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WO2018222987A1 - Constructions ciblées - Google Patents

Constructions ciblées Download PDF

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Publication number
WO2018222987A1
WO2018222987A1 PCT/US2018/035592 US2018035592W WO2018222987A1 WO 2018222987 A1 WO2018222987 A1 WO 2018222987A1 US 2018035592 W US2018035592 W US 2018035592W WO 2018222987 A1 WO2018222987 A1 WO 2018222987A1
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WO
WIPO (PCT)
Prior art keywords
protein
targeted construct
receptor
moiety
binding
Prior art date
Application number
PCT/US2018/035592
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English (en)
Inventor
Sudhakar Kadiyala
Richard Wooster
Mark T. Bilodeau
Benoît MOREAU
Brian H. White
Original Assignee
Tarveda Therapeutics, Inc.
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Filing date
Publication date
Application filed by Tarveda Therapeutics, Inc. filed Critical Tarveda Therapeutics, Inc.
Publication of WO2018222987A1 publication Critical patent/WO2018222987A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—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 Fc-receptors, e.g. CD16, CD32, CD64
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/705—Fusion polypeptide containing domain for protein-protein interaction containing a protein-A fusion

Definitions

  • This invention is generally in the field of targeted constructs comprising protein- binding moieties that bind to a protein (such as FcRn, albumin, and IgG) in vivo for drug delivery and drug half-life extension.
  • a protein such as FcRn, albumin, and IgG
  • therapeutic small molecule drugs and therapeutic peptide drugs are generally directed towards improving the pharmaceutical properties of the drugs and, in some cases, improving their poor pharmacokinetics and other factors.
  • therapeutic peptides ⁇ 100 amino acids
  • macromolecule drug carriers such as polyethylene glycol (PEG), antibody Fc domain and human serum albumin (HSA or albumin) to extend their half-life, so that the therapeutic potential of the peptides may be realized without frequent administration and/or high doses.
  • macromolecule drug carriers also have an enhanced permeability and retention (EPR) effect and accumulate preferentially in tumor tissues and inflame tissues.
  • EPR enhanced permeability and retention
  • the macromolecule drug carriers may introduce steric hindrance and comprise in vivo efficacy.
  • macromolecule drug carriers may be detrimental to the penetration of the drugs into a target tissue.
  • protein-binding moieties such as antibody mimic molecules that bind to a protein, such as neonatal Fc receptor (FcRn), albumin, or IgG, in vivo.
  • targeted constructs comprising such protein-binding moieties.
  • the targeted constructs may further comprise a targeting moiety, an active agent, and/or a conjugate comprising a targeting moiety and an active agent.
  • the targeted constructs are useful for improving the delivery of active agents such as tumor cytotoxic agents to tumor tissue and tumor cells via both passive and active targeting mechanism.
  • the targeted constructs may be used to increase the circulation time of active agents and/or to enable routes of administration other than injection, such as oral administration.
  • Methods of making the protein-binding moieties and targeted constructs are provided. Methods are also provided for treating a disease or condition, the method comprising administering a therapeutically effective amount of the targeted constructs to a subject in need thereof.
  • the targeted constructs are targeted to a cancer or hyperproliferative disease, for example, lymphoma (e.g., non-Hodgkin's lymphoma), renal cell carcinoma, prostate cancer, ovarian cancer, breast cancer, colorectal cancer,
  • neuroendodrine cancer endometrial cancer, pancreatic cancer leukemia, lung cancer, glioblastoma multiforme, stomach cancer, liver cancer, sarcoma, bladder cancer, testicular cancer, esophageal cancer, head and neck cancer, and leptomeningeal carcinomatosis.
  • the present disclosure provides a method for targeted delivery of active agent, extending the serum half-life of an active agent, and/or modifying pharmacokinetic and pharmacodynamic properties of an active agent.
  • the present disclosure also provides protein- binding moieties such as antibody mimic molecules or small molecules that bind to a protein, such as the neonatal Fc receptor (FcRn), albumin, or IgG, in vivo.
  • protein-binding moieties may bind to such a protein via covalent bonds or non-covalent interactions, such as complexation, hydrogen bonds, ionic bonds, van der Waals interactions, and hydrophobic bonds.
  • the targeted constructs may further comprise a targeting moiety, an active agent, and/or a conjugate comprising a targeting moiety and an active agent.
  • the targeted constructs deliver an active agent to a diseased tissue such as tumor tissues and to improve pharmacological and/or pharmacokinetic properties of the active agent.
  • the amount of the active agent delivered at an action site is increased and/or the active agent's systemic toxicity is decreased.
  • These targeted constructs combine passive targeting of macromolecules and active targeting of the targeting moiety. They also have extended half- life and increased penetration in diseased tissues. For example, the serum half-life of the targeted constructs is longer than the active agent alone. Controlled and sustained release of the active agent can be achieved with targeted constructs of the present disclosure.
  • routes of administration other than injection such as oral administration, is enabled.
  • the protein-binding moieties are antibody mimetic molecules that bind to a protein, such as FcRn, albumin, or IgG, in vivo to extend serum half- lives.
  • Targeted constructs comprising such antibody mimetic molecules are also provided.
  • the targeted constructs may further comprise a targeting moiety, an active agent, and/or a conjugate comprising a targeting moiety and an active agent.
  • An antibody mimetic refers to molecules that bind to antigens and/or receptors, but are not antibodies.
  • An antibody mimetic may mimic the binding and/or functional site of an antibody.
  • Examples of an antibody mimetic include antibody fragments, artificial peptides or proteins with a molecular weight of about 3 to about 60 kDa.
  • the antibody mimetic may comprise between 4 and 500 amino acid residues, between 5 and 300 amino acid residues, between 10 and 200 amino acid residues, or between 20 and 150 amino acid residues, for example about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acid residues.
  • the molecular weight may between about 3 to about 10 kDa, about 10 to about 20 kDa, about 20 to about 30 kDa, about 30 to about 40 kDa, about 40 to about 50 kDa, or about 50 to about 60 kDa.
  • the protein-binding moieties are small molecules that bind to a protein, such as FcRn, albumin, or IgG, in vivo to extend serum half-lives.
  • the small molecule may react with a functional group on the protein to form a covalent bond.
  • the small molecule may bind to the protein via non-covalent interactions.
  • Targeted constructs comprising such small molecules and a conjugate comprising a targeting moiety and an active agent are also provided.
  • a small molecule refers to an organic group that is less than 2000 g/mol in molecular weight, less than 1500 g/mol, less than 1000 g/mol, less than 800 g/mol, or less than 500 g/mol. Small molecules may also be small peptides with less than 20 amino acids. They are non-polymeric and/or non-oligomeric.
  • the targeted constructs may comprise at least one protein- binding moiety connected to an active agent with an optional internal linker moiety.
  • the targeted constructs may comprise at least one protein- binding moiety connected to a targeting moiety with an optional internal linker moiety.
  • the targeted constructs may comprise at least one conjugate of an active agent connected to a targeting moiety with an optional internal linker moiety.
  • the conjugate is connected to at least one protein-binding moiety by an external linker.
  • An internal linker moiety or an internal linker refers to a linker in a conjugate, wherein the internal linker connects an active agent and a targeting moiety, or connects a protein-binding moiety and a targeting moiety or an active agent.
  • the internal linker may be non-cleavable or cleavable.
  • the internal linker can be cleaved in an intracellular fashion after uptake of the targeted constructs by the targeted receptor, including but not limited to, pH-sensitive linkers for release in organelles with lowered pH, or linkers cleaved by intracellular proteases such as Cathepsin B, or disulfide linkers.
  • An external linker refers to the linker that is not an integral part of a conjugate, wherein the external linker connects the conjugate with a protein-binding moiety.
  • the external linker may be non-cleavable or cleavable.
  • the external linker can be cleaved in an extracellular fashion, including but not limited to, pH-sensitive linkers for release in the lower pH of the tumor environment, or linkers cleaved by extracellular proteases such as matrix metalloproteinases, or hypoxia-activated linkers.
  • the internal linker and the external linker may be the same or different.
  • the internal linker and the external linker are both cleavable, but are different from each other.
  • the external linker is less stable than the internal linker in conditions expected to be operative in the tumor, i.e., tumor microenvironment. Conditions operative in the tumor may include low pH, a reducing environment, or in the presence of certain enzymes such as matrix metalloproteinases.
  • the external linker is cleavable in excellular manner and the internal linker is cleavable in intracellular manner. As a result, the external linker is cleaved in an extracellular manner in a tumor
  • a conjugate is released from a targeted construct assembly, or separated from a protein, an engineered protein, or a pharmacokinetic modulating unit before the release of an active agent.
  • the conjugates penetrate deeper and faster into the tumor than the targeted construct assembly, because the conjugates have a smaller molecular weight once released.
  • the active agent in the conjugate is only released inside cells.
  • the relative stabilities or cleavage rates of the internal linker and the external linker can be evaluated in a tumor microenvironment.
  • the cleavage rate of the external linker may be at least 25%, at least 50%, at least 100%>, at least 150%, at least 2 folds, at least 3 folds, at least 4 folds, at least 5 folds faster than the internal linker in a tumor microenvironment.
  • a "cleavable" linker refers to any linker which can be cleaved physically or chemically. Examples for physical cleavage may be cleavage by light, radioactive emission or heat, while examples for chemical cleavage include cleavage by re- dox-reactions, hydrolysis, pH-dependent cleavage or cleavage by enzymes.
  • Macromolecules refer to large molecules with molecule weight over 10 KDa, including but not limited to proteins, lipids, nucleic acids, polysaccharides, nanoparticles, polymers, and dendrimers.
  • Pharmacokinetics describes what the body does to a drug. It may refer to the movement of drug into, through, and out of the body— the time course of its absorption, bioavailability, distribution, metabolism, and excretion. Drug pharmacokinetics determines the onset, duration, and intensity of a drug's effect.
  • the serum half-life of a molecule when connected to the protein-binding moiety of the present invention can be extended to be, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 400, or 500 hours.
  • the targeted construct comprising at least one conjugate has a plasma clearance rate less than about 5%, 10%>, 20%, 30%>, 40%, or 50% of the clearance rate of the conjugate itself without a protein-binding moiety (i.e., not connected to any small molecule or any antibody mimetic molecule).
  • the targeted construct comprising at least one conjugate has a plasma area under the curve (AUC) that is at least about 25%, 50%, 75%, 100%, 200%, or 500%) more than the AUC of the conjugate itself without a protein-binding moiety (i.e., not connected to any small molecule or any antibody mimetic molecule).
  • AUC plasma area under the curve
  • controlled release refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to affect a therapeutic outcome.
  • sustained release refers to a pharmaceutical composition or compound that conforms to a release rate over a specific period of time. The period of time may include, but is not limited to, hours, days, weeks, months and years.
  • toxicity refers to the capacity of a substance or composition to hit off targets and/or be harmful or poisonous to a cell, tissue, organ tissue, vasculature, or cellular environment. Low toxicity refers to a reduced capacity of a substance or composition to be harmful or poisonous to a cell, tissue, organ tissue or cellular environment. Such reduced or low toxicity may be relative to a standard measure, relative to a treatment or relative to the absence of a treatment.
  • Toxicity may further be measured relative to a subject's weight loss where weight loss over 15%, over 20% or over 30% of the body weight is indicative of toxicity.
  • Other metrics of toxicity may also be measured such as patient presentation metrics including lethargy and general malaiase.
  • Neutropenia or thrombocytopenia may also be metrics of toxicity.
  • Biomarkers of toxicity include elevated AST or ALT levels, neurotoxicity, kidney damage, GI damage and the like.
  • the toxicity of targeted constructs comprising at least one conjugate containing a targeting moiety linked to an active agent for cells that do not express the target of the targeting moiety is predicted to be decreased compared to the toxicity of the active agent alone. Without committing to any particular theory, applicants believe that this feature is because of the ability of the conjugated active agent to enter a cell is decreased compared the ability to enter a cell of the active agent alone. Accordingly, the conjugates comprising an active agent as described herein generally have reduced toxicity for cells that do not express the target of the targeting moiety and at least the same or increased toxicity for cells that express the target of the targeting moiety compared to the active agent alone.
  • the targeted constructs of the present invention may improve half life of the active agent and prevent the active agent from degradation and/or compromise before the active agent reaches the target site.
  • the protein-binding moieties in the present application may be any peptide, protein, nucleic acid (e.g., an aptamer), or small molecule. They bind to a protein reversibly or irreversibly.
  • the protein may be a naturally occurring protein, such as a serum or plasma protein, or a fragment thereof. Particular examples include Fc neonatal receptor (FcRn), thyroxine-binding protein, transthyretin, a 1 -acid glycoprotein (AAG), transferrin, fibrinogen, albumin, an immunoglobulin, ⁇ -2-macroglobulin, a lipoprotein, or fragments thereof.
  • the protein-binding moieties may bind to such a protein via covalent bonds or non-covalent interactions, such as hydrogen bonds, ionic bonds, van der Waals interactions, and hydrophobic bonds.
  • the protein-binding moieties are antibody mimetics.
  • Antibodies have been used in drug conjugates because of their can bind to a diversified group of targets, such as proteins, peptides, nucleic acids, sugars, etc.
  • targets such as proteins, peptides, nucleic acids, sugars, etc.
  • antibodies or their derivatives are large and possess molecular complexity. They may be difficult to produce. Further, antibodies may fail to penetrate deep into a tumor due to their sizes.
  • antibody mimetics are designed to bind to a protein in vivo to extend serum circulation half-lives and to improve pharmacokinetic and pharmacodynamic properties.
  • the antibody mimetic is selected from the group consisting of a monobody, e.g., an AD ECTINTM (Bristol-Myers Squibb, New York, New York), an Affibody® (Affibody AB, Sweden), an Affilin®, an AnticalinTM, avimers (avidity multimers), a DARPinTM, a FynomerTM, a CentyrinTM, Humabody® (fully human VH domain building blocks by Crescendo Biologies), and a Kunitz domain peptide.
  • a monobody e.g., an AD ECTINTM (Bristol-Myers Squibb, New York, New York), an Affibody® (Affibody AB, Sweden), an Affilin®, an AnticalinTM, avimers (avidity multimers), a DARPinTM, a FynomerTM, a CentyrinTM, Humabody® (fully human VH
  • the antibody mimetic may itself have ability to bind proteins, such as
  • KD values refers to the equilibrium dissociation constant that measures the affinity or the strength of binding between a molecule to another molecule. KD values may be calculated as the ratio between the off-rate (k 0 ff) and the on-rate
  • the binding between the antibody mimetic molecule and the protein may be measured in an experiment in which the protein, or a correctly folded fragment thereof, is immobilized on a sensor chip of an instrument, and the sample containing the antibody mimetic molecule to be tested is passed over the chip.
  • the antibody mimetic molecule to be tested is immobilized on a sensor chip of the instrument, and a sample containing the protein, or a correctly folded fragment thereof, is passed over the chip.
  • KD values between the antibody mimetic molecule and the protein may be determined by surface plasmon resonance (SPR) or Enzyme-Linked Immunosorbent Assay (ELISA) methods.
  • the binding affinity may be determined as the binding affinity relative to some reference molecule.
  • the antibody mimetic molecule may comprise between 4 and 500 amino acid residues, between 5 and 300 amino acid residues, between 10 and 200 amino acid residues, or between 20 and 150 amino acid residues, for example about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acid residues.
  • Fc neonatal Fc receptor also known as Fc neonatal receptor (FcRn)
  • FcRn Fc neonatal receptor
  • FcRn is expressed in many different cell types and organs at diverse body sites. It also binds albumin and plays an important role in homeostatic regulation of both IgG and albumin.
  • FcRn binds IgG at pH around 6 and not at physiological pH (around 7.4) (Mezo et al., PNAS, vol.105:2337-2342 (2008), the contents of which are incorporated herein by reference in their entirety).
  • the antibody mimetic binds to FcRn.
  • the binding to FcRn is pH dependent and is reversible.
  • the antibody mimetic binds to FcRn at pH below 7 (e.g., in acidic endosomes or acidic tumor microenvironments, where pH is around 6.0 - around 6.9) and does not bind to or dissociated from FcRn at pH above 7 (e.g., at extracellular pH or physiological pH of around 7.4).
  • the FcRn-binding antibody mimetic molecules do not inhibit the bindings of IgG and/or albumin to FcRn and do not inhibit the function of IgG and albumin.
  • the FcRn-binding antibody mimetic in these embodiments may bind to a non-IgG and non-albumin competitive site on FcRn.
  • the antibody mimetic molecules bind to FcRn both in vitro and in vivo.
  • the antibody mimetic molecules bind to FcRn at pH around 6.0.
  • the KD value of the interaction between the FcRn-binding antibody mimetic molecule and FcRn at pH around 6.0 may be less than 1 ⁇ 10 "6 M, such as less than 1 ⁇ 10 "7 M, less than 1 x 10 "8 M, less than 1 10 "9 M, or less than 1 10 "10 M.
  • An FcRn-binding antibody mimetic molecule would bind, or remain bound, to FcRn in acidic pH conditions, such as pH around 6.0, for example in a lysosome. Not willing to be bound by any theory, if such an FcRn- binding antibody mimetic molecule enters an increasingly acidic intracellular environment, it would be recycled to the plasma membrane through its interaction with FcRn, and thus avoid degradation.
  • the antibody mimetic molecules do not bind to FcRn at pH around 7.4.
  • the KD value of the interaction between the FcRn-binding antibody mimetic molecule and FcRn at pH around 7.4 may be at least 1 ⁇ 10 "8 M, such as at least 1 ⁇ 10 "7 M, such as at least 1 ⁇ 10 "6 M, or at least 1 ⁇ 10 "5 M.
  • the KD value of the interaction between the FcRn-binding antibody mimetic molecule and FcRn at around pH around 7.4 is higher than the KD value of said interaction at pH around 6.0.
  • the FcRn-binding antibody mimetic molecule would bind to FcRn with a higher affinity at pH around 6.0 than at pH around 7.4.
  • the KD value of said interaction at pH around 7.4 is at least 2 times higher, at least 5 times higher, at least 10 times higher, at least 50 times higher, or at least 100 times higher than the KD value of said interaction at pH around 6.0.
  • the FcRn-binding antibody mimetic is an affibody molecule.
  • Affibody molecules are a class of small affinity proteins, consisting of 58 amino acids, with no cysteine residues in their framework. They generally have good thermostability and stability to proteolysis.
  • Affibody molecules that bind to FcRn in a pH-dependent matter may be selected from a combinatorial library displayed on phase as disclosed in Seijsing et al., PNAS, vol.111(48): 17110 (2014), the contents of which are incorporated herein by reference in their entirety. Any FcRn-binding polypeptide comprising the sequence
  • EX2X3X4AX6X7 EIRWLPNL X16X17X18QRX21 AFIX25X26LX28X29, SEQ ID NO: 1075 disclosed in US20160031967, the contents of which are incorporated herein by reference in their entirety, may be used as an antibody mimetic in the present disclosure.
  • FcRn-binding affibody molecules include SEQ ID NO: 1-353 in
  • the FcRn antibody mimetic is a nanofitin® (also known as affinity) (Affilogic).
  • Nanofitin refers to a single-chain antibody mimetic that are much smaller than antibodies. Nanofitins are small and stable, lack disulfide bridges, and can be produced at high levels. The molecular weight of nanofitins are below lOKDa, preferably around 7KDa. Because of their small size and short half-life, nanofitins may both accumulate specifically at the site of the tumor and be cleared from the serum rapidly, therefore reducing off-target toxicity compared to long lasting antibodies. Conjugates comprise nanofitins may deliver an active agent deeper into a tumor. Nanofitins may bind intracellular targets and affect intracellular protein-protein interaction.
  • Nanofitins are derived from scaffold proteins engineered via combinational mutation/selection process disclosed in WO 2008/068637 (page 2, line 16 to page 9, line 2; examples page 12 to page 34; figures 1 to 21 and sequence listing), the contents of which are incorporated herein by reference in their entirety.
  • the scaffold proteins are chosen from proteins that bind a wide range of structurally different ligands.
  • One such family is proteins with oligonucleotide/oligosaccharide binding fold (OB-fold), a five-stranded ⁇ -barrel capped by an amphiphilic a-helix.
  • OB-fold proteins recognize nucleic acids, oligosaccharides, proteins, and metallic ions.
  • Combinatorial mutation/selection process to generate nanofitins comprises obtaining a combinatorial library corresponding to the randomization of a number of residues involved in the binding of a starting OB-fold protein with its native ligand, followed by a selection, in said library, of variants which bind specifically to a target of interest.
  • Non-limitative examples of OB-fold proteins which can be used in producing nanofitins include: Sac7d from Sulfolobus acidocaldarius (PDB lazp; UNIPROT or
  • GenBank P13123 or Q4JC17 and the truncated form of Sac7d, and Sac7b, Sac7e from Sulfolobus acidocaldarius (UNIPROT or GenBank P13125), Sso7d from Sulfolobus solfataricus (PDB M4; UNIPROT or GenBank P39476 or P81550), DBP 7 from Sulfolobus tokodaii (UNIPROT or GenBank Q96X56), Ssh7b from Sulfolobus shibatae (UNIPROT or GenBank 059632), Ssh7a from Sulfolobus shibatae (UNIPROT or GenBank P61990, 059631, P80170, Q9UW18), p7ss from Sulfolobus solfataricus (UNIPROT or GenBank P61991, 059631, P80170, Q9UW18), the N-terminal domain of SEB, the chain A of
  • PDB IbcpD pertussis
  • GenBank P0A3R5 the Cholera Toxin of Vibrio cholera
  • PB 3chbD the Cholera Toxin of Vibrio cholera
  • GenBank D0UTQ9 the Heat-labile toxin of E. coli
  • PB ItiiD the Heat-labile toxin of E. coli
  • Verotoxin-1 /Shiga toxin B-pentamer of E.
  • Staphylococcus aureus (PDB law7A; UNIPROT or GenBank A0FIN2), the Major cold- shock protein of E.coli (PDB ljmc; UNIPROT or GenBank P0A9Y1), the Initiation translation factor 5a of Pyrobaculum aerophylum (PDB lbkb; UNIPROT or GenBank P56635), the SI RNA-binding domain of PNPase of E.coli (PDB lsro; UNIPROT or GenBank P05055), the human Initiation translation factor 1, elFla (PDB ld7qA; UNIPROT or GenBank P47813), the Initiation translation factor 1, IF1 of E.coli (PDB lah9; UNIPROT or GenBank P69224), , the RNA guanylyltransferase of Chlorella virus, PBCV-1 (PDB lckmA; UNIPROT or GenBank Q84424), the
  • Staphylococcus aureus (PDB lsnc; UNIPROT or GenBank gi/224650), the DNA helicase RuvA subunit, N-terminal domain of E.coli (PDB lhjp; UNIPROT or GenBank P0A811), the Gene V protein of Pseudomonas bacteriophage pf3 (PDB lpfsA; UNIPROT or GenBank P03672), the Gene V protein of Filamentous bacteriophage fl, Ml 3 (PDB lgvp; UNIPROT or GenBank D0U157), the Gene 32 protein (gp32) core of Bacteriophage T4 (PDB lgpc; UNIPROT or GenBank B3IYU0), and the Inorganic pyrophosphatase of Thermus thermophilus (PDB 2prd; UNIPROT or GenBank Q72H95).
  • OB-folds domains originating from toxins may
  • nanofitins comprise 66 amino acid residues and are derived from the DNA binding protein Sac7d of Sulfolobus acidocaldarius.
  • Sac7d is chemically and thermally stable and is resistant to extreme pH. Its molecular organization is simple, comprising only 66 amino acids, lacking a disulfide bridge, and possessing only one structural domain (the OB-fold).
  • the binding face of Sac7d can be modified to recognize various targets without chaning its favorable biophysical properties.
  • the binding area of nanofitins that are Sac7d derivatives is located on the surface and is composed of 14 residues (i.e., residues 7-9, 21, 22, 24, 26, 29, 31, 33, 40, 42, 44, and 46), which can be modified to alter specificity (Mouratou, et al., Proc. Natl. Acad. Sci. USA 104: 17983-8 (2007), the contents of which are incorporated herein by reference in their entirety).
  • the residues of Sac7d which can be substituted are V2, K3, K5, K7, Y8, K9, G10, E14, T17, K21, K22, W24, V26, G27, K28, M29, S31, T33, D36, N37, G38, K39, T40, R42, A44, S46, E47, K48, D49, A50 and P51.
  • the residues of Sac7d which can be deleted are: A59, R60, A61 and E64.
  • Insertions of 1 to 15 amino acid residues can be performed in loop 3, for example in the region of residues 25 to 30 of Sac7d, preferably between residues 27 and 28. Insertions of 1 to 15 amino acid residues can be performed in loop 4, for example in the region of residues 35 to 40 of Sac7d, preferably between residues 37 and 38. Insertions of 1 to 20 amino acid residues can also be performed in loop 1, for example in the region of residues 7 to 12 of Sac7d, preferably between residues 9 and 10.
  • the nanofitins bind to FcRn with high affinity at pH around 6 and do not bind FcRn at pH above 7, e.g., around 7.4, and wherein the nanofitins comprise 66 amino acid residues and are derived from the DNA binding protein Sac7d.
  • Such nanofitins may be selected from the methods in Examples 1-10 of WO 2008/068637.
  • the antibody mimetic molecules may bind to a serum protein, such as albumin (in particular human serum albumin), serum immunoglobulins (such as IgG, with a molecular weight of around 150 kDa), thyroxine-binding protein, transferring, or fibrinogen or a fragment thereof.
  • a serum protein such as albumin (in particular human serum albumin), serum immunoglobulins (such as IgG, with a molecular weight of around 150 kDa), thyroxine-binding protein, transferring, or fibrinogen or a fragment thereof.
  • the antibody mimetic is a single-domain antibody molecule consisting of a single monomeric variable antibody domain, such as nanobodies® (Ablynx).
  • Nanobodies consist of heavy chains only and lack light chains. They comprise at least one single variable domain (VHH) of camelidae antibody and optionally constant domains (CH2, CH3).
  • VHH domain has a molecular weight of 12-15 kDa, has nano- to picomolar affinities, and can be linked together easily.
  • Any single domain anti-serum protein antibody in WO2004041865 (such as SEQ ID NOs: 1 to 4, and 28 to 40), or any amino acid sequence that binds to a serum protein disclosed in US20100216187, the contents of which are incorporated herein by reference in their entirety, may be used as an antibody mimetic molecule in the present disclosure.
  • the antibody mimetic is a monomer, such as a domain in an Avimer (Amgen/Avidia). Any monomer domain disclosed in Example 4 of US20040175756, or immunoglobulin-binding monomer domain disclosed in US7820790 (such as SEQ ID NO. 26, 930, 931, or 932), the contents of each of which are incorporated herein by reference in their entirety, may be used as an antibody mimetic molecule in the present disclosure.
  • a protein-binding moiety may be a small molecule.
  • the binding between the small molecule and the protein may happen in vivo.
  • the binding may be reversible or irreversible.
  • the small molecule may bind to the protein via a covalent bond or via non-covalent interactions.
  • Small molecules when used as a protein-binding moiety in targeted constructs to extend half-lives, do not significantly increase the molecular weight of the targeted constructs, which can be beneficial to achieve good penetration (e.g., diffusion into a tumor tissue). Further, the small size of a targeted construct enables a wide selection of
  • the small molecules may react with a functional group on a protein to form a covalent bond.
  • the functional group on the protein may be on a lysine or cysteine side chain.
  • Cysteine often exists as a disulfide pair in native proteins.
  • Thiol- alkylation reagents such as a-halocarbonyl derivatives
  • Michael acceptors such as maleimide derivatives
  • the functional group is on human serum albumin (HSA or albumin).
  • HSA human serum albumin
  • Albumin is the most abundant plasma protein (35-50 g/L in human serum) with a molecular weight of 66.5 KDa and an effective diameter of 7.2 nm (Kratz, J. of Controlled Release, vol.132: 171, (2008), the contents of which are incorporated herein by reference in their entirety).
  • Albumin has a half-life of about 19 days.
  • Albumin preferentially accumulates in malignant and inflamed tissues due to leaky capillaries and an absent or defective lymphatic drainage system.
  • Albumin accumulates in tumors such as solid tumors also because albumin is a major energy and nutrition source for tumor growth.
  • the functional group may be the cysteine-34 position of albumin that has an accessible free thiol group.
  • the small molecules that react with a functional group on albumin may comprise a disulfide group, a vinylcarbonyl group, a vinyl acetylene group, an aziridine group, an acetylene group or any of the following groups:
  • R is CI, Br, F, mesylate, tosylate, 0-(4-nitrophenyl), O-pentafluorophenyl, or derivatives thereof.
  • the disulfide group, the vinylcarbonyl group, the vinyl acetylene group, the aziridine group, and the acetylene group may be substituted.
  • the small molecules that react with cysteine on albumin comprise a heteroaromatic methylsulfone group, such as methyl sulfonyl benzothiazole (MSBT), Compounds 4a-d disclosed in Toda et al., Angew Chem Int Ed Engl, vol.52(48), 2013), or derivatives thereof.
  • MSBT methyl sulfonyl benzothiazole
  • the functional group is on transthyretin.
  • Transthyretin is a 55 KDa serum protein that has an in vivo half-life of around 48 h.
  • Small molecules that react with a functional group on transthyretin may be selected from AGIO (structure shown below) or its derivative disclosed by Penchala et al. in Nature Chemical Biology, vol.11 :793, (2015), or formula (I), (II), (III) or (IV) (structures shown below) disclosed in US Pat. No. 5714142 to Blaney et al., the contents of each of which are incorporated herein by reference in their entirety. Any transthyretin-selective ligand disclosed on pages 5-8 of Blaney et al.
  • tetraiodothyroacetic acid 2,4,6-triiodophenol
  • flufenamic acid diflunisal
  • milrinone EMD 21388
  • the small molecule that react with a functional group on transthyretin may be an AGIO analog, such as analogs 2, 3 (TLHE1), 4 ((TLHE2) disclosed in Penchala et al., Nature Chemical Biology, vol.11 :793 (2015), the contents of which are incorporated herein by reference in their entirety.
  • TLHE1 has high affinity and selectivity to human transthyretin (hTTR), is stable in serum, and has low cytotoxicity.
  • the small molecule may be any protein binding moiety disclosed in US 9216228 to Kratz, the contents of which are incorporated herein by reference in their entirety, such as a maleimide group, a halogenacetamide group, a halogenacetate group, a pyridylthio group, a vinylcarbonyl group, an aziridine group, a disulfide group, a substituted or unsubstituted acetylene group, and a hydroxysuccinimide ester group.
  • the small molecule may comprise a disulfide group. The disulfide group undergoes an exchange with a thiol group on a protein or an engineered protein or a polymer or
  • derivatives/analogs/mimics thereof, such as albumin to form a disulfide bond that connects the small molecule and the protein.
  • the small molecule may bind to a serum protein via non- covalent interactions.
  • the small molecule may be saturated fatty acids that bind to albumin with weak affinities (10 "4 to 10 "5 M).
  • Non-limiting examples of such fatty acids may include myristic acid (a fatty acid with 14 carbon atoms) and palmitic acid (a fatty acid with 16 carbon atoms).
  • small molecules include a naphthalene acylsulfonamide group, a diphenylcyclohexanol phosphate ester group, a 6-(4- (4-iodophenyl) butanamido)hexanoate group ('Albu'-tag), a series of peptides having the core sequence of DICLPRWGCLW including SA21 (a cyclic peptide with 18 amino acids Ac-RLIEDICLPRWGCLWEDD- H 2 ) disclosed by Dennis et al. in J. Biol. Chem., vol.277:35035 (2002), the contents of which are incorporated herein by reference in their entirety.
  • SA21 a cyclic peptide with 18 amino acids Ac-RLIEDICLPRWGCLWEDD- H 2
  • a small molecule may comprise a structure of:
  • the small molecule protein-binding moiety may comprise any peptide-fatty acid albumin-binding ligand disclosed in Zorzi et al., Nature
  • peptide-fatty acid albumin-binding ligands comprise a fatty acid connected to a short peptide, e.g., a heptapeptide, via an amino acid side chain.
  • the fatty acid may be linked to the short peptide via its carboxylic group to the side chain of lysine.
  • the fatty acid binds to albumin with an affinity in the micromolar range and the short peptide enhances the affinity by forming additional contacts to albumin.
  • the peptide-fatty acid ligands may have a general structure of: ⁇
  • Lys, n 12 (myristic acid), 14 (palmitic acid), or 16 (stearic acid).
  • any albumin-binding functional group disclosed in US 9670482 (Bicycle Therapeutics), the contents of which are incorporated herein in their entirety, may be used as a protein-binding moiety in the present application.
  • the small molecule comprises a fluorene ring and binds to albumin non- covalently and/or reversibly.
  • the small molecule comprises a fluorenylmethyloxycarbonyl (FMOC) group.
  • FMOC fluorenylmethyloxycarbonyl
  • the small molecule comprises at least one amino acid attached to FMOC, such as Lys, Trp, Gly, or Phe.
  • the small molecule may comprise Fmoc-Lys-, Fmoc-Gly-, Fmoc-Phe-, Fmoc-GGSGD-, Fmoc- GSGD-, Fmoc-WGGGA, or Fmoc-Trp-GGG.
  • Targeted Constructs Comprising Protein-binding Moieties and Targeting Moieties
  • targeted constructs comprising at least one protein-binding moiety of the present disclosure and at least one targeting moiety (X) are provided.
  • the protein-binding moiety and the targeting moiety may be connected with an optional internal linker moiety (Y): (Protein-binding moiety)n-(Y)p-(X)m, n>l, m>l, and p>0
  • the protein-binding moiety and/or the targeting moiety is an antibody mimetic. In some embodiments, the protein-binding moiety and/or the targeting moiety is not an antibody mimetic. In some embodiments, the protein-binding moiety is a small molecule.
  • the targeted constructs may contain one or more targeting moieties (also known as targeting ligands).
  • Targeting ligands or moieties can be peptides, antibody mimetics, nucleic acids (e.g., aptamers), polypeptides (e.g., antibodies), glycoproteins, small molecules, carbohydrates, or lipids.
  • Targeting ligands may also have therapeutic functions.
  • the targeting moiety, X can be a peptide such as somatostatin, octreotide, LHRH, an EGFR-binding peptide, RGD-containing peptides, a protein scaffold such as a fibronectin domain, an aptide or bipodal peptide, a single domain antibody, a stable scFv, or a bispecific T-cell engager, nucleic acid (e.g., aptamer), polypeptide (e.g., antibody or its fragment), glycoprotein, small molecule, carbohydrate, or lipid.
  • the targeting moiety, X can be an aptamer being either RNA or DNA or an artificial nucleic acid; small molecules;
  • carbohydrates such as mannose, galactose and arabinose; vitamins such as ascorbic acid, niacin, pantothenic acid, carnitine, inositol, pyridoxal, lipoic acid, folic acid (folate), riboflavin, biotin, vitamin B 12, vitamin A, E, and K; a protein or peptide that binds to a cell- surface receptor such as a receptor for thrombospondin, tumor necrosis factors (TNF), annexin V, interferons, cytokines, transferrin, GM-CSF (granulocyte-macrophage colony- stimulating factor), or growth factors such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), (platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF).
  • VEGF vascular endothelial growth factor
  • HGF hepatocyte growth factor
  • the targeting moiety or moieties in the targeted constructs are present at a predetermined molar weight percentage from about 1% to about 10%, or about 10% to about 20%, or about 20% to about 30%, or about 30% to about 40%, or about 40% to about 50%, or about 50% to about 60%, or about 60% to about 70%, or about 70% to about 80%, or about 80% to about 90%, or about 90% to about 99%, such that the sum of the molar weight percentages of the components of the targeted construct is 100%.
  • the amount of targeting moieties of the targeted construct may also be expressed in terms of proportion to the protein-binding moiety, for example, in a ratio of targeting moiety to protein-binding moiety of about 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3 : 1, 2: 1, 1 : 1, 1 :2, 1 :3, 1 :4; 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, or 1 : 10.
  • the targeting moiety comprises an imaging probe, such as a radio label (e.g., a radioisotope).
  • a radio label e.g., a radioisotope
  • Non-limiting examples of radioisotopes include 1-131, Re- 186, Re-188, Y-90, Bi-212, At-211, Sr-89, Ho-166, Sm-153, Cu-67, Cu-64, Lu-177, Ac-225, Bi-213, Th-227, Pb-212, Ra-223, and P-32.
  • the targeting moiety is a protein scaffold.
  • the protein scaffold may be an antibody-derived protein scaffold.
  • Non-limiting examples include single domain antibody (dAbs), nanobody, single-chain variable fragment (scFv), antigen-binding fragment (Fab), Avibody, minibody, CH2D domain, Fcab, and bispecific T-cell engager (BiTE) molecules.
  • dAbs single domain antibody
  • scFv single-chain variable fragment
  • Fab antigen-binding fragment
  • Avibody minibody
  • CH2D domain CH2D domain
  • Fcab bispecific T-cell engager
  • BiTE bispecific T-cell engager
  • scFv is a stable scFv, wherein the scFv has hyperstable properties.
  • the nanobody may be derived from the single variable domain (VHH) of camelidae antibody.
  • the protein scaffold may be a nonantibody-derived protein scaffold, wherein the protein scaffold is based on nonantibody binding proteins.
  • the protein scaffold may be based on enginnered Kunitz domains of human serine protease inhibitors (e.g., LAC1-D1), DARPins (designed ankyrin repeat domains), avimers created from multimerized low-density lipoprotein receptor class A (LDLR-A), anticalins derived from lipocalins, knottins constructed from cysteine-rich knottin peptides, affibodies that are based on the Z-domain of staphylococcal protein A, adnectins or monobodies and pronectins based on the 10 th or 14 th extracellular domain of human fibronectin III, Fynomers derived from SH3 domains of human Fyn tyrosine kinase, or nanofitins (formerly Affitins) derived from the DNA
  • the protein scaffold may be any protein scaffold disclosed in Mintz and Crea, BioProcess, vol.11(2):40-48 (2013), the contents of which are
  • any of the protein scaffolds disclosed in Tables 2-4 of Mintz and Crea may be used as a targeting moiety of the conjugate of the invention.
  • the protein scaffold may be based on a fibronectin domain.
  • the protein scaffold may be based on fibronectin type III (FN3) repeat protein.
  • the protein scaffold may be based on a consensus sequence of multiple FN3 domains from human Tenascin-C (hereinafter "Tenascin"). Any protein scaffold based on a fibronectin domain disclosed in US Pat. No. 8569227 to Jacobs et al., the contents of which are incorporated herein by reference in their entirety, may be used as a targeting moiety of the conjugate of the invention.
  • the targeting moiety or targeting ligand may be any molecule that can bind to luteinizing-hormone-releasing hormone receptor (LHRHR).
  • LHRHR luteinizing-hormone-releasing hormone receptor
  • Such targeting ligands can be peptides, antibody mimetics, nucleic acids (e.g., aptamers), polypeptides (e.g., antibodies), glycoproteins, small molecules, carbohydrates, or lipids.
  • the targeting moiety is LURH or a LURH analog.
  • Luteinizing-hormone-releasing hormone also known as gonadotropin- releasing hormone (GnRH) controls the pituitary release of gonadotropins (LH and FSH) that stimulate the synthesis of sex steroids in the gonads.
  • LHRH is a 10-amino acid peptide that belongs to the gonadotropin-releasing hormone class. Signaling by LHRH is involved in the first step of the hypothalamic-pituitary-gonadal axis.
  • An approach in the treatment of hormone-sensitive tumors directed to the use of agonists and antagonists of LHRH (A.V. Schally and A.M. Comaru-Schally. Sem.
  • LHRH agonists when substituted in position 6, 10, or both are much more active than LHRH and also possess prolonged activity.
  • LHRH agonists are approved for clinical use, e.g., Leuprolide, triptorelin, nafarelin and goserelin.
  • Some human tumors are hormone dependent or hormone-responsive and contain hormone receptors. Certain of these tumors are dependent on or responsive to sex hormones or growth factors, or have components that are dependent or responsive to such hormones.
  • Mammary carcinomas contain estrogen, progesterone, glucocorticoid, LHRH, EGF IGF-I and somatostatin receptors.
  • Peptide hormone receptors have been detected in acute leukaemia, prostate-, breast-, pancreatic, ovarian-, endometrial cancer, colon cancer and brain tumors (M.N. Pollak, et al., Cancer Lett. 38 223-230 1987; F. Pekonen, et al., Cancer Res., 48 1343- 1347, 1988; M.
  • LHRH binds to human breast cancer cell membranes, and also to the cell membranes of pancreatic cancer. It has been demonstrated that biologically active peptides such a melanotropin (MSH), epidermal growth factor, insulin and agonistic and antagonist analogs of LHRH (L Jennes, et. al., Peptides 5 215-220, 1984) are internalized b their target cells by endocytosis.
  • MSH melanotropin
  • epidermal growth factor epidermal growth factor
  • insulin agonistic and antagonist analogs of LHRH
  • the conjugates of the invention can employ any of the large number of known molecules that recognize the LHRH receptor, such as known LHRH receptor agonists and antagonists.
  • the LHRH analog portion of the conjugate contains between 8 and 18 amino acids.
  • LHRH binding molecules useful in the present invention are described herein. Further non-limiting examples are analogs of pyroGlu-His-Trp-Ser-Tyr- Gly-Leu-Arg-Pro-Gly-NH2, leuprolide, triptorelin, nafarelin, buserelin, goserelin, cetrorelix, ganirelix, azaline-B, degarelix and abarelix. [0091] Methods for synthesizing LHRH peptides and analogs are well documented and are within the ability of a person of ordinary skill in the art as exemplified in the references listed supra. Further synthetic procedures are provided in the following examples.
  • a tumor expressing a LFIRH receptor includes a neoplasm of the lung, breast, prostate, colon, brain, gastrointestinal tract, neuroendocrine axis, liver, or kidney (see Schaer et al., Int. J. Cancer, 70:530-537, 1997; Chave et al., Br. J. Cancer 82(1): 124-130, 2000; Evans et al., Br. J. Cancer 75(6):798-803, 1997).
  • the targeting moiety e.g., LHRH analog
  • the targeting moiety used in the invention is hydrophilic, and is therefore water soluble.
  • such targeted constructs are used in treatment paradigms in which this feature is useful, e.g., compared to conjugates comprising hydrophobic analogs.
  • Hydrophilic analogs described herein can be soluble in blood, cerebrospinal fluid, and other bodily fluids, as well as in urine, which may facilitate excretion by the kidneys. This feature can be useful, e.g., in the case of a composition that would otherwise exhibit undesirable liver toxicity.
  • the invention also discloses specific hydrophilic elements (e.g., incorporation of a PEG linker, and other examples in the art) for incorporation into peptide analogs, allowing modulation of the analog's hydrophilicity to adjust for the chemical and structural nature of the various conjugated cytotoxic agents.
  • specific hydrophilic elements e.g., incorporation of a PEG linker, and other examples in the art
  • the targeting moiety may be any somatostatin receptor 2 (SSTR2) ligand disclosed in WO2016004048, the contents of which are incorporated herein by reference in their entirety.
  • SSTR2 somatostatin receptor 2
  • the targeting moiety is an antibody mimetic such as a monobody, e.g., an AD ECTINTM (Bristol-Myers Squibb, New York, New York), an Affibody® (Affibody AB, Sweden), Affilin (Navigo Proteins, Halle, Germany), nanofitin, affitin (such as those described in WO2012/085861), an AnticalinTM, an avimers (avidity multimers), a DARPinTM, a FynomerTM, CentyrinTM, a Humabody® (fully human VH domain building blocks by Crescendo Biologies), Kunitz domain or an Abdurin peptide.
  • such mimetics are artificial peptides or proteins with a molar mass of about 3 to 20 kDa. Nucleic acids and small molecules may be antibody mimetic.
  • the targeting moiety is an Abdurin peptide. It is an engineered antibody domain molecule comprising at least one protein-binding domain derived from a CH2 domain or CH2-like domain of an immunoglobulin (such as IgG, IgA, IgD), or a CH3 domain or CH3-like domain of IgE or IgM, comprising at least one mutation.
  • the mutation may be an N-terminal truncation of at least one amino acid and/or a C-terminal truncation of at least one amino acid.
  • the molecular weight of an Abdurin peptide is usually less than about 15 kD. Any peptide disclosed in US8580927, US9527903, US9156917,
  • a targeting moiety can be an aptamer, which is generally an oligonucleotide (e.g., DNA, RNA, or an analog or derivative thereof) that binds to a particular target, such as a polypeptide.
  • the targeting moiety is a polypeptide (e.g., an antibody that can specifically bind a tumor marker).
  • the targeting moiety is an antibody or a fragment thereof.
  • the targeting moiety is an Fc fragment of an antibody.
  • a targeting moiety may be a non-immunoreactive ligand.
  • the non-immunoreactive ligand may be insulin, insulin-like growth factors I and II, lectins, apoprotein from low density lipoprotein, etc. as disclosed in US 20140031535 to Jeffrey, the contents of which are incorporated herein by reference in their entirety.
  • Any protein or peptide comprising a lectin disclosed in WO2013181454 to Radin, the contents of which are incorporated herein by reference in their entirety, may be used as a targeting moiety.
  • the conjugate of the invention may target a hepatocyte intracellularly and a hepatic ligand may be used as a targeting moiety.
  • a hepatic ligand disclosed in US 20030119724 to Ts'o et al., the contents of which are incorporated herein by reference in their entirety, such as the ligands in Fig. 1, may be used.
  • the hepatic ligand specifically binds to a hepatic receptor, thereby directing the conjugate into cells having the hepatic receptor.
  • a targeting moiety may interact with a protein that is overexpressed in tumor cells compared to normal cells.
  • the targeting moiety may bind to a chaperonin protein, such as Hsp90, as disclosed in US 20140079636 to Chimmanamada et al., the contents of which are incorporated herein by reference in their entirety.
  • the targeting moiety may be an Hsp90 inhibitor, such as ganetespib or a ganetespib analog (e.g., 4-(5- hydroxy-4-(l-(2-(piperidin-4-yl)ethyl)-lH-indol-5-yl)-4H-l,2,4-triazol-3-yl)-6- isopropylbenzene-l,3-diol), geldanamycins, macbecins, tripterins, tanespimycins, and radicicols.
  • the targeted construct may have a terminal half-life of longer than about 72 hours and a targeting moiety may be selected from Table 1 or 2 of US
  • the targeting moiety may be an antibody targeting delta-like protein 3 (DLL3) in disease tissues such as lung cancer, pancreatic cancer, skin cancer, etc., as disclosed in WO2014125273 to Hudson, the contents of which are incorporated herein by reference in their entirety.
  • DLL3 antibody targeting delta-like protein 3
  • the targeting moiety may also any targeting moiety in
  • the targeting moiety binds to glypican-3 (GPC-3) and directs the conjugate to cells expressing GPC-3, such as hepatocellular carcinoma cells.
  • a target of the targeting moiety may be a marker that is exclusively or primarily associated with a target cell, or one or more tissue types, with one or more cell types, with one or more diseases, and/or with one or more developmental stages.
  • a target can comprise a protein (e.g., a cell surface receptor,
  • transmembrane protein, glycoprotein, etc. a transmembrane protein, glycoprotein, etc.), a carbohydrate (e.g., a glycan moiety, glycocalyx, etc.), a lipid (e.g., steroid, phospholipid, etc.), and/or a nucleic acid (e.g., a DNA, RNA, etc.).
  • a carbohydrate e.g., a glycan moiety, glycocalyx, etc.
  • a lipid e.g., steroid, phospholipid, etc.
  • a nucleic acid e.g., a DNA, RNA, etc.
  • targeting moieties may be peptides for regulating cellular activity.
  • the targeting moiety may bind to Toll Like Receptor (TLR).
  • TLR Toll Like Receptor
  • It may be a peptide derived from vaccinia virus A52R protein such as a peptide comprising SEQ ID No. 13 as disclosed in US 7557086, a peptide comprising SEQ ID No. 7 as disclosed in US 8071553 to Hefeneider, et al., or any TLR binding peptide disclosed in WO 2010141845 to McCoy, et al., the contents of each of which are incorporated herein by reference in their entirety.
  • the A52R derived synthetic peptide may significantly inhibit cytokine production in response to both bacterial and viral pathogen associated molecular patterns, and may have application in the treatment of inflammatory conditions that result from ongoing toll-like receptor activation.
  • targeting moieties many be amino acid sequences or single domain antibody fragments for the treatment of cancers and/or tumors.
  • targeting moieties may be an amino acid sequence that binds to Epidermal Growth Factor Receptor 2 (HER2).
  • HER2 Epidermal Growth Factor Receptor 2
  • Targeting moieties may be any HER2 -binding amino acid sequence described in US 20110059090, US8217140, and US 8975382 to Revets, et al., the contents of each of which are incorporated herein by reference in their entirety.
  • the targeting moiety may be a domain antibody, a single domain antibody, a VHH, a humanized VHH or a camelized VH.
  • targeting moieties may be peptidomimetic macrocycles for the treatment of disease.
  • targeting moieties may be peptidomimetic
  • GHRH growth hormone-releasing hormone
  • the peptidomimetic macrocycle targeting moiety may be prepared by introducing a cross-linker between two amino acid residues of a polypeptide as described in US 20120149648 and US 20130072439 to Nash et al., the contents of each of which are incorporated herein by reference in their entirety.
  • Nash et al. teaches that the peptidomimetic macrocycle may comprise a peptide sequence that is derived from the BCL-2 family of proteins such as a BH3 domain.
  • the peptidomimetic macrocycle may comprise a BID, BAD, BIM, BIK, NOXA, PUMA peptides.
  • targeting moieties may be polypeptide analogues for transport to cells.
  • the polypeptide may be an Angiopep-2 polypeptide analog. It may comprise a polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID No.97 as described in US 20120122798 to Castaigne et al., the contents of which are incorporated herein by reference in their entirety.
  • polypeptides may transport to cells, such as liver, lung, kidney, spleen, and muscle, such as Angiopep-4b, Angiopep-5, Angiopep-6, and Angiopep-7 polypeptide as described in EP 2789628 to Beliveau et al., the contents of each of which are incorporated herein by reference in their entirety.
  • targeting moieties may be homing peptides to target liver cells in vivo.
  • the melittin delivery peptides that are administered with RNAi polynucleotides as described in US 8501930 Rozema, et al., the contents of which are incorporated herein by reference in their entirety may be used as targeting moieties.
  • delivery polymers provide membrane penetration function for movement of the RNAi polynucleotides from the outside the cell to inside the cell as described in US 8313772 to Rozema et al., the contents of each of which are incorporated herein by reference in their entirety. Any delivery peptide disclosed by Rozema et al. may be used as targeting moeities.
  • targeting moieties may be structured polypeptides to target and bind proteins.
  • polypeptides with sarcosine polymer linkers that increase the solubility of structured polypeptides as described in WO 2013050617 to Tite, et al., the contents of which are incorporated herein by reference in their entirety, may be used as targeting moieties.
  • polypeptide with variable binding activity produced by the methods described in WO 2014140342 to Stace, et al., the contents of which are incorporated herein by reference in their entirety. The polypeptides may be evaluated for the desired binding activity.
  • modifications of the targeting moieties affect a compound's ability to distribute into tissues.
  • a structure activity relationship analysis was completed on a low orally bioavailable cyclic peptide and the permeability and clearance was determined as described in Rand, AC, et al., Medchemcomm. 2012, 3(10): 1282-1289, the contents of which are incorporated herein by reference in their entirety.
  • Any of the cyclic peptide disclosed by Rand et al., such as N-methylated cyclic hexapeptides, may be used as targeting moieties.
  • targeting moieties may be a polypeptide which is capable of internalization into a cell.
  • targeting moieties may be an Alphabody capable of internalization into a cell and specifically binding to an intracellular target molecule as described in US 20140363434 to Lasters, et al., the contents of which are incorporated herein by reference in their entirety.
  • an 'Alphabody' or an 'Alphabody structure' is a self-folded, single-chain, triple-stranded, predominantly alpha-helical, coiled coil amino acid sequence, polypeptide or protein.
  • the Alphabody may be a parallel
  • targeting moieties may be any organic compound that can be used to modulate the expression of the alphabody or an anti-parallel Alphabody.
  • targeting moieties may be any organic compound that can be used to promote the production of the alphabody or an anti-parallel Alphabody.
  • targeting moieties may be any organic compound that can be used to promote the production of the alphabody or an anti-parallel Alphabody.
  • targeting moieties may be any organic compound selected from any organic radicals, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids,
  • Alphabody in the single-chain Alphabody library used for the screening for and/or selection of one or more Alphabodies that specifically bind to a target molecule of interest as described in WO 2012092970 to Desmet et al., the contents of which are incorporated herein by reference in their entirety.
  • targeting moieties may consist of an affinity-matured heavy chain-only antibody.
  • targeting moieties may be any VH heavy chain-only antibodies produced in a transgenic non-human mammal as described in US 20090307787 to Grosveld et al., the contents of which are incorporated herein by reference in their entirety.
  • targeting moieties may bind to the hepatocyte growth factor receptor "HGFr" or "cMet".
  • targeting moieties may be a polypeptide moiety that is conjugated to a detectable label for diagnostic detection of cMet as described in US 9000124 to Dransfield et al., the contents of which are incorporated herein by reference in their entirety.
  • targeting moieties may bind to human plasma kallikrein and may comprise BPTI-homologous Kunitz domains, especially LACI homologues, to bind to one or more plasma (and/or tissue) kallikreins as described in WO 1995021601 to Markland et al., the contents of which are incorporated herein by reference in their entirety.
  • targeting moieties are evolved from weak binders and anchor-scaffold conjugates having improved target binding and other desired pharmaceutical properties through control of both synthetic input and selection criteria. Any target binding element identified in US 20090163371 to Stern et al., the contents of which are incorporated herein by reference in their entirety, may be used as a targeting moiety.
  • targeting moieties may be macrocyclic compounds that bind to inhibitors of apoptosis as described in WO 2014074665 to Borzilleri et al., the contents of which are incorporated herein by reference in their entirety.
  • targeting moieties may comprise pre-peptides that encode a chimeric or mutant lantibiotic.
  • targeting moieties may be pre-tide that encode a chimera that was accurately and efficiently converted to the mature lantibiotic, as demonstrated by a variety of physical and biological activity assays as described in
  • targeting moieties may comprise a leader peptide of a recombinant manganese superoxide dismutase (rMnSOD-Lp).
  • rMnSOD-Lp which delivers cisplatin directly into tumor cells as described in Borrelli, A., et al., Chem Biol DrugDes. 2012, 80(1):9-16, the contents of which are incorporated herein by reference in their entirety, may be used a targeting moiety.
  • the targeting moiety may be an antibody for the treatment of glioma.
  • an antibody or antigen binding fragment which specifically binds to JAMM-B or JAM-C as described in US8007797 to Dietrich et al., the contents of which are incorporated herein by reference in their entirety, may be used as a targeting moiety.
  • JAMs are a family of proteins belonging to a class of adhesion molecules generally localized at sites of cell-cell contacts in tight junctions, the specialized cellular structures that keep cell polarity and serve as barriers to prevent the diffusion of molecules across intercellular spaces and along the basolateral-apical regions of the plasma membrane.
  • the targeting moiety may be a target interacting modulator.
  • nucleic acid molecules capable of interacting with proteins associated with the Human Hepatitis C virus or corresponding peptides or mimetics capable of interfering with the interaction of the native protein with the HIV accessory protein as described in WO 2011015379 and US 8685652, the contents of each of which are incorporated herein by reference in their entirety, may be used as a targeting moiety.
  • the targeting moiety may bind with biomolecules.
  • any cystine-knot family small molecule polycyclic molecular scaffolds were designed as peptidomimetics of FSH and used as peptide-vaccine as described in US7863239 to Timmerman, the contents the contents of which are incorporated herein by reference in their entirety, may be used as targeting moieties.
  • the targeting moiety may bind to integrin and thereby block or inhibit integrin binding.
  • any highly selective disulfide-rich dimer molecules which inhibit binding of a4B7 to the mucosal addressin cell adhesion molecule (MAdCAM) as described in WO 2014059213 to Bhandari, the contents of which are incorporated herein by reference in their entirety, may be used as a targeting moiety.
  • MAdCAM mucosal addressin cell adhesion molecule
  • Any inhibitor of specific integrins-ligand interactions may be used as a targeting moiety.
  • the conjugates comprising such target moieties may be effective as anti-inflammatory agents for the treatment of various autoimmune diseases.
  • the targeting moiety may comprise novel peptides.
  • any cyclic peptide or mimetic that is a serine protease inhibitor as described in WO 2013172954 to Wang et al., the contents of which are incorporated herein by reference in their entirety, may be used as a targeting moiety.
  • targeting moieties may comprise a targeting peptide that is used in the reduction of cell proliferation and the treatment of cancer.
  • a peptide composition inhibiting the trpv6 calcium channel as described in US 20120316119 to Stewart, the contents of which are incorporated herein by reference in their entirety, may be used as a targeting moiety.
  • the targeting moiety may comprise a cyclic peptide.
  • any cyclic peptides exhibit various types of action in vivo, as described in
  • cyclic peptide targeting moieties may be used as targeting moieties.
  • Such cyclic peptide targeting moieties have a stabilized secondary structure and may inhibit biological molecule interactions, increase cell membrane permeability and the peptide's half-life in blood serum.
  • the targeting moiety may consist of a therapeutic peptide.
  • peptide targeting moieties may be an AP-1 signaling inhibitor, such as a peptide analog comprising SEQ ID No. 104 of US8946381B2 to Fear that is used for the treatment of wounds, a peptide comprising SEQ ID No. 108 in US8822409B2 to Milech, et al.
  • the targeting moiety may be any biological modulator isolated from biodiverse gene fragment libraries as described in
  • the targeting moiety may consist of a characterized peptide.
  • a characterized peptide any member of the screening libraries created from bioinformatic source data to theoretically predict the secondary structure of a peptide as described in EP 1987178 to Watt et al., any peptide identified from peptide libraries that are screened for antagonism or inhibition of other biological interactions by a reverse hybrid screening method as described by EP1268842 to Hopkins, et al., the contents of each of which are incorporated herein by reference in their entirety, may be used as a targeting moiety.
  • targeting moieties may be cell-penetrating peptides.
  • any cell- penetrating peptides linked to a cargo that are capable of passing through the blood brain barrier as described by US20140141452A1 to Watt, et al., the contents of which are incorporated herein by reference, may be used a targeting moiety.
  • the targeting moiety may comprise a LHRH antagonist, agonist, or analog.
  • the targeting moiety may be Cetrorelix, a decapeptide with a terminal acid amide group (AC-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Cit-Leu-Arg-Pro- D-Ala-NH2) as described in US 4800191, US 6716817, US 6828415, US 6867191, US 7605121, US 7718599, US 7696149 (Zentaris Ag), or pharmaceutically active decapeptides such as SB-030, SB-075 (cetrorelix) and SB-088 disclosed in EP 0 299 402 (Asta Pharma), the contents of each of which are incorporated herein by reference in their entirety.
  • the targeting moiety may be LHRH analogues such as D-/L-Mel (4-[bis(2- chloroethyl)amino]-D/L-phenylalanine), cyclopropanealkanoyl, aziridine-2-carbonyl, epoxyalkyl, l,4-naphthoquinone-5-oxycarbonyl-ethyl, doxorubicinyl (Doxorubicin, DOX), mitomicinyl (Mitomycin C), esperamycinyl or methotrexoyl, as disclosed in US 6214969 to Janaky et al., the contents of which are incorporated herein by reference in their entirety.
  • LHRH analogues such as D-/L-Mel (4-[bis(2- chloroethyl)amino]-D/L-phenylalanine), cyclopropanealkanoyl, aziridine-2-carbonyl, epoxyalky
  • the targeting moiety may be any cell-binding molecule disclosed in US 7741277 or US 7741277 to Guenther et al. (Aeterna Zentaris), the contents of which are incorporated herein by reference in their entirety, such as octamer peptide, nonamer peptide, decamer peptide, luteinizing hormone releasing hormone (LHRH), [D- Lys6]-LHRH, LHRH analogue, LHRH agonist, Triptorelin ([D-Trp6]-LHRH), LHRH antagonist, bombesin, bombesin analogue, bombesin antagonist, somatostatin, somatostatin analogue, serum albumin, human serum albumin (HSA).
  • LHRH luteinizing hormone releasing hormone
  • [D- Lys6]-LHRH LHRH analogue
  • LHRH agonist Triptorelin
  • LHRH antagonist bombesin, bombesin analogue
  • targeting moieties may bind to growth hormone secretagogue (GHS) receptors, including ghrelin analogue ligands of GHS receptors.
  • GHS growth hormone secretagogue
  • targeting moieties may be any triazole derivatives with improved receptor activity and bioavailability properties as ghrelin analogue ligands of growth hormone secretagogue receptors as describe by US8546435 to Aicher, at al. (Aeterna Zentaris), the contents of which are incorporated herein by reference in their entirety.
  • the targeting moiety X is an aptide or bipodal peptide.
  • X may be any D-Aptamer-Like Peptide (D-Aptide) or retro-inverso Aptide which specifically binds to a target comprising: (a) a structure stabilizing region comprising parallel, antiparallel or parallel and antiparallel D-amino acid strands with interstrand noncovalent bonds; and (b) a target binding region I and a target binding region II comprising randomly selected n and m D-amino acids, respectively, and coupled to both ends of the structure stabilizing region, as disclosed in US Pat. Application No.
  • X may be any bipodal peptide binder (BPB) comprising a structure stabilizing region of parallel or antiparallel amino acid strands or a combination of these strands to induce interstrand non-covalent bonds, and target binding regions I and II, each binding to each of both termini of the structure stabilizing region, as disclosed in US Pat. Application No. 20120321697 to Jon et al., the contents of which are incorporated herein by reference in their entirety.
  • BBP bipodal peptide binder
  • X may be an intracellular targeting bipodal- peptide binder specifically binding to an intracellular target molecule, comprising: (a) a structure-stabilizing region comprising a parallel amino acid strand, an antiparallel amino acid strand or parallel and antiparallel amino acid strands to induce interstrand non-covalent bonds; (b) target binding regions I and II each binding to each of both termini of the structure-stabilizing region, wherein the number of amino acid residues of the target binding region I is n and the number of amino acid residues of the target binding region II is m; and (c) a cell-penetrating peptide (CPP) linked to the structure-stabilizing region, the target binding region I or the target binding region II, as disclosed in US Pat. Application No.
  • CPP cell-penetrating peptide
  • X may be any bipodal peptide binder comprising a ⁇ -hairpin motif or a leucine- zipper motif as a structure stabilizing region comprising two parallel amino acid strands or two antiparallel amino acid strands, and a target binding region I linked to one terminus of the first of the strands of the structure stabilizing region, and a target binding region II linked to the terminus of the second of the strands of the structure stabilizing region, as disclosed in US Pat. Application No. 20110152500 to Jon et al., the contents of which are incorporated herein by reference in their entirety.
  • X may be any bipodal peptide binder targeting KPI as disclosed in WO2014017743 to Jon et al, any bipodal peptide binder targeting cytokine as disclosed in WO2011132939 to Jon et al., any bipodal peptide binder targeting transcription factor as disclosed in WO201132941 to Jon et al., any bipodal peptide binder targeting G protein-coupled receptor as disclosed in WO2011132938 to Jon et al., any bipodal peptide binder targeting receptor tyrosine kinase as disclosed in WO2011132940 to Jon et al., the contents of each of which are incorporated herein by reference in their entireties.
  • X may also be bipodal peptide binders targeting cluster differentiation (CD7) or an ion channel.
  • the targeting moiety may be a cyclic peptide or a modified cyclic peptide.
  • the cyclic peptide targeting moieties may be prepared by cyclization of linear peptides via reaction with a small rigid entity (cyclization scaffold) that comprises at least 2 reactive groups as anchor points. The reactive groups form covalent bonds with thiols of the cysteines in the peptide and attach the cyclization scaffold to the peptide. The peptide folds around the cyclization scaffold and loses flexibility while adopting a well-defined three-dimensional structure, enhancing the stability of the peptide.
  • the cyclization scaffold may be any molecular scaffold disclosed in
  • the cyclic peptides are prepared by the method disclosed in WO2012057624 (Pepscan Systems), the contents of which are incorporated herein in their entirety. Molecules with formula (I), (II), (III), or (IV) in WO2012057624 may be used as cyclization scaffolds to prepare cyclic peptides, such as peptides having structures of formulas (V), (VI), (VII), (IX), (X), (XI), (XII), or (XIII).
  • the target moiety is a bicyclic peptide or a modified bicyclic peptide.
  • the bicyclic peptide targeting moieties may comprise between 9 and 15 amino acids in size and have molecular weight of around 1.5-2 kDa.
  • the bicyclic peptides may comprise at least 2 loops and a molecular scaffold which connects the loops.
  • the molecular scaffold is any molecule which is able to connect the peptide at multiple points to cyclize the peptides and form the loops. Each loop may comprise 5 or 6 amino acids.
  • the bicyclic peptides may be protease resistant.
  • the bicyclic peptides may target CD38, AVB3, CAIX, EphA3, membrane type 1 metalloprotease (MTl-MMP),
  • MMP9 metalloprotease 9
  • PSMA prostate-specific membrane antigen
  • VEGF vascular endothelial growth factor
  • FAPa fibroblast activation protein alpha
  • T Fa tumor necrosis factor alpha
  • TNFp tumor necrosis factor alpha
  • Cathepsin G Cathepsin G
  • Cathepsin S IL-17
  • IL-9 kallikrein-related peptidase 7
  • KLK5 heat shock protein 90
  • Chymase or neutrophil elastase.
  • Non-limiting examples of the bicyclic peptides or modified bicyclic peptides include: structured peptides comprising a polypeptide linked to a molecular scaffold and is cyclized to form at least 2 separate loops disclosed in WO2010089117; peptides targeting Kallikrein with at least two polypeptide loops as disclosed in US20140256596,
  • the bicyclic peptide may be made by combining linear polypeptides displayed on a genetic display system with a purification resin such that the display system is bound to the resin and treating the bound display system with a reducing agent; exposing the bound display system to a molecular scaffold; removing unreacted molecular scaffold from the bound display system; and eluting the display system from the purification resin.
  • the genetic display system may be a phage display.
  • the molecular scaffold may be tris-(bromomethyl)benzene (TBMB).
  • the target, target cell or marker is a molecule that is present exclusively or predominantly on the surface of malignant cells, e.g., a tumor antigen.
  • a marker is a prostate cancer marker.
  • the target can be an intra-cellular protein.
  • a marker is a breast cancer marker, a colon cancer marker, a rectal cancer marker, a lung cancer marker, a pancreatic cancer marker, a ovarian cancer marker, a bone cancer marker, a renal cancer marker, a liver cancer marker, a neurological cancer marker, a gastric cancer marker, a testicular cancer marker, a head and neck cancer marker, an esophageal cancer marker, or a cervical cancer marker.
  • the targeting moiety directs the conjugates to specific tissues, cells, or locations in a cell.
  • the target can direct the conjugate in culture or in a whole organism, or both.
  • the targeting moiety binds to a receptor that is present on the surface of or within the targeted cell(s), wherein the targeting moiety binds to the receptor with an effective specificity, affinity and avidity.
  • the targeting moiety targets the conjugate to a specific tissue such as the liver, kidney, lung or pancreas.
  • the targeting moiety can target the conjugate to a target cell such as a cancer cell, such as a receptor expressed on a cell such as a cancer cell, a matrix tissue, or a protein associated with cancer such as tumor antigen.
  • cells comprising the tumor vasculature may be targeted.
  • Targeting moieties can direct the conjugate to specific types of cells such as specific targeting to hepatocytes in the liver as opposed to Kupffer cells.
  • targeting moieties can direct the conjugate to cells of the reticular endothelial or lymphatic system, or to
  • phagocytic cells such as macrophages or eosinophils.
  • the target is member of a class of proteins such as receptor tyrosine kinases (RTK) including the following RTK classes: RTK class I (EGF receptor family) (ErbB family), RTK class II (Insulin receptor family), RTK class III (PDGF receptor family), RTK class IV (FGF receptor family), RTK class V (VEGF receptors family), RTK class VI (HGF receptor family), RTK class VII (Trk receptor family), RTK class VIII (Eph receptor family), RTK class IX (AXL receptor family), RTK class X (LTK receptor family), RTK class XI (TIE receptor family), RTK class XII (ROR receptor family), RTK class XIII (DDR receptor family), RTK class XIV (RET receptor family), RTK class XV (KLG receptor family), RTK class XVI (RYK receptor family) and RTK class XVII (MuSK receptor family).
  • RTK class I EGF receptor family
  • ErbB family ErbB family
  • the target is a serine or threonine kinase, G-protein coupled receptor, methyl CpG binding protein, cell surface glycoprotein, cancer stem cell antigen or marker, carbonic anhydrase, cytolytic T lymphocyte antigen, DNA methyltransferase, an ectoenzyme, a glycosylphosphatidylinositol-anchored co-receptor, a gly pi can-related integral membrane proteoglycan, a heat shock protein, a hypoxia induced protein, a multi drug resistant transporter, a Tumor-associated macrophage marker, a tumor associated
  • carbohydrate antigen a TNF receptor family member, a transmembrane protein, a tumor necrosis factor receptor superfamily member, a tumour differentiation antigen, a zinc dependent metallo-exopeptidase, a zinc transporter, a sodium-dependent transmembrane transport protein, a member of the SIGLEC family of lectins, or a matrix metalloproteinase.
  • HER-2 HER-2
  • HER-3 EGFR
  • NTSR1 neurotensin receptors
  • the targeting moiety binds a target such as CD 19, CD70, CD56, PSMA, alpha integrin, CD22, CD 138, EphA2, AGS-5, Nectin-4, HER2, GPMNB, CD74 and Le.
  • the target is a protein listed in Table A.
  • the targeted constructs may contain one or more internal linker moieties connecting the protein-binding moieties and targeting moieties.
  • the internal linker moiety Y may be attached to the targeting moiety X and the protein-binding moiety by functional groups independently selected from an ester bond, disulfide, thioether, amide, acylhydrazone, ether, carbamate, carbonate, carbon-carbon bond, and urea.
  • the internal linker moiety can be attached to either the targeting moiety or the protein-binding moiety by a non-cleavable group such as provided by the conjugation between a thiol and a maleimide, or between an azide and an alkyne.
  • the internal linker is independently selected from the group consisting of alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, wherein each of the alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups optionally is substituted with one or more groups, each independently selected from halogen, cyano, nitro, hydroxyl, carboxyl, carbamoyl, ether, alkoxy, aryloxy, amino, amide, carbamate, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, or heterocyclyl, wherein each of the carboxyl, carbamoyl, ether, alkoxy, aryloxy, amino, amide, carbamate, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, cycloalkyl,
  • the linker moiety or moieties in the targeted constructs are present at a predetermined molar weight percentage from about 1% to about 10%, or about 10% to about 20%, or about 20% to about 30%, or about 30% to about 40%, or about 40% to about 50%), or about 50%> to about 60%>, or about 60%> to about 70%>, or about 70%> to about 80%, or about 80% to about 90%, or about 90% to about 99%, such that the sum of the molar weight percentages of the components of the targeted construct is 100%.
  • the internal linker moiety is not cleavable.
  • the internal linker moiety comprises a cleavable
  • cleavable functionality designed to be cleaved in an intracellular manner.
  • the cleavable functionality may be hydrolyzed in vivo or may be designed to be hydrolyzed enzymatically, for example by Cathepsin B, or may be a pH-sensitive linker.
  • a "cleavable" linker refers to any linker which can be cleaved physically or chemically. Examples for physical cleavage may be cleavage by light, radioactive emission or heat, while examples for chemical cleavage include cleavage by re- dox-reactions, hydrolysis, pH-dependent cleavage or cleavage by enzymes.
  • the internal linker moiety may be selected from dicarboxylate derivatives of succinic acid, glutaric acid or diglycolic acid.
  • the internal linker moiety may be cleavable and is cleaved to release the targeting moiety.
  • the internal linker may be cleaved by an enzyme.
  • the cleaving of the internal linker moiety is non-enzymatic.
  • targeted constructs comprising at least one protein-binding moiety of the present disclosure and at least one active agent (Z) are provided.
  • the protein- binding moiety and the active agent may be connected with an optional internal linker moiety (Y) as described above: (Protein-binding moiety )n-(Y)p-(Z)m, n>l, m>l, and p>0.
  • the protein-binding moiety is an antibody mimetic described herein. In some embodiments, the protein-binding moiety is a small molecule described herein.
  • the optional internal linker moiety is cleavable.
  • the active agent may be active when the optional internal linker is cleaved and the active agent is released from the targeted constructs.
  • the active agent is connected to the protein-binding moiety with a non-cleavable bond or a non-cleavable linker.
  • the active agent may be active when it's connected with the protein-binding moiety.
  • the targeted constructs may contain at least one active agent.
  • the active agent can be a therapeutic, prophylactic, diagnostic, or nutritional agent.
  • a variety of active agents are known in the art and may be used in the targeted constructs described herein.
  • the active agent can be a protein or peptide, small molecule, nucleic acid or nucleic acid molecule, lipid, sugar, glycolipid, glycoprotein, lipoprotein, or combination thereof.
  • the active agent is an antigen, an adjuvant, radioactive, an imaging agent (e.g., a fluorescent moiety) or a polynucleotide.
  • the active agent of the targeted constructs comprises a predetermined molar weight percentage from about 1% to about 10%, or about 10% to about 20%, or about 20% to about 30%, or about 30% to about 40%, or about 40% to about 50%, or about 50%) to about 60%, or about 60% to about 70%, or about 70% to about 80%, or about 80%) to about 90%), or about 90% to about 99%, such that the sum of the molar weight percentages of the components of the targeted construct is 100%.
  • the amount of active agent(s) of the targeted construct may also be expressed in terms of proportion to the protein- binding moiety(s).
  • the present teachings provide a ratio of active agent to protein-binding moiety of about 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3 : 1, 2: 1, 1 : 1, 1 :2, 1 :3, 1 :4; 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, or 1 : 10.
  • the active agent can be a cancer therapeutic.
  • Cancer therapeutics include, for example, death receptor agonists such as the TNF-related apoptosis- inducing ligand (TRAIL) or Fas ligand or any ligand or antibody that binds or activates a death receptor or otherwise induces apoptosis.
  • TRAIL TNF-related apoptosis- inducing ligand
  • Suitable death receptors include, but are not limited to, TNFRl, Fas, DR3, DR4, DR5, DR6, LTpR and combinations thereof.
  • Cancer therapeutics such as chemotherapeutic agents, cytokines, chemokines, and radiation therapy agents can be used as active agents.
  • Chemotherapeutic agents include, for example, alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor agents. Such agents typically affect cell division or DNA synthesis and function.
  • Additional examples of therapeutics that can be used as active agents include monoclonal antibodies and the tyrosine kinase inhibitors e.g. imatinib mesylate, which directly targets a molecular abnormality in certain types of cancer (e.g., chronic myelogenous leukemia, gastrointestinal stromal tumors).
  • Chemotherapeutic agents include, but are not limited to cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, vincristine, vinblastine, vinorelbine, vindesine, taxol and derivatives thereof, irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, epipodophyllotoxins, trastuzumab, cetuximab, and rituximab, bevacizumab, and combinations thereof. Any of these may be used as an active agent in a conjugate.
  • the active agent is a small molecule having a molecular weight preferably ⁇ about 5 kDa, more preferably ⁇ about 4 kDa, more preferably about 3 kDa, most preferably ⁇ about 1.5 kDa or ⁇ about 1 kDa.
  • the small molecule active agents used in this invention include cytotoxic compounds (e.g., broad spectrum), angiogenesis inhibitors, cell cycle progression inhibitors, PBK/m-TOR/AKT pathway inhibitors, MAPK signaling pathway inhibitors, kinase inhibitors, protein chaperones inhibitors, HDAC inhibitors, PARP inhibitors, Wnt/Hedgehog signaling pathway inhibitors, RNA polymerase inhibitors and proteasome inhibitors.
  • the small molecule active agents in some embodiments the active agent is an analog, derivative, prodrug, or pharmaceutically acceptable salt thereof.
  • Broad spectrum cytotoxins include, but are not limited to, DNA-binding or alkylating drugs, microtubule stabilizing and destabilizing agents, platinum compounds, and topoisom erase I or II inhibitors.
  • Exemplary DNA-binding or alkylating drugs include, CC-1065 and its analogs, anthracyclines (doxorubicin, epirubicin, idarubicin, daunorubicin) and its analogs, alkylating agents, such as calicheamicins, dactinomycins, mitomycins, pyrrolobenzodiazepines, trioxacarcins and the like.
  • anthracyclines doxorubicin, epirubicin, idarubicin, daunorubicin
  • alkylating agents such as calicheamicins, dactinomycins, mitomycins, pyrrolobenzodiazepines, trioxacarcins and the like.
  • trioxacarcins include Trioxacarcins DC- 45-A2, DC-45-A1, A, D, C7"-epi-C, and C disclosed in Nicolaou et al., JACS, vol.138:3118 (2016), and trioxacarcin A, DC-45-A1 and structural analogues disclosed in Fig. 1 of Magauer et al., Nature Chemistry, vol.5:886 (2013), the contents of each of which are incorporated herein by reference in their entirety.
  • doxorubicin analogs include nemorubicin metabolite or analog drug moiety disclosed in US 20140227299 to Cohen et al., the contents of which are incorporated herein by reference in their entirety.
  • Exemplary CC-1065 analogs include duocarmycin SA, duocarmycin CI, duocarmycin C2, duocarmycin B2, DU-86, KW-2189, bizelesin, seco-adozelesin, and those described in U.S. Patent Nos. 5,475,092; 5,595,499; 5,846,545; 6,534,660; 6,586,618;
  • Doxorubicin and its analogs include PNU-159682 and those described in U.S. Patent No.6,630,579 and nemorubicin metabolite or analog drugs disclosed in US 20140227299 to Cohen et al., the contents of which are incorporated herein by reference in their entirety.
  • Calicheamicins include those described in U.S. Patent Nos. 5,714,586 and
  • Duocarmycins include those described in U.S. Patent Nos.5,070,092; 5,101,038; 5, 187,186; 6,548,530; 6,660,742; and 7,553,816 B2; and Li et al., Tet Letts., 50:2932 - 2935 (2009).
  • Pyrrolobenzodiazepines include SG2057 and those described in Denny, Exp. Opin. Ther. Patents., 10(4):459-474 (2000), Anti-Cancer Agents in Medicinal Chemistry, 2009, 9, 1-31; WO 2011/130613 Al; EP 2 789 622 Al; Blood 2013, 122, 1455; J. Antimicrob.
  • microtubule stabilizing and destabilizing agents include taxane compounds, such as paclitaxel, docetaxel, cabazitaxel; maytansinoids, auristatins and analogs thereof, tubulysin A and B derivatives, vinca alkaloid derivatives, epothilones, PM060184 and cryptophycins.
  • Exemplary maytansinoids or maytansinoid analogs include maytansinol and maytansinol analogs, maytansine or DM1 and DM4 are those described in U.S. Patent Nos. 5,208,020; 5,416,064; 6,333.410; 6,441,163; 6,716,821; RE39, 151 and 7,276,497.
  • the cytotoxic agent is a maytansinoid, another group of anti-tubulin agents (ImmunoGen, Inc.; see also Chari et al., 1992, Cancer Res. 52: 127-131), maytansinoids or maytansinoid analogs.
  • Suitable maytansinoids include maytansinol and maytansinol analogs.
  • suitable maytansinoids are disclosed in U.S. Patent Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533; 6,333,410; 5,475,092; 5,585,499; and
  • auristatins include auristatin E (also known as a derivative of dolastatin- 10), auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin F and dolastatin.
  • auristatin E also known as a derivative of dolastatin- 10
  • AEB auristatin EB
  • AEFP auristatin EFP
  • MMAE monomethyl auristatin E
  • MMAF monomethyl auristatin F
  • dolastatin dolastatin.
  • Suitable auristatins are also described in U.S. Publication Nos. 2003/0083263, 2011/0020343, and
  • Exemplary tubulysin compounds include compounds described in U.S. Patent Nos. 7,816,377; 7,776,814; 7,754,885; U.S. Publication Nos. 2011/0021568; 2010/004784;
  • Exemplary vinca alkaloids include vincristine, vinblastine, vindesine, and navelbine (vinorelbine).
  • Suitable Vinca alkaloids that can be used in the present invention are also disclosed in U.S. Publication Nos. 2002/0103136 and 2010/0305149, and in U.S. Patent No. 7,303,749 Bl, the disclosures of which are incorporated herein by reference in their entirety.
  • Exemplary epothilone compounds include epothilone A, B, C, D, E and F, and derivatives thereof. Suitable epothilone compounds and derivatives thereof are described, for example, in U.S. Patent Nos. 6,956,036; 6,989,450; 6,121,029; 6, 117,659; 6,096,757;
  • Exemplary platinum compounds include cisplatin (PLATINOL®), carboplatin (PARAPLATIN®), oxaliplatin (ELOX ATINE®), iproplatin, ormaplatin, and tetraplatin.
  • topoisomerase I inhibitors include camptothecin, camptothecin, derivatives, camptothecin analogs and non-natural camptothecins, such as, for example, CPT- 11 (irinotecan), SN-38, topotecan, 9-aminocamptothecin, rubitecan, gimatecan, karenitecin, silatecan, lurtotecan, exatecan, diflomotecan, belotecan, lurtotecan and S39625.
  • camptothecin compounds that can be used in the present invention include those described in, for example, J. Med. Chem., 29:2358-2363 (1986); J. Med. Chem., 23 :554 (1980); J. Med. Chem., 30: 1774 (1987).
  • topoisomerase II inhibitors include azonafide and etoposide.
  • Additional agents acting on DNA include Lurbinectedin (PM01183), Trabectedin (also known as ecteinascidin 743 or ET-743) and analogs as described in WO 200107711, WO 2003014127.
  • Angiogenesis inhibitors include, but are not limited to, MetAP2 inhibitors.
  • Exemplary MetAP2 inhibitors include fumagillol analogs, meaning any compound that includes the fumagillin core structure, including fumagillamine, that inhibits the ability of MetAP-2 to remove H2-terminal methionines from proteins as described in Rodeschini et al., /. Org. Chem., 69, 357-373, 2004 and Liu, et al., Science 282, 1324-1327, 1998.
  • fumagillol analogs are disclosed in /. Org. Chem., 69, 357, 2004; J. Org. Chem., 70, 6870, 2005; European Patent Application 0 354 787; /. Med.
  • Exemplary cell cycle progression inhibitors include CDK inhibitors such as BMS- 387032 and PD0332991; Rho-kinase inhibitors such as GSK429286; checkpoint kinase inhibitors such as AZD7762; aurora kinase inhibitors such as AZD1152, MLN8054 and MLN8237; PLK inhibitors such as BI 2536, BI6727 (Volasertib), GSK461364, ON-01910 (Estybon); and KSP inhibitors such as SB 743921, SB 715992 (ispinesib), MK-0731, AZD8477, AZ3146 and ARRY-520.
  • CDK inhibitors such as BMS- 387032 and PD0332991
  • Rho-kinase inhibitors such as GSK429286
  • checkpoint kinase inhibitors such as AZD7762
  • aurora kinase inhibitors such as AZD1152, MLN8054
  • Exemplary PI3K/m-TOR/AKT signaling pathway inhibitors include
  • PI3K phosphoinositide 3 -kinase
  • Exemplary PI3 kinase inhibitors are disclosed in U.S. Patent No. 6,608,053, and include BEZ235, BGT226, BKM120, CALIOI, CAL263, demethoxyviridin, GDC-0941, GSK615, IC87114, LY294002, Palomid 529, perifosine, PF-04691502, PX-866,
  • Exemplary AKT inhibitors include, but are not limited to, AT7867, MK-2206, Perifosine, GSK690693, Ipatasertib, AZD5363, TIC 10, Afuresertib, SC79, AT13148, PHT- 427, A-674563, and CCT128930.
  • Exemplary MAPK signaling pathway inhibitors include MEK, Ras, INK, B-Raf and p38 MAPK inhibitors.
  • Exemplary MEK inhibitors are disclosed in U.S. Patent No. 7,517,994 and include GDC-0973, GSK1120212, MSC1936369B, AS703026, R05126766 and R04987655, PD0325901, AZD6244, AZD 8330 and GDC-0973.
  • Exemplary B-raf inhibitors include CDC-0879, PLX-4032, and SB590885.
  • Exemplary B p38 MAPK inhibitors include BIRB 796, LY2228820 and SB202190
  • RTK Receptor tyrosine kinases
  • Exemplary inhibitors of ErbB2 receptor include but not limited to AEE788 (NVP-AEE 788), BIBW2992 (Afatinib), Lapatinib, Erlotinib (Tarceva), and Gefitinib (Iressa).
  • multitargeted kinase inhibitors include AP24534 (Ponatinib) that targets FGFR, FLT-3, VEGFR-PDGFR and Bcr-Abl receptors; ABT-869 (Linifanib) that targets FLT-3 and VEGFR- PDGFR receptors; AZD2171 that targets VEGFR-PDGFR, Flt-1 and VEGF receptors; CHR-258 (Dovitinib) that targets VEGFR-PDGFR, FGFR, Flt-3, and c-Kit receptors.
  • AP24534 Panatinib
  • ABT-869 Liifanib
  • AZD2171 that targets VEGFR-PDGFR, Flt-1 and VEGF receptors
  • CHR-258 Dovitinib
  • Exemplary kinase inhibitors include inhibitors of the kinases ATM, ATR, CFD l, CHK2, WEE1, and RSK.
  • Exemplary protein chaperone inhibitors include HSP90 inhibitors.
  • Exemplary HSP90 inhibitors include Ganetespib, 17AAG derivatives, BIIB021, BIIB028, SNX-5422, NVP-AUY-922, and KW-2478.
  • HDAC inhibitors include Belinostat (PXD101), CUDC-101,
  • Exemplary PARP inhibitors include neratinib (HKI-272), iniparib (BSI 201), olaparib (AZD-2281), ABT-888 (Veliparib), rucaparib (AG014699, CEP 9722, niraparib (MK-4827), KU-0059436 (AZD2281), talazoparib (BMN-673), 3- aminobenzamide, A- 966492, E7016, BGB-290 and AZD2461
  • Exemplary Wnt/Hedgehog signaling pathway inhibitors include vismodegib (RG3616/GDC-0449), cyclopamine (11-deoxojervine) (Hedgehog pathway inhibitors), and XAV-939 (Wnt pathway inhibitor).
  • Exemplary RNA polymerase inhibitors include amatoxins.
  • Exemplary amatoxins include a- amanitins, ⁇ - amanitins, ⁇ - amanitins, ⁇ -amanitins, amanullin, amanullic acid, amaninamide, amanin, and proamanullin.
  • Other amanitin compounds that can be used in the present invention include those described in, for example WO2014135282, WO2016142049, and EP2872479 the contents of each of which are incorporated herein by reference in their entirety.
  • Exemplary proteasome inhibitors include bortezomib, carfilzomib, ONX 0912, CEP- 18770, and MLN9708.
  • the drug of the invention is a non-natural camptothecin compound, vinca alkaloid, kinase inhibitor (e.g., PI3 kinase inhibitor (GDC-0941 and PI- 103)), MEK inhibitor, KSP inhibitor, RNA polymerse inhibitor, PARP inhibitor, docetaxel, paclitaxel, doxorubicin, duocarmycin, tubulysin, auristatin or a platinum compound.
  • kinase inhibitor e.g., PI3 kinase inhibitor (GDC-0941 and PI- 103)
  • MEK inhibitor e.g., PI3 kinase inhibitor (GDC-0941 and PI- 103)
  • MEK inhibitor e.g., PI3 kinase inhibitor (GDC-0941 and PI- 103)
  • MEK inhibitor e.g., PI3 kinase inhibitor (GDC-0941 and PI- 103)
  • KSP inhibitor
  • the drug is a derivative of SN-38, vindesine, vinblastine, PI- 103, AZD 8330, auristatin E, auristatin F, a duocarmycin compound, tubulysin compound, or ARRY- 520.
  • the drug used in the invention is a combination of two or more drugs, such as, for example, PI3 kinases and MEK inhibitors; broad spectrum cytotoxic compounds and platinum compounds; PARP inhibitors and platinum compounds; broad spectrum cytotoxic compounds and PARP inhibitors.
  • drugs such as, for example, PI3 kinases and MEK inhibitors; broad spectrum cytotoxic compounds and platinum compounds; PARP inhibitors and platinum compounds; broad spectrum cytotoxic compounds and PARP inhibitors.
  • the active agent can be a cancer therapeutic.
  • the cancer therapeutics may include death receptor agonists such as the TNF-related apoptosis-inducing ligand (TRAIL) or Fas ligand or any ligand or antibody that binds or activates a death receptor or otherwise induces apoptosis.
  • TRAIL TNF-related apoptosis-inducing ligand
  • Suitable death receptors include, but are not limited to, TNFRl, Fas, DR3, DR4, DR5, DR6, LTpR and combinations thereof.
  • the active agent can be a DNA minor groove binder such as lurbectidin and trabectidin.
  • the active agent can be E3 ubiquitin ligase inhibitors, adeubiquitinase inhibitors or an NFkB pathway inhibitor.
  • the active agent can be a phosphatase inhibitor including inhibitors of PTP1B, SHP2, LYP, FAP-1, CD45, STEP, MKP-1, PRL, LMWPTP or CDC25.
  • the active agent can be an inhibitor of tumor metabolism, such as an inhibitor of GAPDH, GLUT1, HK II, PFK, GAPDH, PK, LDH or MCTs.
  • the active agent can target epigenetic targets including EZH2, MIX, DOTl-like protein (DOTIL), bromodomain-containing protein 4 (BRD4), BRD2, BRD3, NUT, ATAD2, or SMYD2.
  • DOTIL DOTl-like protein
  • BRD4 bromodomain-containing protein 4
  • BRD2, BRD3, NUT ATAD2, or SMYD2.
  • the active agent can target the body's immune system to help fight cancer, including molecules targeting IDOl, ID02, TDO, CD39, CD73, A2A antagonists, STING activators, TLR agonists (TLR 1-13), ALK5, CBP/EP300 bromodomain, ARG1, ARG2, iNOS, PDE5, P2X7, P2Y11, COX2, EP2 Receptor, or EP4 receptor.
  • the active agent can target Bcl-2, IAP, or fatty acid synthase.
  • the active agent can be 20-epi-l,25 dihydroxy vitamin D3, 4- ipomeanol, 5-ethynyluracil, 9-dihydrotaxol, abiraterone, acivicin, aclarubicin, acodazole hydrochloride, acronine, acylfulvene, adecypenol, adozelesin, aldesleukin, all-tk antagonists, altretamine, ambamustine, ambomycin, ametantrone acetate, amidox, amifostine,
  • aminoglutethimide aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitors, antagonist D, antagonist G, antarelix, anthramycin, anti-dorsalizing morphogenetic protein-1, antiestrogen, antineoplaston, antisense
  • oligonucleotides aphidicolin glycinate, apoptosis gene modulators, apoptosis regulators, apurinic acid, ARA-CDP-DL-PTBA, arginine deaminase, asparaginase, asperlin, asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azacitidine, azasetron, azatoxin, azatyrosine, azetepa, azotomycin, baccatin III derivatives, balanol, batimastat, benzochlorins, benzodepa, benzoylstaurosporine, beta lactam derivatives, beta-alethine, betaclamycin B, betulinic acid, BFGF inhibitor, bicalutamide, bisantrene, bisantrene hydrochloride, bisaziridinylsper
  • metalloproteinase inhibitors maytansine, maytansinoid, mertansine (DM1), mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, menogaril, merbarone, mercaptopurine, meterelin, methioninase, methotrexate, methotrexate sodium,
  • metoclopramide metoprine, meturedepa, microalgal protein kinase C inhibitors, MIF inhibitor, mifepristone, miltefosine, mirimostim, mismatched double stranded RNA, mitindomide, mitocarcin, mitocromin, mitogillin, mitoguazone, mitolactol, mitomalcin, mitomycin, mitomycin analogs, mitonafide, mitosper, mitotane, mitotoxin fibroblast growth factor-saporin, mitoxantrone, mitoxantrone hydrochloride, mofarotene, molgramostim, monoclonal antibody, human chorionic gonadotrophin, monophosphoryl lipid
  • a/myobacterium cell wall SK mopidamol, multiple drug resistance gene inhibitor, multiple tumor suppressor 1 -based therapy, mustard anticancer agent, mycaperoxide B, mycobacterial cell wall extract, mycophenolic acid, myriaporone, n-acetyldinaline, nafarelin, nagrestip, naloxone/pentazocine, napavin, naphterpin, nartograstim, nedaplatin, nemorubicin, neridronic acid, neutral endopeptidase, nilutamide, nisamycin, nitric oxide modulators, nitroxide antioxidant, nitrullyn, nocodazole, nogalamycin, n-substituted benzamides, 06- benzylguanine, octreotide, okicenone, oligonucleotides, onapristone, ondansetron, oracin,
  • transduction inhibitors signal transduction modulators, sirolene, single chain antigen binding protein, sizofiran, sobuzoxane, sodium borocaptate, sodium phenylacetate, solverol, somatomedin binding protein, sonermin, sparfosate sodium, sparfosic acid, sparsomycin, spicamycin D, spirogermanium hydrochloride, spiromustine, spiroplatin, splenopentin, spongistatin 1, squalamine, stem cell inhibitor, stem-cell division inhibitors, stipiamide, streptonigrin, streptozocin, stromelysin inhibitors, sulfinosine, sulofenur, superactive vasoactive intestinal peptide antagonist, suradista, suramin, swainsonine, synthetic glycosaminoglycans, talisomycin, tallimustine, tamoxifen methiodide, tauromustine,
  • tetrachlorodecaoxide tetrazomine, thaliblastine, thalidomide, thiamiprine, thiocoraline, thioguanine, thiotepa, thrombopoietin, thrombopoietin mimetic, thymalfasin, thymopoietin receptor agonist, thymotrinan, thyroid stimulating hormone, tiazofurin, tin ethyl etiopurpurin, tirapazamine, titanocene dichloride, topotecan hydrochloride, topsentin, toremifene, toremifene citrate, totipotent stem cell factor, translation inhibitors, trestolone acetate, tretinoin, triacetyluridine, triciribine, triciribine phosphate, trimetrexate, trimetrexate glucuronate, triptorelin
  • the active agent can be an inorganic or organometallic compound containing one or more metal centers. In some examples, the compound contains one metal center.
  • the active agent can be, for example, a platinum compound, a ruthenium compound (e.g., trans- [RuCh (DMSO)4], or /ram , -[RuCl4(imidazole) 2, etc.), cobalt compound, copper compound, or iron compounds.
  • the active agent is a small molecule. In some embodiments, the active agent is a small molecule cytotoxin. In one embodiment, the active agent is cabazitaxel, or an analog, derivative, prodrug, or pharmaceutically acceptable salt thereof. In another embodiment, the active agent is mertansine (DM1) or DM4, or an analog, derivative, prodrug, or pharmaceutically acceptable salt thereof. DM1 or DM4 inhibits the assembly of microtubules by binding to tubulin. Structure of DM1 is shown below:
  • the active agent Z is Monomethyl auristatin E (MMAE), or an analog, derivative, prodrug, or pharmaceutically acceptable salt thereof. Structure of MMAE is shown below:
  • the active agent Z is a sequence-selective DNA minor- groove binding crosslinking agent.
  • Z may be pyrrolobenzodiazepine (PBD), a PBD dimer, or an analog, derivative, prodrug, or pharmaceutically acceptable salt thereof.
  • PBDs pyrrolobenzodiazepines or pyrrolo[2, l-c][l,4]benzodiazepines (PBDs) are a family of sequence-selective DNA minor-groove binding agents.
  • the first example of a PBD monomer is the natural product anthramycin. Synthetic PBDs have been developed by attaching non- covalent minor-groove binding components to the C8-position of the PBD aromatic-ring.
  • PBD dimer Monomeric PBD units have been joined together to afford PBD dimers.
  • PBD dimer is SJG-136.
  • PBD analogs and dimers include, but not limited to, any PBD-based payload disclosed in Mantaj et al., Angew. Chem. Int. Ed, vol.56:462 (2017), the contents of which are incorporated herein by reference in their entirety, such as GWL-78, KMR-28-39, DSB-120, SJG-136 (also known as SG2000, NSC 694501 or BN2629) in Fig. 1 of Mantaj et al.
  • the active agent Z is a pyrrolo[2, l-c][l,4]benzodiazepine compound of Formula (I), as disclosed in US20170050971 (Femtogenix Limited), the contents of which are incorporated herein by reference in their entirety.
  • the active agent Z is a pyrridinobenzodiazepine (PDD) compound of Formula (I), comprising three fused 6-7-6-membered rings linked to aromatic groups, as disclosed in WO2017032983 (Femtogenix Limited), the contents of which are incorporated herein by reference in their entirety.
  • the active agent Z is a
  • PDD pyrridinobenzodiazepine
  • BPD benzopyrridodiazecine
  • the PDD or BPD monomers may be linked to form a dimer.
  • the PDD or BPD monomers may also link to aromatic groups.
  • the active agent Z is a compound of formula (I): A-X1-L-X2-B, as disclosed in WO2017194960 (Femtogenx Limited), the contents of which are incorporated herein by reference in their entirety, wherein A is a group selected from ( ⁇ i L (A2), (A3), (A4) and (A5), ami B is a group selected from (Bl), ( 2), and (B3).
  • the active agent Z is capable of increasing a cancer specific immune response, such as any active agent disclosed in WO2017023779, the contents of which are incorporated herein by reference in their entirety.
  • the active agent Z inhibits the immunosuppressive effect. In some embodiments, the active agent Z binds to a checkpoint receptor on T cells or natural killer cells. Any checkpoint inhibitor disclosed in WO2017023749, the contents of which are incorporated herein by reference in their entirety, may be used as an active agent in the present disclosure.
  • the active agent Z is a topoisomerase I inhibitor, such as camptothecin, irinotecan, SN-38, or an analog, derivative, prodrug, or pharmaceutically acce table salt thereof.
  • the active agent Z is a radioactive agent.
  • radionuclides have emission properties, including ⁇ , ⁇ , ⁇ , and Auger emissions, that may be used for therapeutic and/or diagnostic purposes.
  • the active agent Z may comprise a radioisotope, such as In-I l l, Y-90, Y-86, 1-131, Re-186, Re-188, Y-90, Bi-212, At-21 1, Zr-89, Sr-89, Ho-166, Sm-153, Cu-67, Cu-64, Lu-177, Ac-225, Pb-212, Pb-203, Bi- 213, Th-227, Pb-212, Ra-223, and P-32.
  • a radioisotope such as In-I l l, Y-90, Y-86, 1-131, Re-186, Re-188, Y-90, Bi-212, At-21 1, Zr-89, Sr-89, Ho-166, Sm-153, Cu-67, Cu-64, Lu
  • the active agent Z comprises a chelating agent.
  • the chelating agent may be a metal chelating agent that binds to a metal including a metallic neuclide.
  • the chelating agent may also be a chelating agent that binds to a non-metal active agent.
  • the chelating agent may be acyclic or macrocyclic.
  • Non-limiting examples of chelating agents include l,4,7, 10-tetraazacyclododecane-l,4,7, 10-tetraacetic acid (DOTA); DOTA derivative: D03 A; diethylenetriamine-N,N;N'N",N"-pentaacetic acid (DTPA); DTPA derivatives: 2-(p-SCN-Bz)-6-methyl-DTPA, CHX-A"-DTPA, and the cyclic anhydride of DTPA (CA-DTPA); l,4,7-triazacyclononane-l,4-7-triacetic acid (NOT A); NOT A derivatives (e.g., BCNOTA, p-NCS-Bz-NOTA, BCNOT); 6-hydrazinonicotinamide (HYNIC);
  • EDTA ethylenediamine tetraacetic acid
  • N ⁇ -ethylene-di-J-cysteine N ⁇ -ethylene-di-J-cysteine
  • N,N'-diacetic acid B6SS; Deferoxamine (DFO); l, l,l-tris(aminomethyl)ethane (TAME); tris(aminomethyl)ethane-N,N,N',N',N' ',N' '-hexaacetic acid (TAME Hex); O-hydroxybenzyl iminodiacetic acid; 1,4,7-triazacyclononane (TACN); 1,4,7, 10-tretraazacyclododecane (cyclen); 1,4,7-triazacyclononane-l -succinic acid-4,7-diacetic acid (NOD AS A); 1-(1- carboxy-3-carboxypropyl)-4,7-bis-(carboxymethyl)-l,4,7-triazacyclononane (NODAGA); 1 ,4,7-tris(2-mercaptoethyl)- 1 ,4,7-triazacylclonane (triaza
  • the chelating agents are polyaminocarboxylate agents, such as ethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTP A), l,4,7,10-tetra-azacylcododecane-N,N',N",N"'-tetraacetic acid (DOTA), or derivatives thereof. They can coordinate with metals such as Fe, In, Ga, Zr, Y, Bi, Pb, or Ac.
  • the cheating agents are macrocyclic agents: 1,4,7- Triazacyclononane-N,N',N"-triacetic acid (NOTA), 1,4,7, 10-tetraazacy clododecane- N,N',N",N"'-tetraacetic acid (TETA), 1,4,7,10, 13-pentaazacyclopentadecane- N,N',N",N"',N" "-pentaacetic acid (PEP A), 1,4,7,10, 13,16- hexaazacyclohexadecane- N,N',N",N"',N"",N -hexaacetic acid (HEHA), or derivatives thereof.
  • NOTA 1,4,7- Triazacyclononane-N,N',N"-triacetic acid
  • TETA 10-tetraazacy clododecane- N,N',N",N"'-tetraacetic acid
  • PEP A 1,4,7,10
  • DTP A and derivatives thereof are:
  • DOTA and derivatives thereof are:
  • chelating agents are:
  • derivatives/analogs thereof may be used as an active agent in conjugates of the present invention.
  • the active agent is an immune stimulating moiety.
  • the immune stimulating moiety may target various pathways involved in critical processes of anti-cancer immune responses. These critical processes may include antigen degradation and processing, activation of dendritic cells to present antigenic epitopes, production of cytokines (e.g., interferons), expression of co-stimulatory ligands, induction of a productive T cell response for example within lymph nodes, migration of activated T cells to the tumor microenvironment in response to chemokines and homing receptor expression, having effector T cells (e.g., CD4+ T cells and CD8+ T cells) gain access to antigen expressing tumor cells and maintenance of sufficient functionality of effector T cell to destroy tumor cells.
  • cytokines e.g., interferons
  • co-stimulatory ligands e.g., co-stimulatory ligands
  • induction of a productive T cell response for example within lymph nodes
  • an immune stimulating moiety may be an active agent that can boost or provoke an anti-cancer immune response in a subject. Any compound that can provoke and/or enhance an immune response to destroy tumor cells in a subject may be included in the targeted constructs.
  • agents may be tumor associated antigens (TAAs), antigen epitopes including antigen peptides presented by either MHC (major histocompatibility complex) class I or MHC class II molecules; cytokines,
  • chemokines other immunomodulators, T cell receptors (TCRs), CD (cell differentiation molecules) antigens, antibodies, cytotoxic agents, cell adhesion molecules and any components that are involved in an immune response; or variants thereof.
  • TCRs T cell receptors
  • CD cell differentiation molecules
  • the plural payloads may belong to the same category such as multiple epitope peptides derived from a single TAA, or multiple different tumor associated antigens isolated from a tumor tissue.
  • a plural of immune stimulating moieties having different functionality such as a mix of tumor associated antigens and co-stimulatory factors may be included in the same targeted construct to synergistically enhance the antigen presentation to T cells.
  • An immune stimulating moiety may be a protein, a peptide, a nucleic acid, a sugar, a lipid, a lipoprotein, a glycoprotein, a glycolipid, or a small molecule.
  • Any payload / active agent disclosed in WO2017023779 to Kadiyala et al., the contents of which are incorporated herein by reference in their entirety, may be used as an immune stimulating moiety, such as tumor associated antigens and antigenic peptides; any active agent that can increase the activity of antigen presenting cells (APCs) (such as dendritic cells (DCs)); any active agent that can activate T cells or natural killer (NK) cells; cytokines, chemokines and immunoregulatory molecules; antibodies or fragments thereof; cell surface antigens or fragments thereof; or other immuno-active agents such as a complement component or an immunomodulatory adjuvant.
  • APCs antigen presenting cells
  • DCs dendritic cells
  • NK natural killer cells
  • cytokines, chemokines and immunoregulatory molecules antibodies or fragments thereof; cell surface antigens or fragments thereof; or other immuno-active agents such as a complement component or an immunomodulatory adjuvant.
  • the immune stimulating moiety comprises an antibody, an antibody binding moiety, or an antibody mimetic molecule.
  • antibodies are specialized natural proteins called
  • immunoglobulins that specifically recognize and bind to specific antigens that caused their stimulation. Antibody production by B lymphocytes in vivo and binding to foreign antigens is often critical as a means of signaling other cells to engulf, kill or remove that substance that contains the foreign antigens from the body.
  • An immunoglobulin is a protein comprising one or more polypeptides substantially encoded by the immunoglobulin kappa and lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Also subclasses of the heavy chain are known. For example, IgG heavy chains in humans can be any of IgGl, IgG2, IgG3 and IgG4 subclass.
  • Antibodies may exist as full length intact antibodies or as a number of well- characterized fragments produced by digestion with various peptidases or chemicals, such as F(ab')2, a dimer of Fab which itself is a light chain joined to VH-CHl by a disulfide bond; an Fab' monomer, a Fab fragment with the hinge region; and a Fc fragment, a portion of the constant region of an immunoglobulin.
  • F(ab')2 a dimer of Fab which itself is a light chain joined to VH-CHl by a disulfide bond
  • an Fab' monomer a Fab fragment with the hinge region
  • Fc fragment a portion of the constant region of an immunoglobulin.
  • the immune stimulating moiety may be an antibody, a fragment of an antibody or a derivative thereof.
  • Antibodies may be immuno-specific for a tumor cell antigen or against immuno-modulatory factors.
  • An antibody that can recognize a TAA and/or a TAA antigenic peptide may be a monoclonal antibody or a polyclonal antibody.
  • the antibody may be generated by standard hybridoma techniques, phase display and recombinant techniques.
  • antibodies may recognize tumor antigens that are overexpressed in tumor cells, or tumor antigens associated with Leukaemias and lymphomas such as cell differentiation (CD) antigens, e.g.
  • CD cell differentiation
  • antibodies may recognize non-protein antigens such as glycolipids, e.g., ganglioside, and carbohydrates that are associated with tumors. In other examples, antibodies may recognize any one of TAAs as discussed hereinabove.
  • antibodies that can recognize a specific antigen epitope may include, without limitation, anti-HER2, anti-EGFR as disclosed in US Pat. No.: 9,023,362 and 8,722, 362; anti-FcyRIIB as disclosed in US Pat. No. : 8, 784,808; and antibodies against PSCA (prostate stem cell antigen) as disclosed in US Pat. No. : 8, 404, 817;
  • the immune stimulating moiety may be an agonist antibody that can manipulate a process of a cancer specific immune response.
  • an agonist antibody may be an antibody specific to 4-1BB (CD137) (e.g., PCT patent publication NO. 2006/088464 to Chen et al.; the content of which is incorporated by reference in its entirety). Stimulation of CD137 by agonistic antibody induces vigorous T-cell proliferation and prevents activation-induced cell death, and induces dendritic and NK cell activation as well.
  • the immune stimulating antibody may be an agonist antibody that specifically binds to an costimulatory molecule selected from CD28, B7-1 (CD80), B7-2 (CD86), 4-lBB (CD137), 4-lBB ligand (CD137-L), OX40, OX40L, inducible co-stimulatory ligand (ICOS-L), ICOS, intercellular adhesion molecule (ICAM), CD30, CD30L, CD40, CD27, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, GITR, GITR-L, TLR agonist, B7-H3, B7-H3 ligand, CD226, ICOS, LFA-1, CD2, CD7, LIGHT, KG2D, and DNAM-1.
  • costimulatory molecule selected from CD28, B7-1 (CD80), B7-2 (CD86), 4-lBB (CD137), 4-lBB
  • the immune stimulating antibody may be an antagonist antibody that specifically binds to a coinhibitory molecule selected from CTLA-4, PD-1, PD-L1, PD- L2, TIM-3, LAG-3, BTLA, CD 160, C200R, TIGIT, KLRG-1, KIR, 2B4/CD244, VISTA and Ara2R.
  • a coinhibitory molecule selected from CTLA-4, PD-1, PD-L1, PD- L2, TIM-3, LAG-3, BTLA, CD 160, C200R, TIGIT, KLRG-1, KIR, 2B4/CD244, VISTA and Ara2R.
  • the immune stimulating antibody may be a bispecific antibody (bsAb) or multiple specific antibody (msAb) (Weidle et al., Tumor-Antigen- Binding Bispecific Antibodies for Cancer Treatment, Seminars in Oncology, 2014, 41(5): 653-660).
  • bsAb bispecific antibody
  • msAb multiple specific antibody
  • the term "bispecific antibody” refers to an antibody construct that is capable of redirecting immune effector cells to the tumor microenvironment. Clinical studies of various bsAb constructs have shown spectacular results in terms of immune effector cell retargeting, target dependent activation and the induction of anti-tumor responses.
  • Some examples of bispecific antibodies include bispecific antibody against TIM-3 and PD-1 in WO201159877 to Kuchroo et al., the content of which is incorporated by reference in its entirety.
  • the immune stimulating moiety may be an agonist of RIG- 1-like receptors (RLRs).
  • RLR may be retinoic acid-inducible gene I (RIG-1), Melanoma Differentiation-Associated protein 5 (MDA5), or LGP2.
  • RLRs are important for pathogen sensing of RNA virus infection to initiate and modulate antiviral immunity. They detect viral RNA ligands or processed self RNA in the cytoplasm to trigger innate immunity and control infection.
  • the RLR agonist may be an oligonucleotide, a single- or double-stranded RNA.
  • the RLR agonist may be any RIG-1 agonist disclosed in US20130005028 to Smith et al., the contents of which are incorporated herein by reference in their entirety, such as an RNA molecule comprising a 5' triphosphate moiety.
  • the RLR agonist may be an RNA molecule having a central hairpin and an internal loop as disclosed in WO2017173427 to Alici, the contents of which are incorporated herein by reference in their entirety.
  • the immune stimulating moiety may be an agonist of STING (stimulator of interferon genes).
  • the STING agonist may be a nucleic acid, a protein, a peptide, or a small molecule.
  • the STING agonist is any cyclic dinucleotide disclosed in US20170239283 to Gough et al., the contents of which are incorporated herein by reference in their entirety, such as cyclic diadenylate monophosphate (CD A), cyclic diguanylate monophosphate (CDG), cyclic di-inosine monophosphate (CDI), cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), cyclic adenosine monophosphate-insoine monophosphate, or cyclic guanosine monophosphate-inosine monophosphate.
  • CD A cyclic diadenylate monophosphate
  • CDG cyclic diguanylate monophosphate
  • CDI cyclic
  • the STING agonist is any STING agonist disclosed in WO2015/077354, the contents of which are incorporated herein by reference in their entirety, such as the small molecules in claim 5 and claim 23, dithio-(Rp, Rp)- [cyclic[A(2',5')pA(3',5')p]] (also known as 2'-5', 3'-5' mixed phosphodiester linkage (ML) RR-S2 c-di-AMP or ML RR-S2 CD A)), ML RR-S2-c-di-GMP (ML-CDG), ML RR-S2 cGAMP, or any mixtures thereof.
  • dithio-(Rp, Rp)- [cyclic[A(2',5')pA(3',5')p]] also known as 2'-5', 3'-5' mixed phosphodiester linkage (ML) RR-S2 c-di-AMP or ML RR-S2 CD A
  • the immune stimulating moiety may be an antibody binding moiety.
  • the antibody binding moiety may be a small molecule, a peptide, or an aptamer.
  • the immune stimulating moiety in the present disclosure may comprise any antibody-binding terminus (ABT) that recruits endogenous antibodies disclosed in US9556167 to Spiegel et al. (Kleo Pharmaceuticals), the contents of which are incorporated herein by reference in their entirety.
  • ABT antibody-binding terminus
  • the ABT may be a hapten.
  • the term "hapten" is used to describe a small-molecular-weight inorganic or organic molecule that alone is not antigenic but which when linked to another molecule, such as a targeting moiety of the present disclosure, becomes antigenic.
  • the present disclosure uses a hapten which is reactive (binds to) an endogenous antibody that preexists in the patient prior to initiating therapy with the targeted constructs of the present invention and does not have to be separately raised as part of a treatment regimen.
  • the haptens bind dinitrophenyl (DNP)-specific antibodies.
  • DNP dinitrophenyl
  • the haptens comprise a di- or trinitro phenyl group, or a digalactose hapten (Gal-Gal-Z, preferably Gal -Gal -sugar, preferably Gal-Gal-Glu). Any other small molecule [ABT] disclosed in claim 1 of US9556167 to Spiegel et al. may also be used.
  • the immune stimulating moiety comprises a 2,4-dinitrophenyl (DNP) moiety, also called a DNP antigen.
  • DNP 2,4-dinitrophenyl
  • the DNP antigen is of particular interest for therapeutic applications, because anti-DNP antibodies have been found endogenously in the plasma of most humans.
  • the DNP antigens can direct antibodies to targeted tissues/cells and induce phagocytosis and cytotoxicity in an antibody-dependent, immune-mediated way.
  • the immune stimulating moiety binds to an FC immune receptor and modulates an FC immune receptor, such as an FcyRI (CD64) receptor.
  • FcyRI is a 72 KDa integral membrane glycoprotein with high affinity for the Fc portion of IgG.
  • FcyRI is a cell surface immune receptor responsible for initiating pro-inflammatory responses against antibody-opsonized targets. It is expressed on the surface of numerous immune cells, including monocytes and macrophages. Ligation of this receptor leads to varied proinflammatory responses, which include phagocytosis and reactive oxygen species generation. Targets displaying multiple copies of an FcyRI binding motifs, induce receptor crosslinking and subsequent signaling, which results in a pro-inflammatory response.
  • the immune stimulating moiety may bind to FcyRI receptor and activate a local humor response for induction of immunologic memory against a tumor.
  • the immune stimulating moiety of the present disclosure comprises CP33, a cyclic peptide that was discovered using phage display and can bind to FcyRI, disclosed in WO2014/178878 to Spiegel et al. (Kleo Pharmaceuticals), the contents of which are incorporated herein by reference in their entirety.
  • the immune stimulating moiety binds to a Toll-like Receptor (TLR). Binding to TLR may mediate tumor lysis, stimulate APC activation, generate anti-tumor T-cells which provide for enhanced anticancer activity, including prolonged activity and/or immunologic memory.
  • TLR Toll-like Receptor
  • the immune stimulating moiety may be a TLR agonist, such as a TLR 7/8 agonist.
  • TLR agonists include Rintatolimod, SMP-105, IPH-3102, CBLB502, MGN-1706, IMO-2055, ANA773, OM-174, ISS1018, Agatolimod, 852A, Imiquimod and Cadi-05. Any [TLR] disclosed in US9556167 to Spiegel et al. (Kleo Pharmaceuticals), the contents of which are incorporated herein by reference in their entirety, can be used as the immune stimulating moiety of the present disclosure.
  • the targeted constructs may comprise at least one conjugate of an active agent (Z) connected to a targeting moiety (X) with an optional internal linker moiety (Y).
  • the conjugate is connected to a protein-binding moiety of the present disclosure by an optional external linker: (Protein-binding moiety )n-(External linker)p-(Conjugate)m, n>l, m>l, and p>0.
  • the protein-binding moiety and/or the external linker may be attached to anywhere on the conjugate, such as the targeting moiety or the optional internal linker moiety of the conjugate.
  • the protein-binding moiety is an antibody mimetic described herein. In some embodiments, the protein-binding moiety is a small molecule described herein.
  • the optional internal linker moiety is cleavable.
  • the active agent may be active when the optional internal linker is cleaved and the active agent is released from the targeted constructs.
  • the optional external linker is cleavable.
  • the active agent may be active when the optional external linker is cleaved and the conjugate is released from the targeted constructs.
  • both the optional internal linker and the optional external linker are cleavable.
  • the active agent may be active when both linkers are cleaved.
  • both the optional internal linker and the optional external linker are non-cleavable.
  • the active agent may be active when it is still in the targeted construct.
  • the conjugates can be a conjugate between a single active agent and a single targeting moiety, e.g. a conjugate having the structure X-Y-Z, where X is the targeting moiety, Y is the linker, and Z is the active agent.
  • the conjugate contains more than one targeting moiety, more than one linker, more than one active agent, or any combination thereof.
  • the conjugate can have any number of targeting moieties, linkers, and active agents.
  • the conjugate can have the structure X-Y-Z-Y-X, (X-Y)n-Z, X-(Y-Z) n , X-Y-Zn, (X-Y-Z) n , (X-Y-Z-Y)n-Z where X is a targeting moiety, Y is a linker, Z is an active agent, and n is an integer between 1 and 50, between 2 and 20, for example, between 1 and 5.
  • Each occurrence of X, Y, and Z can be the same or different, e.g. the conjugate can contain more than one type of targeting moiety, more than one type of linker, and/or more than one type of active agent.
  • the conjugate can contain more than one targeting moiety attached to a single active agent.
  • the conjugate can include an active agent with multiple targeting moieties each attached via a different linker.
  • the conjugate can have the structure X-Y-Z-Y- X where each X is a targeting moiety that may be the same or different, each Y is a linker that may be the same or different, and Z is the active agent.
  • the conjugate can contain more than one active agent attached to a single targeting moiety.
  • the conjugate can include a targeting moiety with multiple active agents each attached via a different linker.
  • the conjugate can have the structure Z-Y-X-Y-Z, where X is the targeting moiety, each Y is a linker that may be the same or different, and each Z is an active agent that may be the same or different.
  • the conjugate may comprise pendent or terminal functional groups that allow further modification or conjugation.
  • the pendent or terminal functional groups may be protected with any suitable protecting groups.
  • the active agent (Z), targeting moiety (X), and optional linker (Y) are as described above.
  • the internal linker Y moiety may be X'-R ⁇ -Y'-R ⁇ -Z' and the conjugate can be a compound according to Formula la:
  • X is a targeting moiety defined above; Z is an active agent; X', R 1 , Y', R 2 and Z' are as defined herein.
  • X' is either absent or independently selected from carbonyl, amide, urea, amino, ester, aryl, arylcarbonyl, aryloxy, arylamino, one or more natural or unnatural amino acids, thio or succinimido;
  • R 1 and R 2 are either absent or comprised of alkyl, substituted alkyl, aryl, substituted aryl, polyethylene glycol (2-30 units);
  • Y' is absent, substituted or unsubstituted 1,2-diaminoethane, polyethylene glycol (2-30 units) or an amide;
  • Z' is either absent or independently selected from carbonyl, amide, urea, amino, ester, aryl, arylcarbonyl, aryloxy, arylamino, thio or succinimido.
  • the internal linker can allow one active agent molecule to be linked to two or more ligands, or one ligand to be linked to two or more active agent molecules.
  • the internal linker Y moiety may be A m and the conjugate can be a com ound according to Formula lb:
  • a in Formula la is a spacer unit, either absent or independently selected from the following substituents.
  • the dashed lines represent substitution sites with X, Z or another independently selected unit of A wherein the X, Z, or A can be attached on either side of the substituent:
  • R' is any side chain found in either natural or unnatural amino acids.
  • C in Formula Ic is a branched unit containing three to six functionalities for covalently attaching spacer units, ligands, or active drugs, selected from amines, carboxylic acids, thiols, or succinimides, including amino acids such as lysine, 2,3-diaminopropanoic acid, 2,4-diaminobutyric acid, glutamic acid, aspartic acid, and cysteine.
  • the internal linker moiety may be cleavable and is cleaved to release the active agent.
  • the internal linker may be cleaved by an enzyme.
  • the internal linker moiety may be a polypeptide moiety, e.g. AA in WO2010093395 to Govindan, the contents of which are incorporated herein by reference in their entirety, that is cleavable by intracellular peptidase.
  • Govindan teaches AA in the linker moiety may be a di, tri, or tetrapeptide such as Ala-Leu, Leu- Ala-Leu, and Ala- Leu- Ala-Leu.
  • the cleavable internal linker moiety may be a branched peptide.
  • the branched peptide linker may comprise two or more amino acid moieties that provide an enzyme cleavage site. Any branched peptide linker disclosed in WO 1998019705 to Dubowchik, the contents of which are incorporated herein by reference in their entirety, may be used as an internal linker moiety in the conjugate of the present invention.
  • the linker may comprise a lysosomally cleavable polypeptide disclosed in US 8877901 to Govindan et al., the contents of which are incorporated herein by reference in their entirety.
  • the internal linker may comprise a protein peptide sequence which is selectively enzymatically cleavable by tumor associated proteases, such as any Y and Z structures disclosed in US 6214345 to Firestone et al., the contents of which are incorporated herein by reference in their entirety.
  • the cleaving of the internal linker moiety is non-enzymatic.
  • Any linker disclosed in US 20110053848 to Cleemann et al., the contents of which are incorporated herein by reference in their entirety, may be used.
  • the linker may be a non-biologically active linker represented by formula (I).
  • the internal linker moiety may be a beta-glucuronide linker disclosed in US 20140031535 to Jeffrey, the contents of which are incorporated herein by reference in their entirety.
  • the internal linker may be a self- stabilizing linker such as a succinimide ring, a maleimide ring, a hydrolyzed succinimide ring or a hydrolyzed maleimide ring, disclosed in US20130309256 to Lyon et al., the contents of which are incorporated herein by reference in their entirety.
  • the internal linker may be a human serum albumin (HAS) linker disclosed in US 20120003221 to McDonagh et al., the contents of which are incorporated herein by reference in their entirety.
  • the internal linker may comprise a fullerene, e.g., C 6 o, as disclosed in US 20040241173 to Wilson et al., the contents of which are incorporated herein by reference in their entirety.
  • the internal linker may be a recombinant albumin fused with polycysteine peptide as disclosed in US 8541378 to Ahn et al., the contents of which are incorporated herein by reference in their entirety.
  • the internal linker comprises a heterocycle ring.
  • the internal linker may be any heterocyclic 1,3-substituted five- or six-member ring, such as thiazolidine, disclosed in US 20130309257 to Giulio, the contents of which are incorporated herein by reference in their entirety.
  • the internal linker moiety Y may be a Linker Unit (LU) as described in US2011/0070248, the contents of which are incorporated herein by reference in their entirety.
  • the Ligand Drug Conjugate has formula L-(LU-D) P the targeting moiety X corresponds to L (the Ligand unit) and the active agent Z corresponds to D (the drug unit).
  • the conjugate X— Y— Z can be a conjugate as described in WO2014/134486, the contents of which are incorporated herein by reference in their entirety.
  • the targeting moiety X corresponds to the cell binding agent, CBA in formula ( ⁇ ) or (I) as reproduced here, wherein the internal linker moiety Y and the active agent Z together correspond to the remainder of the formula (in parentheses).
  • the conjugate X— Y— Z can be a conjugate as described in US 7601332, the contents of which are incorporated herein by reference in their entirety, wherein conjugates are described as follows, and the targeting moiety X corresponds to V (the vitamin receptor binding moiety), the active agent Z corresponds to D (drugs and includes analogs or derivatives thereof), and the internal linker moiety Y corresponds to the bivalent linker (L) which can comprise one or more components selected from spacer linkers (Is), releasable linkers (lr), and heteroatom linkers (1H), and combinations thereof, in any order:
  • the weight percentage of the conjugate in the targeted construct is at least about 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%), 45%), or 50%) such that the sum of the weight percentages of the components of the targeted construct is 100%>.
  • the weight percentage of the conjugate in the targeted construct is from about 0.5%> to about 10%>, or about 10%> to about 20%>, or about 20% to about 30%, or about 30% to about 40%, or about 40% to about 50%, or about 50% to about 60%), or about 60%> to about 70%>, or about 70%> to about 80%>, or about 80%> to about 90%), or about 90%> to about 99%> such that the sum of the weight percentages of the components of the targeted construct is 100%>.
  • a conjugate may have a molecular weight of less than about 50,000 Da, less than about 40,000 Da, less than about 30,000 Da, less than about 20,000 Da, less than about 15,000 Da, less than about 10,000 Da, less than about 8,000 Da, less than about 5,000 Da, or less than about 3,000 Da.
  • the conjugate may have a molecular weight of between about 1,000 Da and about 50,000 Da, in some embodiments between about 1,000 Da and about 40,000 Da, in some embodiments between about 1,000 Da and about 30,000 Da, in some embodiments bout 1,000 Da and about 50,000 Da, between about 1,000 Da and about 20,000 Da, in some embodiments between about 1,000 Da and about 15,000 Da, in some embodiments between about 1,000 Da and about 10,000 Da, in some embodiments between about 1,000 Da and about 8,000 Da, in some embodiments between about 1,000 Da and about 5,000 Da, and in some embodiments between about 1,000 Da and about 3,000 Da.
  • the molecular weight of the conjugate may be calculated as the sum of the atomic weight of each atom in the formula of the conjugate multiplied by the number of each atom.
  • the conjugate may comprise a bicyclic peptide or a modified bicyclic peptide as a targeting moiety.
  • the conjugate may be any conjugate disclosed in WO2016067035, the contents of which are incorporated herein by reference in their entirety, wherein the conjugate comprises a bicyclic peptide ligand that binds to membrane type 1 metalloprotease (MTl-MMP) and a cytotoxic agent such as DM1, a maytansinoid with formula (II).
  • the conjugate may have the structure of formula (IV), (V), (V) a , (V) b , (V) c , (V) d , BT17BDC-9, BT17BDC-17, BT17BDC-18, BT17BDC-19, or
  • the conjugate may also be any conjugate disclosed in WO2013050617, the contents of which are incorporated herein by reference in their entirety, wherein the bicyclic peptide ligand is attached to an effector with a polysarcosine linker.
  • the conjugate comprises a somatostatin receptor 2 (SSTR2) ligand.
  • SSTR2 ligand may be somatostatin, octreotide, Tyr 3 -octreotate (TATE), vapreotide, cyclo(AA-Tyr-DTrp-Lys-Thr-Phe) where AA is ⁇ - ⁇ -Me lysine or N-Me glutamic acid, pasireotide, lanreotide, or seglitide.
  • the conjugate comprises a neurotensin receptor 1 (NTSR1) ligand.
  • the NTSR1 ligand may be neurotensin, neurotensin (6-13), or any neurotensin derivative/analog.
  • the neurotensin derivate/analog may have stronger binding to NTSR1 than neurotensin.
  • the neurotensin analog may be NMeArg-Arg-Pro- Tyr-Tle-Leu-OH or DArg-Arg-Pro-Tyr-Ile-TMSAla-OH.
  • the conjugate comprises DNA alkylators as payloads, such as pyrndinobenzodiazepines (PDDs) disclosed in WO2017032983 and WO2016198869.
  • PPDs pyrndinobenzodiazepines
  • the conjugate comprises a SSTR2 ligand (e.g., TATE) and a DNA alkylator.
  • the conjugate may have a structure of Conjugate 1 or Conjugate 2.
  • the conjugate comprises a NTSR1 ligand (e.g., DArg-Arg-Pro- Tyr-Ile-TMSAla-OH) and a DNA alkylator.
  • the conjugate may have a structure of Conjugate 3 or Conjugate 4.
  • the optional external linker in the targeted construct is independently selected from the group consisting of alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, wherein each of the alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups optionally is substituted with one or more groups, each independently selected from halogen, cyano, nitro, hydroxyl, carboxyl, carbamoyl, ether, alkoxy, aryloxy, amino, amide, carbamate, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, heterocyclyl, wherein each of the carboxyl, carbamoyl, ether, alkoxy, aryloxy, amino, amide, carbamate, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl,
  • the external linker moiety is not cleavable.
  • the external linker moiety comprises a cleavable functionality designed to be cleaved in an extracellular manner.
  • the external linker may be cleavable in tumor microenvironment, e.g., by a pH-dependent or hypoxia-dependent cleavage that relies upon conditions in the tumor microenvironment or by extracellular proteases such as matrix metalloproteinases.
  • an internal linker Y as described above may be employed as an external linker.
  • the targeted construct of the present invention comprises a masking moiety (MM).
  • MM may be attached to any suitable position of the rest of the targeted construct.
  • MM may be attached to the active agent, the targeting moiety, or the protein-binding moiety.
  • MM may be attached to the active agent, the targeting moiety, or the protein-binding moiety directly, via a non- cleavable linker, or via a cleavable moiety (CM).
  • CM cleavable moiety
  • CM cleavable moiety
  • the binding of the active agent or the targeting moiety to its target is inhibited or hindered by MM.
  • the binding may be sterically hindered by the presence of MM or may be inhibited by the charge of MM. Leaving of MM upon cleavage of CM, a conformation change, or a chemical transformation may unmask the targeted constructs.
  • the masking/unmasking process may be reversible or irreversible.
  • the active agent or the targeting moiety might be less accessible to its target when CM is uncleaved.
  • CM Upon cleavage of CM, MM no longer interferes with the binding of the active agent or the targeting moiety to its target, thereby activating the targeted construct of the present invention.
  • MM prevents binding of the targeted constructs of the present invention at nontreatment sites.
  • Such targeted constructs can further provide improved biodistribution characteristics.
  • MM may be selected from a plurality of polypeptides based on its ability to inhibit binding of the active agent or the targeting moiety to the target in an uncleaved state and allow binding of the active agent or the targeting moiety to the target in a cleaved state.
  • CM may locate between the active agent or the targeting moiety and MM in the targeted construct, or may locate within MM.
  • CM may be cleaved by an enzyme such as protease.
  • CM may comprise a peptide that may be a substrate for an enzyme selected from the group consisting of MMP1, MMP2, MMP3, MMP8, MMP9, MMP14, plasmin, PSA, PSMA, CATHEPSIN D, CATHEPSIN K, CATHEPSIN S, ADAMIO, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE.
  • CM may comprise a protease substrate such as a plasmin substrate, a caspase substrate or a matrix metalloprotease (MMP) substrate (e.g., a substrate of MMP-1, MMP-2, MMP-9, or MMP- 14).
  • MMP matrix metalloprotease
  • CM may be cleaved by a reducing agent capable of reducing a disulfide bond between a cysteine-cysteine pair.
  • CM may comprise a cysteine- cysteine pair capable of forming a reducible disulfide bond.
  • Reducing agents of particular interest include cellular reducing agents such as proteins or other agents that are capable of reducing a disulfide bond under physiological conditions, e.g., glutathione, thioredoxin, NADPH, flavins, and ascorbate.
  • cellular reducing agents such as proteins or other agents that are capable of reducing a disulfide bond under physiological conditions, e.g., glutathione, thioredoxin, NADPH, flavins, and ascorbate.
  • the targeting moiety may be any activatable binding polypeptides (ABPs) disclosed in US9169321 to Daugherty et al. (CytomX), the contents of which are incorporated herein by reference in their entirety.
  • the targeting moiety may be an enzyme activatable binding polypeptide (ABP) that binds CTLA-4, VEGF, or VCAM-1.
  • the targeting moiety may be an activatable binding polypeptide (ABP) that binds epidermal growth factor disclosed in US 9120853 to Lowman et al., an ABP that binds Jagged 1 or Jagged 2 disclosed in US9127053 to West et al., activatable anti-CD3 antibodies disclosed in WO2016014974 to Irving et al., activatable antibodies that bind to interleukin-6 receptor (IL6R) disclosed in WO2014052462 to West et al., activatable proproteins disclosed in US20150203559 to Stagliano et al., any modified antibody or activatable antibody disclosed in US20140024810 to Stagliano et al., WO2015089283 to Desnoyers et al., WO2015066279 to Lowman et al., WO2015048329 to Moore et al., US20150079088 to Lowman et al., WO2014197612 to
  • the masking moiety or CM may be acid-cleavable and the conjugate becomes unmasked in the acidic tumor microenvironment.
  • the masking moiety is a photocleavable moiety.
  • the binding of the immune stimulating moiety or the targeting moiety to its target is reversibly inhibited by the photocleavable moiety.
  • a "photocleavable moiety” means any agent that can be removed application of radiation of a chosen frequency or wavelength. Any frequency of radiation may be suitable, including infra-red (IR) radiation, visible light, ultra-violet (UV) radiation, microwaves, gamma rays, etc. depending on the type of photocleavable moiety.
  • the photocleavable moiety may be located at or about the binding site of the immune stimulating moiety or the targeting moiety.
  • the photocleavable moiety may be attached to any suitable position of the rest of the targeted construct.
  • the photocleavable moiety may be attached to the immune stimulating moiety or the targeting moiety.
  • the photocleavable moiety is bound to the internal linker between the immune stimulating moiety and the targeting moiety.
  • the photocleavable moiety may be a reagent which couples to hydroxy or amino residues present in the targeted moiety.
  • the photocleavable moiety is cleaved by UV radiation.
  • the photocleavable moiety may comprise a 2-nitrophenyl moiety, which may also be substituted at other positions around the benzene ring.
  • a l-(2- nitrophenyl) ethyl ( PE) residue or moiety may be used.
  • the photocleavable moiety may be photocleavable esters, amides, carbonates and the like generated from a wide range of alcohols by reactions with phosgene, diphosgene, dicyclohexyl carbodiimide (DCCI) or the like.
  • the photocleavable moiety may be substituted arylalkanols are employed, particularly nitorphenyl methyl alcohol, 1 -nitrophenyl ethan-l-ol and substituted analogues.
  • the targeted constructs may comprise any photocleavable moiety disclosed in WO1996034892 to Self et al., the contents of which are incorporated herein by reference in their entirety.
  • TBM may be an antibody component that retain the active site and bind to a tumor cell marker.
  • TBM may also be any antibody component made against suitable cells such as T-cells, cytotoxic T-cell clones, cytotoxic T-cells and activated peripheral blood lymphocytes, CD3+ lymphocytes, CD 16+ lymphocytes, Fc gamma Rl 11, the low affinity Fc gamma receptor for polymorphonuclear leucocytes, macrophages and large granular lymphocytes, B-lymphocyte markers, myeloid cells, T Lymphocyte CD2, CD3, CD4, CD8, dengue virus, lymphokine activated killer (LAK) cells, NK cells or monocytes.
  • TBM may be a monoclonal antibody anti-CD-3 OKT3 against T-cells, or a monoclonal antibody that binds to tumor antigen carcinoembrionic antigen (CEA).
  • CEA tumor antigen carcinoembrionic antigen
  • the masking moiety or the photocleavable moiety is present at a predetermined molar weight percentage from about 1% to about 10%, or about 10% to about 20%, or about 20% to about 30%, or about 30% to about 40%, or about 40% to about 50%), or about 50% to about 60%, or about 60% to about 70%, or about 70% to about 80%, or about 80% to about 90%, or about 90% to about 99% such that the sum of the molar weight percentages of the components of the targeted construct is 100%.
  • the amount of masking moiety or the photocleavable moiety may also be expressed in terms of proportion to the active agent or the targeting moiety, for example, in a ratio of about 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3 : 1, 2: 1, 1 : 1, 1 :2, 1 :3, 1 :4; 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, or 1 : 10.
  • compositions are administered to humans, human patients, healthy volunteers, or any other subjects.
  • active ingredient generally refers to the targeted constructs of the present invention to be delivered as described herein.
  • compositions are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to animals, e.g. mammals, rodents, or avians. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation.
  • Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.
  • Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology.
  • preparatory methods include the step of bringing the active ingredient into association with one or more excipients and/or one or more other accessory ingredients including solvents and aqueous solutions, and then, if necessary and/or desirable, dissolving, dividing, sterilizing, filling or shaping and/or packaging the product into a desired single- or multi-use units.
  • a pharmaceutical composition in accordance with the invention may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • compositions in accordance with the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.05% and 100%, e.g., between 0.1 and 75%, between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.
  • the targeted constructs of the present invention can be formulated using one or more excipients to: (1) increase stability; (2) permit the sustained or delayed release (e.g., from a depot formulation of the monomaleimide); (3) alter the biodistribution (e.g., target the monomaleimide compounds to specific tissues or cell types); (4) alter the release profile of the monomaleimide compounds in vivo.
  • excipients include any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, and
  • Excipients of the present invention may also include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, hyaluronidase, nanoparticle mimetics and combinations thereof.
  • formulations of the invention may include one or more excipients, each in an amount that together increases the stability of the targeted constructs of the present invention.
  • compositions may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • a pharmaceutically acceptable excipient includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • Remington's The Science and Practice of Pharmacy 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety) discloses various excipients
  • a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure.
  • an excipient is approved for use in humans and for veterinary use.
  • an excipient is approved by United States Food and Drug Administration.
  • an excipient is pharmaceutical grade.
  • an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
  • compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical compositions.
  • Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and/or combinations thereof.
  • Exemplary granulating and/or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation- exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked polyvinylpyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, etc., and/or combinations thereof.
  • crospovidone cross-linked polyvinylpyrrolidone
  • sodium carboxymethyl starch sodium starch
  • Exemplary surface active agents and/or emulsifiers include, but are not limited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g.
  • natural emulsifiers e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin
  • colloidal clays e.g. bentonite [aluminum silicate
  • stearyl alcohol cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol
  • carbomers e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer
  • carrageenan cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate
  • TWEEN®20 polyoxyethylene sorbitan [TWEENn®60], polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate [SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate [SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ®45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether
  • polyvinylpyrrolidone di ethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLUORINC®F 68, POLOXAMER®188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations thereof.
  • Exemplary binding agents include, but are not limited to, starch (e.g. cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol,); natural and synthetic gums (e.g. acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose,
  • hydroxypropyl methylcellulose microcrystalline cellulose, cellulose acetate, polyvinylpyrrolidone), magnesium aluminum silicate (Veegum®), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid;
  • polymethacrylates are polymethacrylates; waxes; water; alcohol; etc.; and combinations thereof.
  • Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and/or other preservatives.
  • Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and/or sodium sulfite.
  • Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate.
  • EDTA ethylenediaminetetraacetic acid
  • citric acid monohydrate disodium edetate
  • dipotassium edetate dipotassium edetate
  • edetic acid fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate.
  • antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/or thimerosal.
  • Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and/or sorbic acid.
  • Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol.
  • Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and/or phytic acid.
  • Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium
  • Exemplary buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium
  • phosphate potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, etc., and/or combinations thereof.
  • Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.
  • oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl my ri state, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana
  • oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and/or combinations thereof.
  • Excipients such as cocoa butter and suppository waxes, retinoid-like excipient (e.g. excipients that resemble vitamin A), coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator.
  • the formulation for controlled release and/or targeted delivery may also include at least one controlled release coating.
  • Controlled release coatings include, but are not limited to, OP ADR Y®, polyvinylpyrrolidone/vinyl acetate copolymer, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, EUDRAGIT RL®, EUDRAGIT RS® and cellulose derivatives such as
  • ethylcellulose aqueous dispersions (AQUACOAT® and SURELEASE®).
  • the controlled release and/or targeted delivery formulation may comprise at least one degradable polyester which may contain polycationic side chains.
  • Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co- L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof.
  • the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
  • the targeted constructs of the present invention may be administered by any route which results in a therapeutically effective outcome. These include, but are not limited to enteral, gastroenteral, epidural, oral, transdermal, epidural (peridural), intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intravesical infusion, intravitreal, (through the eye), intracavernous injection, ( into the base of the penis), intravaginal administration, intrauterine, extra- amniotic administration, transdermal
  • the formulations described herein contain an effective amount of the targeted constructs of the present invention in a pharmaceutical carrier appropriate for administration to an individual in need thereof.
  • The may be administered parenterally (e.g., by injection or infusion).
  • the formulations or variations thereof may be administered in any manner including enterally, topically (e.g., to the eye), or via pulmonary administration. In some embodiments, the formulations are administered topically.
  • the targeted constructs of the present invention can be formulated for parenteral delivery, such as injection or infusion, in the form of a solution, suspension or emulsion.
  • the formulation can be administered systemically, regionally or directly to the organ or tissue to be treated.
  • Parenteral formulations can be prepared as aqueous compositions using techniques known in the art.
  • such compositions can be prepared as injectable formulations, for example, solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w/o) emulsions, oil-in-water (o/w) emulsions, and microemulsions thereof, liposomes, or emulsomes.
  • injectable formulations for example, solutions or suspensions
  • solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection emulsions, such as water-in-oil (w/o) emulsions, oil-in-water (o/w) emulsions, and microemulsions thereof, liposomes, or emulsomes.
  • emulsions such as water-in-oil (w/o) emulsions,
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), and
  • Suitable surfactants may be anionic, cationic, amphoteric or nonionic surface active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions.
  • anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate.
  • Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine.
  • nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG- 1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide.
  • amphoteric surfactants include sodium N-dodecyl-P-alanine, sodium N-lauryl- ⁇ - iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.
  • the formulation can contain a preservative to prevent the growth of
  • Suitable preservatives include, but are not limited to, parabens,
  • the formulation may also contain an antioxidant to prevent degradation of the active agent(s) or targeted constructs.
  • the formulation is typically buffered to a pH of 3-8 for parenteral administration upon reconstitution.
  • Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers. If using 10% sucrose or 5% dextrose, a buffer may not be required.
  • Suitable water-soluble polymers include, but are not limited to,
  • polyvinylpyrrolidone dextran, carboxymethylcellulose, and polyethylene glycol.
  • compositions for parenteral administration can be in the form of a sterile aqueous solution or suspension of targeted constructs.
  • Acceptable solvents include, for example, water, Ringer's solution, phosphate buffered saline (PBS), and isotonic sucrose, dextrose or sodium chloride solution.
  • PBS phosphate buffered saline
  • the formulation may also be a sterile solution, suspension, or emulsion in a nontoxic, parenterally acceptable diluent or solvent such as 1,3- butanediol.
  • the formulation is distributed or packaged in a liquid form.
  • formulations for parenteral administration can be packed as a solid, obtained, for example by lyophilization of a suitable liquid formulation. The solid can be reconstituted with an appropriate carrier or diluent prior to administration.
  • Solutions, suspensions, or emulsions for parenteral administration may be buffered with an effective amount of buffer necessary to maintain a pH suitable for ocular
  • Suitable buffers are well known by those skilled in the art and some examples of useful buffers are acetate, borate, carbonate, citrate, and phosphate buffers.
  • Solutions, suspensions, or emulsions for parenteral administration may also contain one or more tonicity agents to adjust the isotonic range of the formulation.
  • Suitable tonicity agents are well known in the art and some examples include glycerin, sucrose, dextrose, mannitol, sorbitol, sodium chloride, and other electrolytes.
  • Solutions, suspensions, or emulsions for parenteral administration may also contain one or more preservatives to prevent bacterial contamination of the ophthalmic preparations.
  • Suitable preservatives are known in the art, and include
  • PHMB polyhexamethylenebiguanidine
  • BAK benzalkonium chloride
  • Purite® stabilized oxychloro complexes
  • phenylmercuric acetate chlorobutanol
  • sorbic acid chlorhexidine
  • benzyl alcohol parabens, thimerosal, and mixtures thereof.
  • Solutions, suspensions, or emulsions for parenteral administration may also contain one or more excipients known art, such as dispersing agents, wetting agents, and suspending agents.
  • the targeted constructs of the present invention can be formulated for topical administration to a mucosal surface.
  • Suitable dosage forms for topical administration include creams, ointments, salves, sprays, gels, lotions, emulsions, liquids, and transdermal patches.
  • the formulation may be formulated for transmucosal transepithelial, or transendothelial administration.
  • the compositions contain one or more chemical penetration enhancers, membrane permeability agents, membrane transport agents, emollients, surfactants, stabilizers, and combination thereof.
  • the targeted constructs can be administered as a liquid formulation, such as a solution or suspension, a semi-solid formulation, such as a lotion or ointment, or a solid formulation.
  • the targeted constructs are formulated as liquids, including solutions and suspensions, such as eye drops or as a semi-solid formulation, to the mucosa, such as the eye or vaginally or rectally.
  • “Surfactants” are surface-active agents that lower surface tension and thereby increase the emulsifying, foaming, dispersing, spreading and wetting properties of a product. Suitable non-ionic surfactants include emulsifying wax, glyceryl monooleate,
  • the non-ionic surfactant is stearyl alcohol.
  • Emmulsifiers are surface active substances which promote the dispersion of one liquid in another and promote the formation of a stable mixture, or emulsion, of oil and water or water in oil. Common emulsifiers are: anaionic, cataionic and nonionic surfactants or micttures of surfactants, certain animal and vegetable oils, and various polar surface active compounds.
  • Suitable emulsifiers include acacia, anionic emulsifying wax, calcium stearate, carbomers, cetostearyl alcohol, cetyl alcohol, cholesterol, diethanolamine, ethylene glycol palmitostearate, glycerin monostearate, glyceryl monooleate, hydroxpropyl cellulose, hypromellose, lanolin, hydrous, lanolin alcohols, lecithin, medium-chain triglycerides, methylcellulose, mineral oil and lanolin alcohols, monobasic sodium phosphate,
  • the emulsifier is glycerol stearate.
  • Suitable classes of penetration enhancers include, but are not limited to, fatty alcohols, fatty acid esters, fatty acids, fatty alcohol ethers, amino acids, phospholipids, lecithins, cholate salts, enzymes, amines and amides, complexing agents (liposomes, cyclodextrins, modified celluloses, and diimides), macrocyclics, such as macrocylic lactones, ketones, and anhydrides and cyclic ureas, surfactants, N-methyl pyrrolidones and derivatives thereof, DMSO and related compounds, ionic compounds, azone and related compounds, and solvents, such as alcohols, ketones, amides, polyols (e.g., glycols). Examples of these classes are known in the art.
  • the present invention provides methods comprising administering the targeted constructs of the present invention to a subject in need thereof.
  • the targeted constructs of the present invention may be administered to a subject using any amount and any route of administration effective for preventing or treating or imaging a disease, disorder, and/or condition (e.g., a disease, disorder, and/or condition relating to working memory deficits).
  • a disease, disorder, and/or condition e.g., a disease, disorder, and/or condition relating to working memory deficits.
  • the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like.
  • compositions in accordance with the invention are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present invention may be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or
  • compositions in accordance with the present invention may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic, diagnostic, prophylactic, or imaging effect.
  • the desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks.
  • the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).
  • split dosing regimens such as those described herein may be used.
  • a "split dose” is the division of single unit dose or total daily dose into two or more doses, e.g, two or more administrations of the single unit dose.
  • a "single unit dose” is a dose of any therapeutic administed in one dose/at one time/single route/single point of contact, i.e., single administration event.
  • a "total daily dose” is an amount given or prescribed in 24 hr period. It may be administered as a single unit dose.
  • the monomaleimide compounds of the present invention are administed to a subject in split doses.
  • the monomaleimide compounds may be formulated in buffer only or in a formulation described herein.
  • a pharmaceutical composition described herein can be formulated into a dosage form described herein, such as a topical, intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal,
  • injectable e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal,
  • Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs.
  • liquid dosage forms may comprise inert diluents commonly used in the art including, but not limited to, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art including, but not limited to,
  • compositions may be mixed with solubilizing agents such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
  • solubilizing agents such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
  • Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art and may include suitable dispersing agents, wetting agents, and/or suspending agents.
  • Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, a solution in 1,3-butanediol.
  • the acceptable vehicles and solvents that may be employed include, but are not limited to, water, Ringer's solution, U.S.P., and isotonic sodium chloride solution.
  • Sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • monomaleimide compound may be accomplished by dissolving or suspending the
  • Injectable depot forms are made by forming microencapsule matrices of the monomaleimide compounds in biodegradable polymers such as polylactide- polyglycolide. Depending upon the ratio of monomaleimide compounds to polymer and the nature of the particular polymer employed, the rate of monomaleimide compound release can be controlled. Examples of other biodegradable polymers include, but are not limited to, poly(orthoesters) and poly(anhydrides). Depot injectable formulations may be prepared by entrapping the monomaleimide compounds in liposomes or microemulsions which are compatible with body tissues.
  • Formulations described herein as being useful for pulmonary delivery may also be used for intranasal delivery of a pharmaceutical composition.
  • Another formulation suitable for intranasal administration may be a coarse powder comprising the active ingredient and having an average particle from about 0.2 um to 500 um.
  • Such a formulation may be administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nose.
  • Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of active ingredient, and may comprise one or more of the additional ingredients described herein.
  • a pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for buccal administration.
  • Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may, for example, contain about 0.1% to 20% (w/w) active ingredient, where the balance may comprise an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein.
  • formulations suitable for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising active ingredient.
  • Such powdered, aerosolized, and/or aerosolized formulations when dispersed, may have an average particle and/or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein.
  • Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the targeted constructs and conjugates can be made by many different synthetic procedures.
  • the conjugates can be prepared from internal linker moieties having one or more reactive coupling groups or from one or more internal linker precursors capable of reacting with a reactive coupling group on an active agent or targeting moiety to form a covalent bond.
  • the conjugates can be prepared from an internal linker moiety precursor capable of reacting with a reactive coupling group on an active agent or targeting moiety to form the internal linker covalently bonded to the active agent or targeting moiety.
  • the internal linker moiety precursor can be a diacid or substituted diacid.
  • Diacids as used herein, can refer to substituted or unsubstituted alkyl, heteroalkyl, aryl, or heteroaryl compounds having two or more carboxylic acid groups, preferably having between 2 and 50, between 2 and 30, between 2 and 12, or between 2 and 8 carbon atoms.
  • Suitable diacids can include oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, phthalic acid, iso-phthalic acid, terepthalic acid, and derivatives thereof.
  • the internal linker moiety precursor can be an activated diacid derivative such as a diacid anhydride, diacid ester, or diacid halide.
  • the diacid anhydride can be a cyclic anhydride obtained from the intramolecular dehydration of a diacid or diacid derivative such as those described above.
  • the diacid anhydride can be malonic anhydride, succinic anhydride, glutaric anhydride, adipic anhydride, pimelic anhydride, phthalic anhydride, diglycolic anhydride, or a derivative thereof; preferably succinic anhydride, diglycolic anhydride, or a derivative thereof.
  • the diacid ester can be an activated ester of any of the diacids described above, including methyl and butyl diesters or bis-(p-nitrophenyl) diesters of oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, phthalic acid, iso-phthalic acid, terepthalic acid, and derivatives thereof.
  • the diacid halide can include the corresponding acid fluorides, acid chlorides, acid bromides, or acid iodides of the diacids described above.
  • the diacid halide is succinyl chloride or diglycolyl chloride.
  • a therapeutic agent having a reactive (-OH) coupling group and a targeting moiety having a reactive (- H2) coupling group can be used to prepare a conjugate having a disuccinate linker according to the following general scheme.
  • the conjugates can be prepared by providing an active agent having a hydroxyl group and reacting it with a succinic anhydride linker precursor to form the conjugate of active agent— succinate- SSPy.
  • a targeting moiety with an available - H2 group is reacted with a coupling reagent and the active agent— succinate- SSPy to form the targeting moiety— internal linker— active agent conjugate.
  • the coupling reaction can be carried out under esterification conditions known to those of ordinary skill in the art such as in the presence of activating agents, e.g.,
  • carbodiimides such as diisopropoylcarbodiimide (DIPC)
  • DIPC diisopropoylcarbodiimide
  • DMAP dimethylaminopyridine
  • This reaction can be carried out in an appropriate solvent, such as dichloromethane, chloroform or ethyl acetate, at a temperature or between about 0° C and the reflux temperature of the solvent (e.g., ambient temperature).
  • the coupling reaction is generally performed in a solvent such as pyridine or in a chlorinated solvent in the presence of a catalyst such as DMAP or pyridine at a temperature between about 0° C and the reflux temperature of the solvent (e.g., ambient temperature).
  • the coupling reagent is selected from the group consisting of 4-(2-pyridyldithio)-butanoic acid, and a carbodiimide coupling reagent such as DCC in a chlorinated, ethereal or amidic solvent (such as ⁇ , ⁇ -dimethylformamide) in the presence of a catalyst such as DMAP at a temperature between about 0°C and the reflux temperature of the solvent (e.g., ambient temperature).
  • a carbodiimide coupling reagent such as DCC in a chlorinated, ethereal or amidic solvent (such as ⁇ , ⁇ -dimethylformamide) in the presence of a catalyst such as DMAP at a temperature between about 0°C and the reflux temperature of the solvent (e.g., ambient temperature).
  • the conjugates can be prepared by coupling an active agent and/or targeting moiety having one or more reactive coupling groups to an internal linker moiety having complimentary reactive groups capable of reacting with the reactive coupling groups on the active agent or targeting moiety to form a covalent bond.
  • an active agent or targeting moiety having a primary amine group can be coupled to an internal linker having an isothiocyonate group or another amine-reactive coupling group.
  • the internal linker contains a first reactive coupling group capable of reacting with a
  • the reactive coupling groups on the internal linker can be protected with a suitable protecting group during part of the synthesis.
  • the conjugates may be synthesized with 'click chemistry' of the copper ion-catalyzed acetylene-azide cycloaddition reaction.
  • the conjugates may be synthesized with 'click chemistry' of the copper ion-catalyzed acetylene-azide cycloaddition reaction.
  • the targeting moiety comprises L2, wherein L2 comprises a targeting moiety-coupling end and one or more acetylene or azide groups at the other end.
  • the active agent moiety comprises LI, wherein LI comprises a defined PEG with azide or acetylene at one end, complementary to the acetylene or azide moiety in L2, and a reactive group such as carboxylic acid or hydroxyl group at the other end. 'Click chemistry' between L2 and LI yields a conjugate comprising the targeting moiety and the active agent.
  • the conjugates may be synthesized with thiol-ene 'click chemistry' .
  • US 20130323169 to Xu et al. the contents of which are
  • conjugates can then be modified to include at least one protein-binding moiety described herein.
  • the targeted constructs of the present invention or formulations containing the targeted constructs of the present invention can be administered to treat any
  • the formulations can be used for immunization.
  • the formulations may be delivered to various body parts, such as but not limited to, brain and central nervous system, eyes, ears, lungs, bone, heart, kidney, liver, spleen, breast, ovary, colon, pancreas, muscles, gastrointestinal tract, mouth, skin, to treat diseases associated with such body parts.
  • Formulations may be administered by injection, orally, or topically, typically to a mucosal surface (lung, nasal, oral, buccal, sublingual, vaginally, rectally) or to the eye (intraocularly or transocularly).
  • the targeted constructs of the present invention may be combined with at least one other active agent to form a composition.
  • the at least one active agent may be a therapeutic, prophylactic, diagnostic, or nutritional agent. It may be a small molecule, protein, peptide, lipid, glycolipid, glycoprotein, lipoprotein, carbohydrate, sugar, or nucleic acid.
  • the targeted constructs of the present invention and the at least one other active agent may have the same target and/or treat the same disease.
  • the targeted constructs of the present invention and the at least one other active agent may be administered simultaneously or sequentially. They may be present as a mixture for simultaneous administration, or may each be present in separate containers for sequential administration.
  • spontaneous administration is not specifically restricted and means that the targeted constructs and the at least one other active agent are substantially administered at the same time, e.g. as a mixture or in immediate subsequent sequence.
  • the term "sequential administration,” as used herein, is not specifically restricted and means that the targeted constructs and the at least one other active agent are not administered at the same time but one after the other, or in groups, with a specific time interval between administrations.
  • the time interval may be the same or different between the respective administrations of the targeted constructs and the at least one other active agent and may be selected, for example, from the range of 2 minutes to 96 hours, 1 to 7 days or one, two or three weeks.
  • the time interval between the administrations may be in the range of a few minutes to hours, such as in the range of 2 minutes to 72 hours, 30 minutes to 24 hours, or 1 to 12 hours. Further examples include time intervals in the range of 24 to 96 hours, 12 to 36 hours, 8 to 24 hours, and 6 to 12 hours.
  • more than one targeted construct of the present invention may be combined to form a composition.
  • the targeted constructs may comprise different conjugates, wherein the conjugates may have different active agents, different linkers, and/or different targeting moieties.
  • the targeted constructs may have different compositions, different active agent loadings, and/or different molecular weights.
  • the targeted constructs in the composition may be administered simultaneously or sequentially. They may be present as a mixture for simultaneous administration, or may each be present in separate containers for sequential administration. Pharmacokinetic properties of the composition, such as Cmax, may be modulated by adjusting the weight percent ratio of the targeted constructs in the composition.
  • cancer embraces any disease or malady characterized by uncontrolled cell proliferation, e.g., hyperproliferation. Cancers may be characterized by tumors, e.g., solid tumors or any neoplasm.
  • the targeted constructs of the present invention have been found to inhibit cancer and/or tumor growth. They may also reduce, including cell proliferation, invasiveness, and/or metastasis, thereby rendering them useful for the treatment of a cancer.
  • the targeted constructs of the present invention may be used to prevent the growth of a tumor or cancer, and/or to prevent the metastasis of a tumor or cancer.
  • compositions of the present teachings may be used to shrink or destroy a cancer.
  • the targeted constructs of the present invention are useful for inhibiting proliferation of a cancer cell including but not limited to mammalian cancer cells.
  • the mammalian cancer cells are human cancer cells.
  • the targeted constructs of the present invention are useful for inhibiting cellular proliferation, e.g., inhibiting the rate of cellular proliferation, preventing cellular
  • the targeted constructs of the present invention can inhibit cellular proliferation of a cancer cell or both inhibiting proliferation and/or inducing cell death of a cancer cell.
  • the cancers treatable by methods of the present teachings generally occur in mammals.
  • Mammals include, for example, humans, non-human primates, dogs, cats, rats, mice, rabbits, ferrets, guinea pigs horses, pigs, sheep, goats, and cattle.
  • mammals include, for example, humans, non-human primates, dogs, cats, rats, mice, rabbits, ferrets, guinea pigs horses, pigs, sheep, goats, and cattle.
  • the cancer is lung cancer, breast cancer, e.g., mutant BRCA1 and/or mutant BRCA2 breast cancer, non-BRCA-associated breast cancer, colorectal cancer,
  • the cancer is lung cancer.
  • the cancer is human lung carcinoma, ovarian cancer, pancreatic cancer or colorectal cancer.
  • the targeted constructs of the present invention or formulations containing the targeted constructs of the present invention can be used for the selective tissue delivery of a therapeutic, prophylactic, or diagnostic agent to an individual or patient in need thereof.
  • Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic.
  • conjugates contained within the targeted constructs of the present invention are released in a controlled manner. The release can be in vitro or in vivo.
  • conjugates contained within the targeted constructs of the present invention administered to a subject may be protected from a subject's body, and the body may also be isolated from the conjugate until the conjugates are released from the targeted constructs of the present invention.
  • the conjugates are released from the targeted constructs of the present invention prior to the targeted constructs being delivered into the body, for example, a treatment site, of a subject.
  • the conjugate may be released over an extended period of time or by bursts (e.g., amounts of the conjugate are released in a short period of time, followed by a periods of time where substantially no conjugate is released).
  • the conjugates can be released over 6 hours, 12 hours, 24 hours, or 48 hours. In certain embodiments, the conjugates are released over one week or one month.
  • the targeted constructs of the present invention may be administered to tumors with a high level of enhanced permeability and retention (EPR) effect.
  • tumors with a high level of enhanced permeability and retention effect may be identified with imaging techniques.
  • imaging techniques As a non-limited example, iron oxide
  • nanoparticle magnetic resonance imaging may be administered to a patient and EPR effects are measured.
  • the targeted constructs of the present invention may be administered to a subject selected with the method disclosed in WO2015017506, the contents of which are incorporated herein by reference in their entirety, the method comprising:
  • kits and devices for conveniently and/or effectively carrying out methods of the present invention.
  • kits will comprise sufficient amounts and/or numbers of components to allow a user to perform multiple treatments of a subject(s) and/or to perform multiple experiments.
  • the present invention provides kits for inhibiting tumor cell growth in vitro or in vivo, comprising the targeted constructs of the present invention or a combination of the targeted constructs of the present invention, optionally in combination with any other active agents.
  • the kit may further comprise packaging and instructions and/or a delivery agent to form a formulation composition.
  • the delivery agent may comprise a saline, a buffered solution, or any delivery agent disclosed herein.
  • the amount of each component may be varied to enable consistent, reproducible higher concentration saline or simple buffer formulations.
  • the components may also be varied in order to increase the stability of the targeted constructs of the present invention in the buffer solution over a period of time and/or under a variety of conditions.
  • the present invention provides for devices which may incorporate the targeted constructs of the present invention. These devices contain in a stable formulation available to be immediately delivered to a subject in need thereof, such as a human patient. In some embodiments, the subject has cancer.
  • Non-limiting examples of the devices include a pump, a catheter, a needle, a transdermal patch, a pressurized olfactory delivery device, iontophoresis devices, multi- layered microfluidic devices.
  • the devices may be employed to deliver targeted constructs of the present invention according to single, multi- or split-dosing regiments.
  • the devices may be employed to deliver the targeted constructs of the present invention across biological tissue, intradermal, subcutaneously, or intramuscularly.
  • conjugate is also meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted.
  • the compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated.
  • Tautomeric forms result from the swapping of a single bond with an adjacent double bond and the concomitant migration of a proton.
  • Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge.
  • Examples prototropic tautomers include ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, amide - imidic acid pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, such as, IH- and 3H-imidazole, 1H-, 2H- and 4H- 1,2,4-triazole, IH- and 2H- isoindole, and IH- and 2H-pyrazole.
  • Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.
  • Compounds of the present disclosure also include all of the isotopes of the atoms occurring in the intermediate or final compounds.
  • “Isotopes” refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei.
  • isotopes of hydrogen include tritium and deuterium.
  • the compounds and salts of the present disclosure can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods.
  • subject refers to any organism to which the particles may be administered, e.g., for experimental, therapeutic, diagnostic, and/or prophylactic purposes.
  • Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, guinea pigs, cattle, pigs, sheep, horses, dogs, cats, hamsters, lamas, non-human primates, and humans).
  • treating can include preventing a disease, disorder or condition from occurring in an animal that may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having the disease, disorder or condition; inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and/or condition.
  • Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.
  • a target shall mean a site to which targeted constructs bind.
  • a target may be either in vivo or in vitro.
  • a target may be cancer cells found in leukemias or tumors (e.g., tumors of the brain, lung (small cell and non-small cell), ovary, prostate, breast and colon as well as other carcinomas and sarcomas).
  • a target may refer to a molecular structure to which a targeting moiety or ligand binds, such as a hapten, epitope, receptor, dsDNA fragment, carbohydrate or enzyme.
  • a target may be a type of tissue, e.g., neuronal tissue, intestinal tissue, pancreatic tissue, liver, kidney, prostate, ovary, lung, bone marrow, or breast tissue
  • therapeutic effect is art-recognized and refers to a local or systemic effect in animals, particularly mammals, and more particularly humans caused by a pharmacologically active substance.
  • the term thus means any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of desirable physical or mental development and conditions in an animal or human.
  • modulation is art-recognized and refers to up regulation (i.e., activation or stimulation), down regulation (i.e., inhibition or suppression) of a response, or the two in combination or apart.
  • Parenteral administration means administration by any method other than through the digestive tract (enteral) or non-invasive topical routes.
  • parenteral administration may include administration to a patient intravenously,
  • intradermally intraperitoneally, intrapleurally, intratracheally, intraossiously, intracerebrally, intrathecally, intramuscularly, subcutaneously, subjunctivally, by injection, and by infusion.
  • Topical administration means the non-invasive administration to the skin, orifices, or mucosa. Topical administrations can be administered locally, i.e., they are capable of providing a local effect in the region of application without systemic exposure. Topical formulations can provide systemic effect via adsorption into the blood stream of the individual. Topical administration can include, but is not limited to, cutaneous and
  • transdermal administration transdermal administration, buccal administration, intranasal administration, intravaginal administration, intravesical administration, ophthalmic administration, and rectal
  • Enteral administration means administration via absorption through the gastrointestinal tract. Enteral administration can include oral and sublingual administration, gastric administration, or rectal administration.
  • Pulmonary administration means administration into the lungs by inhalation or endotracheal administration.
  • inhalation refers to intake of air to the alveoli. The intake of air can occur through the mouth or nose.
  • the terms “sufficient” and “effective”, as used interchangeably herein, refer to an amount (e.g., mass, volume, dosage, concentration, and/or time period) needed to achieve one or more desired result(s).
  • a “therapeutically effective amount” is at least the minimum concentration required to effect a measurable improvement or prevention of at least one symptom or a particular condition or disorder, to effect a measurable enhancement of life expectancy, or to generally improve patient quality of life. The therapeutically effective amount is thus dependent upon the specific biologically active molecule and the specific condition or disorder to be treated.
  • Therapeutically effective amounts of many active agents, such as antibodies, are known in the art.
  • the therapeutically effective amounts of compounds and compositions described herein, e.g., for treating specific disorders may be determined by techniques that are well within the craft of a skilled artisan, such as a physician.
  • bioactive agent and “active agent”, as used interchangeably herein, include, without limitation, physiologically or pharmacologically active substances that act locally or systemically in the body.
  • a bioactive agent is a substance used for the treatment (e.g., therapeutic agent), prevention (e.g., prophylactic agent), diagnosis (e.g., diagnostic agent), cure or mitigation of disease or illness, a substance which affects the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a predetermined physiological environment.
  • prodrug refers to an agent, including a nucleic acid or protein that is converted into a biologically active form in vitro and/or in vivo.
  • Prodrugs can be useful because, in some situations, they may be easier to administer than the parent compound.
  • a prodrug may be bioavailable by oral administration whereas the parent compound is not.
  • the prodrug may also have improved solubility in pharmaceutical compositions compared to the parent drug.
  • a prodrug may be converted into the parent drug by various mechanisms, including enzymatic processes and metabolic hydrolysis. Harper, N.J. (1962) Drug Latentiation in Jucker, ed. Progress in Drug Research, 4:221-294; Morozowich et al.
  • biocompatible refers to a material that along with any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause any significant adverse effects to the recipient.
  • biocompatible materials are materials that do not elicit a significant inflammatory or immune response when administered to a patient.
  • biodegradable generally refers to a material that will degrade or erode under physiologic conditions to smaller units or chemical species that are capable of being metabolized, eliminated, or excreted by the subject.
  • the degradation time is a function of composition and morphology. Degradation times can be from hours to weeks or even longer.
  • pharmaceutically acceptable refers to compounds, materials, compositions, and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio, in accordance with the guidelines of agencies such as the U.S. Food and Drug Administration.
  • a “pharmaceutically acceptable carrier”, as used herein, refers to all components of a pharmaceutical formulation that facilitate the delivery of the composition in vivo.
  • Pharmaceutically acceptable carriers include, but are not limited to, diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof.
  • molecular weight generally refers to the mass or average mass of a material. If a polymer or oligomer, the molecular weight can refer to the relative average chain length or relative chain mass of the bulk polymer. In practice, the molecular weight of polymers and oligomers can be estimated or characterized in various ways including gel permeation chromatography (GPC) or capillary viscometry. GPC molecular weights are reported as the weight-average molecular weight (Mw) as opposed to the number-average molecular weight (Mn). Capillary viscometry provides estimates of molecular weight as the inherent viscosity determined from a dilute polymer solution using a particular set of concentration, temperature, and solvent conditions.
  • hydrophilic refers to substances that have strongly polar groups that readily interact with water.
  • hydrophobic refers to substances that lack an affinity for water; tending to repel and not absorb water as well as not dissolve in or mix with water.
  • lipophilic refers to compounds having an affinity for lipids.
  • amphiphilic refers to a molecule combining
  • Amphiphilic material refers to a material containing a hydrophobic or more hydrophobic oligomer or polymer (e.g., biodegradable oligomer or polymer) and a hydrophilic or more hydrophilic oligomer or polymer.
  • targeting moiety refers to a moiety that binds to or localizes to a specific locale.
  • the moiety may be, for example, a protein, nucleic acid, nucleic acid analog, carbohydrate, or small molecule.
  • the locale may be a tissue, a particular cell type, or a subcellular compartment.
  • a targeting moiety can specifically bind to a selected molecule.
  • reactive coupling group refers to any chemical functional group capable of reacting with a second functional group to form a covalent bond.
  • the selection of reactive coupling groups is within the ability of the skilled artisan.
  • Examples of reactive coupling groups can include primary amines (- H2) and amine-reactive linking groups such as isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters.
  • reactive coupling groups can include aldehydes (-COH) and aldehyde reactive linking groups such as hydrazides, alkoxyamines, and primary amines.
  • reactive coupling groups can include thiol groups (-SH) and sulfhydryl reactive groups such as maleimides, haloacetyls, and pyridyl disulfides.
  • reactive coupling groups can include photoreactive coupling groups such as aryl azides or diazirines.
  • the coupling reaction may include the use of a catalyst, heat, pH buffers, light, or a combination thereof.
  • protective group refers to a functional group that can be added to and/or substituted for another desired functional group to protect the desired functional group from certain reaction conditions and selectively removed and/or replaced to deprotect or expose the desired functional group.
  • Protective groups are known to the skilled artisan. Suitable protective groups may include those described in Greene and Wuts., Protective Groups in Organic Synthesis, (1991). Acid sensitive protective groups
  • Base sensitive protective groups include 9-fluorenylmethoxycarbonyl (Fmoc), isobutyrl (iBu), benzoyl (Bz) and phenoxyacetyl (pac).
  • protective groups include acetamidomethyl, acetyl, tert- amyloxycarbonyl, benzyl, benzyloxycarbonyl, 2-(4-biph8nylyl)-2- propy!oxycarbonyl, 2- bromobenzyloxycarbonyl, tert-butyb tert-butyloxycarbonyl, 1- carbobenzoxamido-2,2.2- trifluoroethyl, 2,6-dichlorobenzyl, 2-(3,5-dimethoxyphenyl)-2- propyloxycarbonyl, 2,4- dinitrophenyl, dithiasuccinyl, formyl, 4-methoxybenzenesulfonyl, 4- methoxybenzyl, 4- methylbenzyl, o-nitrophenylsulfenyl, 2-phenyl-2-propyloxycarbonyl, a- 2,4,5- tetramethylbenzyloxycarbonyl,
  • activated ester refers to alkyl esters of carboxylic acids where the alkyl is a good leaving group rendering the carbonyl susceptible to nucleophilic attack by molecules bearing amino groups. Activated esters are therefore susceptible to aminolysis and react with amines to form amides. Activated esters contain a carboxylic acid ester group -CO2R where R is the leaving group.
  • alkyl refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl- substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups.
  • a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chains, C3-C30 for branched chains), 20 or fewer, 12 or fewer, or 7 or fewer.
  • cycloalkyls have from 3-10 carbon atoms in their ring structure, e.g. have 5, 6 or 7 carbons in the ring structure.
  • alkyl (or “lower alkyl) as used throughout the specification, examples, and claims is intended to include both “unsubstituted alkyls” and “substituted alkyls”, the latter of which refers to alkyl moieties having one or more substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • substituents include, but are not limited to, halogen, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, a hosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety.
  • carbonyl such as a carboxyl, alkoxycarbonyl, formyl, or an acyl
  • thiocarbonyl such as a thioester, a
  • lower alkyl as used herein means an alkyl group, as defined above, but having from one to ten carbons, or from one to six carbon atoms in its backbone structure. Likewise, “lower alkenyl” and “lower alkynyl” have similar chain lengths. Throughout the application, preferred alkyl groups are lower alkyls. In some embodiments, a substituent designated herein as alkyl is a lower alkyl.
  • the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.
  • the substituents of a substituted alkyl may include halogen, hydroxy, nitro, thiols, amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN and the like.
  • Cycloalkyls can be substituted in the same manner.
  • substituted refers to all permissible substituents of the compounds described herein.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
  • Illustrative substituents include, but are not limited to, halogens, hydroxyl groups, or any other organic groupings containing any number of carbon atoms, preferably 1-14 carbon atoms, and optionally include one or more heteroatoms such as oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclic structural formats.
  • substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl
  • Heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. It is understood that “substitution” or “substituted” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, or elimination.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
  • Illustrative substituents include, for example, those described herein.
  • the permissible substituents can be one or more and the same or different for appropriate organic compounds.
  • the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms.
  • the substituent is selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone, each of which optionally is substituted with one or more suitable substituents.
  • the substituent is selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone, wherein each of the alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfony
  • substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, thioketone, ester, heterocyclyl, -CN, aryl, aryloxy, perhaloalkoxy, aralkoxy, heteroaryl, heteroaryloxy, heteroarylalkyl, heteroaralkoxy, azido, alkylthio, oxo, acylalkyl, carboxy esters, carboxamido, acyloxy, aminoalkyl, alkylaminoaryl, alky
  • carboxamidoalkylaryl carboxamidoaryl, hydroxyalkyl, haloalkyl, alkylaminoalkylcarboxy, aminocarboxamidoalkyl, cyano, alkoxyalkyl, perhaloalkyl, arylalkyloxyalkyl, and the like.
  • the substituent is selected from cyano, halogen, hydroxyl, and nitro.
  • copolymer generally refers to a single polymeric material that is comprised of two or more different monomers.
  • the copolymer can be of any form, such as random, block, graft, etc.
  • the copolymers can have any end-group, including capped or acid end groups.
  • mean particle size generally refers to the statistical mean particle size (diameter) of the particles in the composition.
  • the diameter of an essentially spherical particle may be referred to as the physical or hydrodynamic diameter of a spherical particle with an equivalent volume.
  • the diameter of a non-spherical particle may refer to the hydrodynamic diameter.
  • the diameter of a non-spherical particle may refer to the largest linear distance between two points on the surface of the particle.
  • Mean particle size can be measured using methods known in the art such as dynamic light scattering (DLS), electron microscopy, laser diffraction, MALDI-TOF, zeta potential measurement, AFM, TEM, SEM X-Ray microanalysis, or nanoparticle tracking analysis.
  • DLS dynamic light scattering
  • MALDI-TOF zeta potential measurement
  • AFM TEM
  • SEM X-Ray microanalysis or nanoparticle tracking analysis.
  • Two populations can be said to have a "substantially equivalent mean particle size" when the statistical mean particle size of the first population of particles is within 20% of the statistical mean particle size of the second population of particles; for example, within 15%, or within 10%.
  • monodisperse and “homogeneous size distribution”, as used interchangeably herein, describe a population of particles, microparticles, or nanoparticles all having the same or nearly the same size.
  • a monodisperse distribution refers to particle distributions in which 90% of the distribution lies within 5% of the mean particle size.
  • polydispersity index is used herein as a measure of the size distribution of an ensemble of particles, e.g., nanoparticles.
  • the polydispersity index can be calculated based on dynamic light scattering measurements.
  • polypeptide generally refer to a polymer of amino acid residues. As used herein, the term also applies to amino acid polymers in which one or more amino acids are chemical analogs or modified derivatives of corresponding naturally-occurring amino acids.
  • protein refers to a polymer of amino acids linked to each other by peptide bonds to form a polypeptide for which the chain length is sufficient to produce tertiary and/or quaternary structure.
  • protein excludes small peptides by definition, the small peptides lacking the requisite higher-order structure necessary to be considered a protein.
  • a "functional fragment" of a protein, polypeptide or nucleic acid is a protein, polypeptide or nucleic acid whose sequence is not identical to the full-length protein, polypeptide or nucleic acid, yet retains at least one function as the full-length protein, polypeptide or nucleic acid.
  • a functional fragment can possess more, fewer, or the same number of residues as the corresponding native molecule, and/or can contain one or more amino acid or nucleotide substitutions.
  • the DNA binding function of a polypeptide can be determined, for example, by filter-binding, electrophoretic mobility shift, or immunoprecipitation assays. DNA cleavage can be assayed by gel electrophoresis.
  • the ability of a protein to interact with another protein can be determined, for example, by co-immunoprecipitation, two-hybrid assays or complementation, e.g., genetic or biochemical. See, for example, Fields et al. (1989) Nature 340:245-246; U.S. Patent No. 5,585,245 and PCT WO 98/44350.
  • linker refers to a carbon chain that can contain heteroatoms (e.g., nitrogen, oxygen, sulfur, etc.) and which may be 1, 2, 3, 4, 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, 46, 47, 48, 49, 50 atoms long.
  • heteroatoms e.g., nitrogen, oxygen, sulfur, etc.
  • Linkers may be substituted with various substituents including, but not limited to, hydrogen atoms, alkyl, alkenyl, alkynl, amino, alkylamino, dialkylamino, trialkylamino, hydroxyl, alkoxy, halogen, aryl, heterocyclic, aromatic heterocyclic, cyano, amide, carbamoyl, carboxylic acid, ester, thioether, alkylthioether, thiol, and ureido groups. Those of skill in the art will recognize that each of these groups may in turn be substituted.
  • linkers include, but are not limited to, pH-sensitive linkers, protease cleavable peptide linkers, nuclease sensitive nucleic acid linkers, lipase sensitive lipid linkers, glycosidase sensitive carbohydrate linkers, hypoxia sensitive linkers, photo-cleavable linkers, heat-labile linkers, enzyme cleavable linkers (e.g., esterase cleavable linker), ultrasound-sensitive linkers, and x-ray cleavable linkers.
  • pH-sensitive linkers protease cleavable peptide linkers
  • nuclease sensitive nucleic acid linkers include lipase sensitive lipid linkers, glycosidase sensitive carbohydrate linkers, hypoxia sensitive linkers, photo-cleavable linkers, heat-labile linkers, enzyme cleavable linkers (e.g., esterase cleavable linker), ultrasound-sensitive linkers, and x-ray cleavable linkers.
  • salts refers to salts of acidic or basic groups that may be present in compounds used in the present compositions.
  • Compounds included in the present compositions that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids.
  • the acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including but not limited to sulfate, citrate, malate, acetate, oxalate, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p- toluenesulfonate and pamoate (i
  • Compounds included in the present compositions that include an amino moiety may form pharmaceutically acceptable salts with various amino acids, in addition to the acids mentioned above.
  • Compounds included in the present compositions, that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations.
  • salts include alkali metal or alkaline earth metal salts and, particularly, calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts.
  • the free base can be obtained by basifying a solution of the acid salt.
  • an addition salt particularly a pharmaceutically acceptable addition salt, may be produced by dissolving the free base in a suitable organic solvent and treating the solution with an acid, in accordance with conventional procedures for preparing acid addition salts from base compounds.
  • Those skilled in the art will recognize various synthetic methodologies that may be used to prepare non-toxic pharmaceutically acceptable addition salts.
  • a pharmaceutically acceptable salt can be derived from an acid selected from 1- hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2- oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, camphoric acid, camphor- 10- sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid
  • octanoic acid carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-l,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isethionic, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic, naphthalene-l,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid,
  • bioavailable is art-recognized and refers to a form of the subject invention that allows for it, or a portion of the amount administered, to be absorbed by, incorporated to, or otherwise physiologically available to a subject or patient to whom it is administered.
  • Antibody mimetics that bind to soluble human FcRn are selected from screening a library of affibodies. Targeted constructs comprising such affibodies are prepared.
  • Affibodies are injected in buffers with various pH values. Binding affinity and kinetics are measured.
  • HSA human serum albumin
  • articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.
  • the invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process.
  • the invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
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  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
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  • Peptides Or Proteins (AREA)

Abstract

L'invention a trait à des fractions de liaison à des protéines, telles que des mimétiques d'anticorps qui se lient à des protéines telles que la FcRn, l'albumine ou les IgG, ainsi qu'à des constructions ciblées comprenant les fractions de liaison à des protéines, qui ont été conçues pour assurer une distribution temporo-spatiale améliorée de l'agent actif et/ou une biodistribution et une demi-vie améliorées. L'invention concerne des procédés de production desdits mimétiques d'anticorps, des constructions ciblées et des compositions pharmaceutiques en comprenant. L'invention concerne en outre des procédés d'administration des compositions pharmaceutiques à un sujet en ayant besoin, par exemple pour traiter ou prévenir le cancer ou des maladies infectieuses.
PCT/US2018/035592 2017-06-01 2018-06-01 Constructions ciblées WO2018222987A1 (fr)

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US201762513882P 2017-06-01 2017-06-01
US201762513842P 2017-06-01 2017-06-01
US62/513,842 2017-06-01
US62/513,882 2017-06-01
US201762535347P 2017-07-21 2017-07-21
US62/535,347 2017-07-21
US201762599352P 2017-12-15 2017-12-15
US62/599,352 2017-12-15
US201862653093P 2018-04-05 2018-04-05
US62/653,093 2018-04-05

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CN111991570A (zh) * 2020-07-24 2020-11-27 北京大学 一种FAP-α特异性肿瘤诊断SPECT显像剂
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CN113777329A (zh) * 2021-09-16 2021-12-10 何秋富 一种视黄醇结合蛋白测定试剂盒及其制备方法
WO2022148974A3 (fr) * 2021-01-08 2022-12-15 Bicycletx Limited Ligands peptidiques bicycliques spécifiques des cellules nk
WO2023205752A3 (fr) * 2022-04-22 2024-05-30 University Of Miami Peptides mimétiques de ligand d'éphrine pour le traitement de maladies neurodégénératives
US12049520B2 (en) 2017-08-04 2024-07-30 Bicycletx Limited Bicyclic peptide ligands specific for CD137
US12318454B2 (en) 2014-10-29 2025-06-03 Bicyclerd Limited Bicyclic peptide ligands specific for MT1-MMP
US12350343B2 (en) 2018-12-13 2025-07-08 Bicycletx Limited Bicyclic peptide ligands specific for MT1-MMP

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Cited By (14)

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Publication number Priority date Publication date Assignee Title
US12318454B2 (en) 2014-10-29 2025-06-03 Bicyclerd Limited Bicyclic peptide ligands specific for MT1-MMP
US12049520B2 (en) 2017-08-04 2024-07-30 Bicycletx Limited Bicyclic peptide ligands specific for CD137
US20210145971A1 (en) * 2018-04-05 2021-05-20 Tarveda Therapeutics, Inc. Hsp90-targeting conjugates and formulations thereof
US12350343B2 (en) 2018-12-13 2025-07-08 Bicycletx Limited Bicyclic peptide ligands specific for MT1-MMP
CN113474045A (zh) * 2018-12-21 2021-10-01 拜斯科技术开发有限公司 Pd-l1特异性的双环肽配体
WO2020191011A1 (fr) * 2019-03-18 2020-09-24 The Trustees Of Columbia University In The City Of New York Lieurs stables non hydrolysables, non clivables pour agents thérapeutiques de précision et leurs utilisations
WO2021003050A3 (fr) * 2019-07-03 2021-02-11 Kleo Pharmaceuticals, Inc. Agent de liaison à cd38 et utilisations associées
CN114401732A (zh) * 2019-07-03 2022-04-26 克莱奥药品有限公司 Cd38结合剂和其用途
WO2022017375A1 (fr) * 2020-07-24 2022-01-27 北京大学 AGENT DE CONTRASTE DE SPECT POUR DIAGNOSTIC DE TUMEUR SPÉCIFIQUE DE FAP-α
CN111991570A (zh) * 2020-07-24 2020-11-27 北京大学 一种FAP-α特异性肿瘤诊断SPECT显像剂
WO2022148974A3 (fr) * 2021-01-08 2022-12-15 Bicycletx Limited Ligands peptidiques bicycliques spécifiques des cellules nk
CN113777329B (zh) * 2021-09-16 2023-07-18 何秋富 一种视黄醇结合蛋白测定试剂盒及其制备方法
CN113777329A (zh) * 2021-09-16 2021-12-10 何秋富 一种视黄醇结合蛋白测定试剂盒及其制备方法
WO2023205752A3 (fr) * 2022-04-22 2024-05-30 University Of Miami Peptides mimétiques de ligand d'éphrine pour le traitement de maladies neurodégénératives

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