2012-05-23 Procédés et compositions pour ligands de ciblage hétérodimères WO2012162418A1 (fr)

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WO2015048724A1 (fr) * 2013-09-30 2015-04-02 The University Of North Carolina At Chapel Hill Procédés et compositions pour système d'auto-assemblage de nanoparticules et de microparticules pour un ciblage multispécifique
US9416170B2 (en) 2011-10-31 2016-08-16 Bristol-Myers Squibb Company Fibronectin binding domains with reduced immunogenicity
WO2016179534A2 (fr) 2015-05-06 2016-11-10 Janssen Biotech, Inc. Domaines de type iii de fibronectines fixant l'antigène membranaire spécifique de la prostate
CN107847594A (zh) * 2015-05-06 2018-03-27 詹森生物科技公司 前列腺特异性膜抗原(psma)双特异性结合剂及其用途
CN108129566A (zh) * 2017-12-31 2018-06-08 中国科学院武汉病毒研究所 靶向间皮素的高亲和力c-型单域抗体及其制备方法与应用
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US11447539B2 (en) 2016-12-14 2022-09-20 Janssen Biotech, Inc. PD-L1 binding fibronectin type III domains
US11628222B2 (en) 2019-10-14 2023-04-18 Aro Biotherapeutics Company CD71 binding fibronectin type III domains
US11781138B2 (en) 2019-10-14 2023-10-10 Aro Biotherapeutics Company FN3 domain-siRNA conjugates and uses thereof
US11857628B2 (en) 2016-12-07 2024-01-02 Molecular Templates, Inc. Shiga toxin A subunit effector polypeptides, Shiga toxin effector scaffolds, and cell-targeting molecules for site-specific conjugation
US11932680B2 (en) 2016-12-14 2024-03-19 Janssen Biotech, Inc. CD8A-binding fibronectin type III domains
WO2024064261A1 (fr) * 2022-09-21 2024-03-28 University Of Houston System Ingénierie de l'administration non cytotoxique de protéines par des lymphocytes t par fusion à npc2

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US10370414B2 (en) 2012-11-30 2019-08-06 Molecular Partners Ag Binding proteins comprising at least two repeat domains against HER2
WO2014083208A1 (fr) * 2012-11-30 2014-06-05 Molecular Partners Ag Protéines de liaison comprenant au moins deux domaines de répétition dirigées contre her2
WO2015048724A1 (fr) * 2013-09-30 2015-04-02 The University Of North Carolina At Chapel Hill Procédés et compositions pour système d'auto-assemblage de nanoparticules et de microparticules pour un ciblage multispécifique
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JP2018515087A (ja) * 2015-05-06 2018-06-14 ヤンセン バイオテツク，インコーポレーテツド 前立腺特異的膜抗原結合フィブロネクチンｉｉｉ型ドメイン
WO2016179534A2 (fr) 2015-05-06 2016-11-10 Janssen Biotech, Inc. Domaines de type iii de fibronectines fixant l'antigène membranaire spécifique de la prostate
US10844111B2 (en) 2015-05-06 2020-11-24 Janssen Biotech, Inc. Prostate specific membrane antigen binding fibronectin type III domains
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KR102625793B1 (ko) * 2015-05-06 2024-01-15 얀센 바이오테크 인코포레이티드 전립선 특이적 막 항원 결합 피브로넥틴 iii형 도메인
AU2016258174B2 (en) * 2015-05-06 2022-01-20 Janssen Biotech, Inc. Prostate specific membrane antigen binding fibronectin type III domains
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JP7307133B2 (ja) 2015-05-06 2023-07-11 ヤンセン バイオテツク，インコーポレーテツド 前立腺特異的膜抗原結合フィブロネクチンｉｉｉ型ドメイン
US11857628B2 (en) 2016-12-07 2024-01-02 Molecular Templates, Inc. Shiga toxin A subunit effector polypeptides, Shiga toxin effector scaffolds, and cell-targeting molecules for site-specific conjugation
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