WO2018222929A1 - Administration ciblée améliorée d'agents thérapeutiques - Google Patents

Administration ciblée améliorée d'agents thérapeutiques Download PDF

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WO2018222929A1
WO2018222929A1 PCT/US2018/035481 US2018035481W WO2018222929A1 WO 2018222929 A1 WO2018222929 A1 WO 2018222929A1 US 2018035481 W US2018035481 W US 2018035481W WO 2018222929 A1 WO2018222929 A1 WO 2018222929A1
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tumor
therapeutic
antibody
targeted
agent
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PCT/US2018/035481
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English (en)
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Jan E. Schnitzer
Philip Sung-Whan OH
Adrian CHRASTINA
Michael Levin
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Prism | Proteogenomics Research Institute For Systems Medicine
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/087—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins the peptide being an annexin, e.g. annexin V

Definitions

  • This passive transendothelial delivery tends to be slow and inefficient, largely because it depends on the concentration gradient of the drug across the vascular EC barrier: the larger the dose, the higher the drug concentration in the blood that is used to generate the driving force needed for faster and greater dose delivery into target diseased tissue.
  • vascular endothelium does form a formidably restrictive blood:tissue interface (Burrows & Thorpe, 1993; L. A. Carver & Schnitzer, 2003; H.F. Dvorak et al., 1991; D. P. McIntosh et al., 2002; J. E. Schnitzer, 1998)
  • the molecular variations present on the surface of vascular ECs in vivo does represent opportunities to target discrete vascular beds, to traverse blood vessels and to gain access to the underlying tissue.
  • the vascular endothelium, and EC surface specifically, is inherently, directly and rather immediately accessible to agents injected into, and circulating, in the blood.
  • Annexin A1 cleaved form of Annexin A1 (AnnA1) as a key delivery target that is selectively concentrated in caveolae of tumor endothelium and that is sufficiently specific and accessible to circulating molecular probes (i.e. antibodies) to enable tumor targeting and active penetration in vivo (Oh et al., 2014).
  • mAnnA1 Monoclonal antibodies against AnnA1 (mAnnA1) that recognizes human, rat and mouse AnnA1, were shown to enter and bind tumor endothelial caveolae and be rapidly, precisely, and actively pumped across the tumor EC barrier, and even concentrate most of the dose only inside rodent mammary tumors within 1-2 hr of intravenous (i.v.) injection (Oh et al., 2014).
  • APP2 aminopeptidase 2
  • mAPP2 monoclonal antibodies against APP2
  • Radionuclide therapy has over 50 years become most effective for hematogenous tumors because the targeting moiety, usually an antibody, has immediate intravenous access to its target, thereby readily delivering the desired therapeutic radionuclide specifically to the tumor cells. Solid tumors lack similar target accessibility and responsiveness, primarily because vascular EC and other barriers limit passive tissue entry of the radioconjugate from blood.
  • Tumor interstitial radiation brachytherapy has evolved as an effective alternative to systemic therapies by directly injecting each tumor with multiple radioactive seeds arranged geometrically to optimize coverage.
  • mechanical implantation of 125 I-loaded seeds into the tumor interstitium is widely applied clinically because of its curative effect, minimal surgical trauma, and few complications.
  • 125 I trapped inside each seed provides a low intensity yet sustained exposure better confined to its immediate space than other radionuclides.
  • 125 I can be quite toxic to individual tumor cells and destroys adjacent tissue within 4 mm of the seeds in a graded fashion.
  • the overall goal is to boost drug delivery into diseased tissue by exploiting a highly precise active transport pathway that could result in improved precision drug delivery and therapy while possibly reducing toxicities for patients.
  • Caveolae pumping system Our search for ways to go beyond passive transvascular delivery led to a breakthrough discovery of a transvascular pumping system in vascular endothelium in lung first and then in solid tumors (Oh et al., 2007; Oh et al., 2014). Caveolae at the EC surface can precisely, rapidly, and actively transcytose a selected targeted moiety, such as specific antibodies, out of the bloodstream and into the underlying tissue (Chrastina, Valadon, Massey, & Schnitzer, 2010; D.P. McIntosh et al., 2002; Oh et al., 2007; Oh et al., 2004; Oh et al., 2014; J. E. Schnitzer, 1998, 2001).
  • Caveolae are ⁇ 60-80 nm omega-shaped plasmalemmal invaginations that are distinct from clathrin-coated vesicles and act as dynamic transport vesicles (Oh, McIntosh, & Schnitzer, 1997; J. E. Schnitzer, 2001; J. E. Schnitzer, J. Liu, et al., 1995; J.E. Schnitzer, McIntosh, Dvorak, Liu, & Oh, 1995; J.E. Schnitzer et al., 1994; J. E. Schnitzer, Oh, & McIntosh, 1996) mediating endocytosis in many cell types and transcytosis, particularly in endothelium (Lucy A.
  • IVM imaging system and new ectopic-orthotopic mammary tumor models.
  • caveolae operate effectively as transvascular pumps, moving the lung-specific CTAs within 60 seconds from blood across the EC barrier and deep into the lung tissue, even against a concentration gradient (Oh et al., 2007).
  • IVM intracranial window model
  • IVM intracranial window model
  • Subcutaneously implanted tumors are the most popular and current IVM standard, but for studying many solid tumors, they lack proper orthotopic stroma and tumor microenvironment. They may not duplicate human disease and appear to respond therapeutically to many single therapies not found to be as effective in clinical trials and cancer patients.
  • EO tumors unlike subcutaneous tumors, exhibit resistance to an array of standard cancer monotherapies (e.g. doxorubicin, cisplatin, paclitaxel), as well as single immune- therapies (e.g.
  • Herceptin, anti-VEGF Herceptin, anti-VEGF.
  • EO tumors advance beyond subcutaneous tumors used in IVM by providing orthotopic tumor microenvironment and by lacking excessive therapeutic sensitivity that does not reflect most solid tumor patients who do not respond to these monotherapies, require combination therapies, or acquire resistance.
  • These drug-resistant models may better represent the hard-to-treat patients.
  • Fluorescence IVM of EO tumors following IV injection of fluorophore-labeled mAnnA1 readily revealed even at low magnifications that mAnnA1 precisely and rapidly accumulated throughout the tumor (Oh et al., 2014).
  • the signal inside the tumor tissue continued to increase within minutes of injection until approaching image saturation at 60 min.
  • the fluorescence signals from the“red” tumor cells and the“green” mAnnA1 clearly overlap; the antibody floods the tumor throughout, but not the surrounding tissue.
  • Caveolae can pump and concentrate targeted antibodies with attached cargo inside tumors and solid tissues, even at low doses and against a concentration gradient. Tumor uptake far exceeds other antibodies and passive transvascular delivery.
  • the ability to pump select therapeutic and imaging agents across EC barriers may enable a shift away from the current, passive transvascular delivery paradigm towards using an active portal to deliver agents directly inside diseased tissue.
  • a targeted therapeutic agent would include a specific antibody (e.g. Herceptin), specific small drug (e.g.
  • tyrosine kinase inhibitor or antibody-drug conjugate
  • typical chemotherapeutic agents e.g., cisplatin, paclitaxel, unconjugated radiopharmaceutical agents, etc.
  • nanocarriers e.g., Doxil
  • Interacting with a specific therapeutic target does not obviate this passive delivery.
  • dependence on passive transvascular delivery necessitates the use of high doses to generate a large concentration gradient across the EC barrier in order to drive more drug from the bloodstream to the inside (interstitium and parenchyma) of the diseased tissue, such as tumors.
  • a dose (if properly and precisely delivered) readily produces >10 mM levels of small drugs and ⁇ 100 ⁇ M of antibodies inside the tumor.
  • Most of the drugs are fully active when directly applied to cells at low ⁇ M to high nM concentration for standard chemotherapeutics and at low to even sub-nM levels with modern targeted agents. Despite this dosage overkill and achieving blood levels above 1 ⁇ M (and even beyond 100 ⁇ M), drugs designed and confirmed to be effective at nanomolar concentrations or less, have difficulty reaching intra-tumoral therapeutic concentrations that enable their full potency.
  • transvascular pumping of targeted agents into tumors and other diseased tissue can overcome this problem and enhance therapeutic potency or therapeutic index at ultra low doses (e.g., ⁇ g/kg to ng/kg dose ranges).
  • ultra low doses e.g., ⁇ g/kg to ng/kg dose ranges.
  • MDR multi-drug resistant
  • Bind refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise,“binding affinity” refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described elsewhere herein.
  • Binding affinity of an antibody can be measured using any suitable technique, e.g., by a radioimmunoassay (RIA) or by Scatchard analysis or by surface plasmon resonance (e.g., via a Biacore).
  • a targeting agent has a dissociation constant (Kd) of 0.1 uM, 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, or 0.001 nM (e.g., 10 -7 M or less, e.g., from 10 -7 M to 10 -13 M.
  • An“affinity matured” antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent antibody that does not possess such alterations. Preferably, such alterations result in improved affinity of the antibody for its target antigen.
  • HVRs hypervariable regions
  • the term“antibody” herein is used in the broadest sense and encompasses various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
  • an“antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
  • full length antibody “intact antibody,” and“whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.
  • Fc region herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
  • A“human antibody” is one that possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a“humanized” antibody comprising non-human antigen-binding residues.
  • A“humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human hypervariable regions (HVRs) and amino acid residues from human framework regions (FRs).
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., complementarity determining regions or“CDRs”) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
  • HVRs e.g., complementarity determining regions or“CDRs”
  • CDRs complementarity determining regions
  • a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
  • A“humanized form” of an antibody e.g., a non-human antibody, refers to an antibody that has undergone humanization.
  • An“effective amount” of an agent e.g., a targeted drug conjugate, an therapeutic agent, a pharmaceutical formulation, etc. refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • An“individual” or“patient” or“subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non- human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.
  • An“isolated” molecule is one that has been separated from a component of its natural environment.
  • an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC).
  • electrophoretic e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatographic e.g., ion exchange or reverse phase HPLC
  • the term“monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical (as assessed at the level of Ig heavy and/or light chain amino acid sequence) and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • polyclonal antibody preparations typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • the modifier“monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including, but not limited to, the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
  • package insert is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
  • Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • pharmaceutical composition refers to a preparation that is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • treatment refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology.
  • Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.
  • A“patentable” composition, machine, method, process, or article of manufacture according to the invention means that the subject matter satisfies all statutory requirements for patentability at the time the analysis is performed. For example, with regard to novelty, non-obviousness, or the like, if later investigation reveals that one or more claims encompass one or more embodiments that would negate novelty, non-obviousness, etc., the claim(s), being limited by definition to“patentable” embodiments, specifically exclude the unpatentable embodiment(s). Also, the claims appended hereto are to be interpreted both to provide the broadest reasonable scope, as well as to preserve their validity.
  • a protein that is“associated with caveolae”,“caveolae-associated”, or the like refers to a cell- surface protein located in proximity or otherwise effecting caveolae function, formation, stability, or activity.
  • caveolae-associated proteins expressed in normal or diseased tissue include aminopeptidase P2 (APP2), annexin A1 (AnnA1) including a truncated 34 kD form of the protein, caveolin-1 (CAV1), caveolin-2 (CAV2), caveolin-3 (CAV3), Cavin-1 (also known as polymerase-1 and transcript release factor) (PTRF), Cavin-2, Cavin-3, EDH2, GlycosylPhosphatidylInositol (GPI)- linked receptors, Pacsin2 (also referred to as syndapin2), Flotillin-1, Flotillin-2 and Cavin-4.
  • APP2 aminopeptidase P2
  • AnnA1 annexin A1
  • CAV1 caveolin-1
  • Additional proteins“associated with caveolae”,“caveolae-associated”, or the like also refer to proteins isolated from luminal vascular endothelial cell membranes enriched for caveolae and include APP2, CD34, OX-45, AnnA1, vascular endothelial growth factor (VEGF) receptors-1 and -2, Tie2, aminopeptidase-N, endoglin, carcino- embryonic antigen-related cell adhesion molecule 1 (CD66), (C-CAM-1) and neuropilin-1, annexin A8, ephrin A5, ephrin A7, myeloperoxidase, nucleolin, transferrin receptor and vitamin D-binding protein.
  • APP2 proteins isolated from luminal vascular endothelial cell membranes enriched for caveolae and include APP2, CD34, OX-45, AnnA1, vascular endothelial growth factor (VEGF) receptors-1 and -2, Tie2, aminopeptidase-N,
  • TI refers to the dose ratio between a drug’s toxic and therapeutic (or prophylactic) effects.
  • TI generally refers to the dose of a drug (or lead compound, drug candidate, or the like) that causes adverse effects at an incidence or of a severity that is incompatible with the desired effect(s) (e.g., the toxic dose in 50% of cells, study animals, or other subjects or patients (TD50)) compared to the minimum effective dose (the minimum effective dose in an assay, in 50% of a patients or a population of study subjects (human or non-human) (ED 50 ).
  • a higher TI is preferable to a lower TI, as a higher TI means that a larger amount of the compound would be required to observe toxic effects as compared to the amount needed to efficacious.
  • the invention provides for increasing a particular therapeutic agent’s TI, preferably by at least a factor of about 10, even more preferably by a factor of 20-10,000 or more. For example, if drug A when untargeted has a TI of X, in the context of the invention the targeted form of drug A will have a TI of at least about 10X, preferably 100X to 10,000X or more.
  • therapeutic window refers to the range of dosages between efficacy and toxicity.
  • therapeutic potency refers to the amount of a compound (e.g., an drug, a lead compound, etc.) required to produce an effect of a given intensity. Highly potent compounds produce a particular effect at low concentrations.
  • the invention provides for increasing a particular therapeutic agent’s therapeutic potency, preferably by at least a factor of about 10, even more preferably by a factor of 20-10,000 or more. For example, if drug A when untargeted has a potency of X, in the context of the invention the targeted form of drug A will have a potency of at least about 10X, preferably 100X to 10,000X or more.
  • compositions or kits or methods of the present invention include additional component(s), composition(s), or method step(s) that do not materially change the basic and novel characteristics of the present invention. Such characteristics include the ability to selectively detect target nucleic acids in biological samples (e.g., whole blood or plasma). Any component(s), composition(s), or method step(s) that have a material effect on the basic and novel characteristics of the present invention would fall outside of this term.
  • the object of this invention is to provide patentable targeted drug conjugates, compositions containing such conjugates, kits containing such conjugates and compositions, and methods for making and using the same.
  • the invention concerns targeted drug conjugates that comprise an active ingredient (i.e., a therapeutic agent) and a targeting agent.
  • the targeted drug conjugate comprises a therapeutic agent conjugated directly or indirectly (i.e., via an intermediate chemical moiety such as a linker, dendrimers, etc.) to a targeting agent
  • the therapeutic agent and targeting agent of a targeted drug conjugate are merely associated in a composition, e.g., as part of a nanoparticle (e.g., a liposome encapsulating the therapeutic agent and having the targeting agent displayed on the liposome’s outer surface).
  • the active ingredient is a therapeutic agent.
  • the potency of the therapeutic agent is enhanced as compared to untargeted forms of the same therapeutic agent such that when formulated into a suitable composition, for example, a pharmaceutical composition comprising the targeted drug conjugate and a pharmaceutically acceptable carrier, at least about 10-fold less (e.g., 10-10,000-fold less, e.g., 20-, 50-, 100-, 500-, 1,000-fold less) of the therapeutic agent is required to exert the desired effect (e.g., a prophylactic or therapeutic effect) than when an effective amount of the therapeutic agent present in an untargeted form is administered to a subject having a disease or condition amenable to treatment thereby.
  • a suitable composition for example, a pharmaceutical composition comprising the targeted drug conjugate and a pharmaceutically acceptable carrier, at least about 10-fold less (e.g., 10-10,000-fold less, e.g., 20-, 50-, 100-, 500-, 1,000-fold less) of the therapeutic agent is required to exert the desired effect (e.g., a prophylactic or therapeutic effect
  • Any suitable assessment can be used to determine whether the amount of the therapeutic agent administered as part of a targeted drug conjugate of the invention is at least about 10-fold less as compared to an untargeted form (i.e., a composition or formulation that does include a targeting agent) of the same therapeutic agent in order to achieve substantially the same therapeutic benefit, including vitro assays, non-human animal models, and treatment of subjects.
  • the therapeutic agent is a small molecule, a peptide, a protein, a nucleic acid, a radionuclide, or a gene delivery vehicle (e.g., a virus, preferably an engineered virus).
  • a gene delivery vehicle e.g., a virus, preferably an engineered virus.
  • Useful therapeutic agents include those that are selected from among chemotherapeutic agents, immune stimulatory agents, anti-neoplastic agents, pro-coagulants, toxins, antibiotics, hormone, enzymes, and lytic agents.
  • the targeting agent specifically binds to an extracellular domain of a protein displayed on an outer surface of a cell membrane of a cell.
  • the targeting agent is a member of a high affinity binding pair.
  • Antibodies and antigen- binding antibody fragments e.g., Fab fragments
  • Fab fragments that target an extracellular domain of a protein species expressed predominantly on the extracellular surface of endothelial cells, for example, caveolae of tumor endothelium (e.g., Annexin A1 (AnnA1)) are representative examples of suitable targeting agents in the context of the invention.
  • the targeting agent is a receptor, a ligand-binding receptor fragment, a receptor ligand, a small molecule, or an aptamer.
  • the targeting agent of the targeted drug conjugate of the composition specifically binds to an extracellular domain of a protein displayed on the outer surface of a cell membrane of a vascular endothelial cell, which protein is capable of mediating active transvascular pumping of the targeted drug conjugate across the cell into underlying diseased tissue.
  • the extracellular domain targeted by the targeting agent is displayed on the surface of the vascular endothelial cell is predominantly located in or is translocated to caveolae.
  • a related aspect of the invention concerns pharmaceutical compositions, which include a targeted drug conjugate composition of the invention wherein the carrier is a pharmaceutically acceptable carrier.
  • kits typically contain a composition of the invention packaged in a suitable container.
  • the kits, or packages also include instructions for use.
  • kit instructions are usually a package insert, which contains not only instructions for use but also information about the pharmaceutically active ingredient of the targeted drug conjugate of the composition packaged in the particular kit.
  • Another aspect of the invention relates to methods of decreasing the amount of a therapeutic agent needed to effect therapy.
  • Such methods comprise administering a targeted drug conjugate composition of the invention to a subject having a disease or condition amenable to treatment by the therapeutic agent deployed therein, thereby decreasing the amount of the particular therapeutic agent needed to treat the disease or condition.
  • a related aspect of the invention involves methods of treating a disease or condition afflicting (or which may later afflict, either for the first time or as a result of recurrence) a subject. Such methods also typically include administering to a subject suspected of or having a disease or condition a targeted drug conjugate composition according to the invention, thereby treating the disease or condition.
  • Subjects that can be treated in accordance with invention include humans or other mammals, for example, bovine, canine, equine, feline, ovine, or porcine animals.
  • the disease or condition to be treated is a non-hematologic cancer, an infection, inflammation, fibrosis, acute injury, infarction, or a pathological malfunction that is none of the foregoing.
  • non-hematologic cancers that can be treated in accordance with the invention include solid cancers, for example, sarcomas, carcinomas, lymphomas, and metastatic lesions.
  • Fig. 1 Destruction of mammary tumors using mAnnA1-doxorubicin ADC.
  • H2B-GFP mammary tumor spheroids were co-implanted into window chambers with mammary fat pad and allowed to vascularize and grow for 10 days.
  • the mice were then injected via the tail vein with the indicated dose and imaged by IVM for 14 days thereafter, as indicated.
  • the dosages are expressed as total doxorubicin injected (either alone or immunoconjugated as indicated) per kg body weight.
  • Fig. 2 Destruction of mammary tumors using mAnnA1-docetaxel ADC.
  • Mammary tumor spheroids H2B-GFP
  • H2B-GFP Mammary tumor spheroids
  • the mice were then injected with the indicated dose and imaged by IVM for 14 days thereafter, as indicated.
  • the dosages are expressed as total docetaxel injected (either alone or immunoconjugated as indicated) per kg body weight.
  • Fig. 3 Conjugated mAnnA1-CMD-cisplatin causes tumor ablation in a dose dependent manner in mammary tumors.
  • Cav1KO (c) and AnnA1KO (e) Mice with H2B-GFP expressing mammary tumors were injected i.v. with the mAnnA1 conjugated to cisplatin (21 ⁇ g/kg) and imaged with fluorescence video- microscopy. Representative static frames were captured at the indicated days after treatment.
  • Fig. 4 Destruction of prostate tumors using mAnnA1 ADC.
  • Fig. 5 Destruction of breast metastatic lesions with mAnnA1-DM1 conjugates.
  • Female rats were intravenously inoculated with 13762 MATB II mammary adenocarcinoma cells to generate ample, well-circumscribed tumors in the lungs.
  • Treatment 100 ug/kg was started approximately 2 weeks after inoculation. Animals received either one dose of 100 ug/kg mAnnA1-DM1 or two doses 1 week apart (marked with *). Body weights of tumor-bearing rats as well as a healthy non-tumor bearing control were tracked over time.
  • Fig. 6 Destruction of mammary tumors using AnnA1 radioimmunotherapy.
  • H2B-GFP mammary tumor spheroids were co-implanted into window chambers with mammary fat pad and allowed to vascularize and grow for 10 days. Then the mice were imaged by IVM for 14 days after being injected via tail vein with 0.3, 1.0 or 3.0 ⁇ g of 125 I-mAnnA1, 3 ⁇ g of control 125I-IgG (Cont IgG), or untreated control (0.0 ⁇ g).
  • Fig. 7. Biodistribution analysis of 125 I-mAnnA1 tumor targeting.
  • Female Fisher rats were injected intravenously via tail vein with 1 x 10 6 13762 Mat B III mammary adenocarcinoma cells. In this metastatic breast cancer model ample, well-circumscribed tumors colonize the lungs approximately 2 weeks after injection.
  • (a-b) Biotinylated mAnnA1 was used to stain paraffin sections of neoplastic and normal lung tissues. Results indicate the perfused antibody preferentially localizes to tumor blood vessels.
  • Tissue targeting index for tumors TTItumor, tissue-to-blood ratio, %ID/g tumor divided by %ID/g blood.
  • TTI Tissue selectivity index
  • m Standardized uptake value.
  • Rapid concentration of mAnnA1 only in to tumors reaches levels well beyond the highest blood levels. Calculating an index measuring pumping efficiency/power of caveolae targeting via mAnnA1 to concentrate 125 I inside tumors.
  • TCPI tissue concentration power index
  • Fig. 8 Biodistribution analysis of 125 I-mAnnA1 targeting in control and tumor-bearing animals. % injected dose (%ID) was determined for organs collected 2 hrs post-injection of 125 I- mAnnA1 in non-tumor-bearing (control) and tumor-bearing rats.
  • Fig.9. In vivo targeting of AnnA1 antibodies.
  • Her2/neu mice with spontaneous tumors were injected intravenously with 125 I-mAnnA1 antibodies (a) or with isotype matched 125 I-IgG (e).
  • SPECT- CT images were acquired at the indicated times. Yellow outline demarcates tumor.
  • ROI The Region of Interest (ROI) was determined for the tumor (outlined in yellow) and liver over time and plotted. ROI was measure using the program LumaGEM_P (GammaMedica) after acquiring images using LumaGEM_A (GammaMedica).
  • FIG. 10 125 I-mAnnA1 increases survival in the 13762 rat metastatic breast cancer model.
  • Female Fisher rats were injected intravenously via tail vein with 1 x 10 4 13762 Mat B III mammary adenocarcinoma cells.
  • Tumor bearing rats received 125 I - mAnnA1 (4.5 ⁇ g at 7 ⁇ Ci/ ⁇ g) treatment or a control vehicle.
  • mVEGF Antibodies to VEGF
  • mAnnA1 antibodies were conjugated to mAnnA1 antibodies and injected into mice implanted mammary tumor spheroids in an IVM EO tumor model system.
  • A Antibody uptake was assessed via image capture at indicated times.
  • B Fluorescent intensity was measured for each conjugate.
  • C The response of the tumor to treatment with the bifunctional dual antibody conjugate was compared to animals injected intravenously with unconjugated mVEGF or untreated (control) and tumor size was assessed over a course of 14 days.
  • D Graph of relative tumor size for each dose of conjugate, including control.
  • FIG. 12 Conjugating Herceptin to mAnnA1 boosts therapeutic potency.
  • A GFP-tagged human BT474 breast tumors grown in IVM model in mice were treated as indicated and observed over 14 days.
  • B Graph of relative tumor size for each dose of conjugate, including control.
  • Fig.13 Low dose precision delivery enables acute inhibition of fibrotic signaling pathway in lung and prophylactic inhibition of lung fibrosis.
  • Rats received intratracheal injections of bleomycin and, concurrently, i.v. injections of either mTGF- ⁇ or mAPP2:mTGF- ⁇ at the indicated doses.
  • A Targeted lung delivery of mAPP2-mTGF- ⁇ inhibits pSMAD signaling. Immunoblot of pSmad2 and Smad2 from whole mouse lung lysates 6 hr following acute lung injury concurrent with i.v. injection of mTBG- ⁇ alone or conjugated to mAPP2.
  • B Lungs were removed and processed for Trichrome stain to examine collagen deposition 2 weeks later.
  • Fig.14 Low dose precision delivery and therapy effective for lung fibrosis model.
  • A Rats received IT bleomycin and 12 days later, i.v. injections of either TGF- ⁇ blocking antibodies alone or conjugated to mAPP2 (both at 0.1 mg/kg). Lungs were removed 1 week later and processed for H&E and Trichrome staining. Trichrome blue shows collagen. All micrographs are at the same magnification.
  • B Histogram of whole lung collagen levels determined by Sircoll biochemical assay in experiments where rats were treated as described above.
  • (*) p ⁇ 0.05; by ranked ANOVAs with the Tukey post hoc test.
  • C Lungs from rats treated as described above were processed for blotting with antibodies specific for pSmad2, Smad, smooth muscle actin (SMA) and beta actin (loading control).
  • Fig.17 IVM showing rapid tumor uptake & enhanced radiotherapeutic efficacy of mAnnA1- targeted dendrimers.
  • Nude mice with EO model expressing H2B-CFP red tumor cells; left column
  • PAMAM G5 dendrimers either conjugated to mAnnA1 or control isotype matched IgG (mIgG).
  • the IVM images (size bar 200 ⁇ m) taken before injection and 1 hr after clearly show rapid tumor uptake of only the mAnnA1- dendrimers. They targeted and crossed the tumor endothelium to flood the tumor as indicated by the accumulation of green signal.
  • B and C The IVM images (size bar 200 ⁇ m) taken before injection and 1 hr after clearly show rapid tumor uptake of only the mAnnA1- dendrimers. They targeted and crossed the tumor endothelium to flood the tumor as indicated by the accumulation of green signal.
  • mice with EO model expressing H2B-GFP green tumor cells were injected iv with 3 ⁇ g of 125 I-dendrimers conjugated to mIgG or linked to mAnnA1.
  • B Fluorescent IVM at low magnification on the days indicated post-treatment shows the radioimmunotherapy was effective only with linkage to caveolae-targeting mAnnA1. Size bar, 50 ⁇ m.
  • the invention is based on precision delivery mediated by the caveolae pumping system as a means to enhance therapeutic efficacy at ultra low doses.
  • caveolae plasmalemmal vesicles called caveolae are abundant on the endothelial cell surface, function in selective endocytosis and transcytosis of nutrients, and provide a means to enter endothelial cells (endocytosis) and/or to penetrate the endothelial cell barrier (transcytosis) for delivery to underlying tissue cells.
  • endocytosis endocytosis
  • transcytosis endothelial cell barrier
  • vascular endothelial cell surface that is in immediate and intimate contact with the circulating blood. This vascular endothelial cell surface provides an inherently accessible, and thus targetable, surface on diseased tissues and organs.
  • Targeting the caveolae pumping system is a viable means to breach the vascular endothelial barrier to improve precision drug delivery and enhance the potency of systemically administered drugs to treat diseases, including for example but not limited to solid cancers and pulmonary fibrosis. DELIVERY OF AGENTS
  • a targeted drug conjugate composition comprising a carrier and targeted drug conjugate further comprising an active ingredient conjugated to a targeting agent is precisely delivered to target disease tissue as a result of the targeting agent specifically binding to an extracellular domain of a protein displayed on the outer surface of a cell membrane of a vascular endothelial cell. It is believed that the therapeutic activity of active agents will be enhanced because of precision delivery directly into targeted diseased tissue. It is further believed that using a highly efficient and rapid transvascular pathway to deliver agents directly into target tissue will concentrate such agents and thereby boost activity even at ultra low doses as a result of precision delivery.
  • the targeted drug conjugate composition comprising an active ingredient that is present in an amount at least about 10-fold less than an effective amount of the active ingredient present in an untargeted drug composition.
  • the targeted drug conjugate composition is used to treat a disease or condition tissue arising from non-hematologic cancers, an infection, inflammation, fibrosis, acute injury, infarction or other pathological malfunction.
  • non-hematologic cancers refers to solid cancers an includes sarcomas, carcinomas, lymphomas and metastatic lesions.
  • non-hematologic cancers that can be targeted include brain, breast, lung, kidney, prostate, ovarian, head and home, and liver tumors and lesions arising from metastases.
  • An agent that specifically binds to a targeted protein is an agent that preferentially or selectively binds to that targeted protein. While certain degree of non-specific interaction may occur between the agent that specifically binds and the targeted protein, nevertheless, specific binding, may be distinguished as mediated through specific recognition of the targeted protein, in whole or part. Typically, specific binding results in a much stronger association between the agent and the targeted protein than between the agent and other proteins, e.g., other vascular proteins.
  • the affinity constant (Ka, as opposed to Kd) of the agent for its cognate is at least 10 6 or 10 7 , usually at least 10 8 , alternatively at least 10 9 , alternatively at least 10 10 , or alternatively at least 10 11 M.
  • “specific” binding may be binding that is sufficiently site- specific to effectively be“specific”: for example, when the degree of binding is greater by a higher degree (e.g., equal to or greater than 10-fold, equal to or greater than 20-fold, or even equal to or greater than 100-fold), the binding may become functionally equivalent to binding solely to the targeted protein at a particular location: directed and effective binding occurs with minimal or no delivery to other tissues.
  • the amount that is functionally equivalent to specific binding can be determined by assessing whether the goal of effective delivery of agents is met with minimal or no binding to other tissues.
  • Representative antibodies include commercially available antibodies (as listed in Linscotts Directory).
  • the agent is or comprises another agent that specifically binds to a targeted protein (a specific binding partner).
  • Representative specific binding partners include an antigen-binding fragment, a receptor, a ligand- binding receptor fragment, a receptor ligand, natural ligands, peptides, small molecules (e.g., inorganic small molecules, organic small molecules, derivatives of small molecules, composite small molecules); aptamers; cells, including modified cells; vaccine-induced or other immune cells; nanoparticles (e.g., lipid or non-lipid based formulations); lipids; lipoproteins; lipopeptides; lipid derivatives; liposomes; modified endogenous blood proteins used to carry chemotherapeutics; a protein (e.g., a recombinant protein or a recombinant modified protein) a carrier protein (e.g., albumin, modified albumin); a lytic agent; a small molecule; other nanoparticles (e.g., albumin-based nanoparticles); transferrins; immunoglobulins; multivalent antibodies; lipids; lipoproteins
  • the agent can also comprise a first component that binds to the targeted protein, as described above, and a second component, that is an active component (e.g., a therapeutic agent or imaging agent, as described in detail below).
  • the agent can be administered by itself, or in a composition (e.g., a pharmaceutical or physiological composition) comprising the agent. It can be administered either in vivo (e.g., to an individual) or in vitro (e.g., to a tissue sample).
  • the methods of the invention can be used not only for human individuals, but also are applicable for veterinary uses (e.g., for other mammals, including domesticated animals (e.g., horses, cattle, sheep, goats, pigs, dogs, cats, etc.) and non-domesticated animals.
  • the agent can be administered by itself, or in a composition (e.g., a physiological or pharmaceutical composition) comprising the agent.
  • a composition e.g., a physiological or pharmaceutical composition
  • the therapeutic targeting agent can be formulated together with a physiologically acceptable carrier or excipient to prepare a pharmaceutical composition.
  • the carrier and composition can be sterile. The formulation should suit the mode of administration.
  • non-specific background and/or scavenger uptake of agents by reticulo-endothelial system may be reduced by overwhelming the system by inhibition and/or competitions with various reagents, including, for example, immunoglobulins, proteins or protein fragments, starches or hydroxyethylstarches, albumins, modified albumins, or other agents.
  • agents can be administered prior to, or concurrently with, the agents of the invention.
  • Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well as combinations thereof.
  • the pharmaceutical preparations can, if desired, be mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active agents.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active agents.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as trigly
  • compositions include, but are not limited to, intradermal, intramuscular, intraperitoneal, intraocular, intravenous, subcutaneous, topical, oral and intranasal.
  • Other suitable methods of introduction can also include rechargeable or biodegradable devices, particle acceleration devises (gene guns) and slow release polymeric devices.
  • the compositions can be administered into a specific tissue, or into a blood vessel serving a specific tissue (e.g., the carotid artery to target brain).
  • the pharmaceutical compositions can also be administered as part of a combinatorial therapy with other agents, either concurrently or in proximity (e.g., separated by hours, days, weeks, months).
  • the activity of the compositions may be potentiated by other agents administered concurrently or in proximity.
  • compositions for intravenous administration typically are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water.
  • an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • nonsprayable forms viscous to semi-solid or solid forms comprising a carrier compatible with topical application and having a dynamic viscosity preferably greater than water
  • Suitable formulations include but are not limited to solutions, suspensions, emulsions, creams, ointments, powders, enemas, lotions, sols, liniments, salves, aerosols, etc., which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc.
  • auxiliary agents e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc.
  • the agent may be incorporated into a cosmetic formulation.
  • sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant, e.g., pressurized air.
  • a pressurized volatile, normally gaseous propellant e.g., pressurized air.
  • Agents described herein can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • methods are available for treating a disease or condition, such as but not limited to non-hematologic cancers or other pathologies in an individual, by administering a targeted therapeutic agent.
  • treatment can refer to ameliorating symptoms associated with the non-hematologic cancer, an infection, inflammation, fibrosis, acute injury, infarction or other pathological malfunction; to reducing, preventing or delaying metastasis of the neoplasms such as cancer; to reducing the number, volume, and/or size of one or more solid tumors; and/or to lessening the severity, duration or frequency of symptoms of a disease or pathology.
  • a targeted drug composition is used.
  • a targeted drug composition refers to a composition comprising a carrier and targeted drug conjugate that comprises an active ingredient conjugated to a targeting agent.
  • the active ingredient is a therapeutic agent, which is present in the composition in an amount that is at least about 10-fold or 100-fold less than when an effective amount of the therapeutic agent is present in an untargeted composition used to treat a disease or condition amenable to treatment by the therapeutic agent.
  • the targeting agent specifically binds to an extracellular domain of a protein displayed on an outer surface of a cell membrane of the cell.
  • angiogenesis or the development of other neovasculature, atherosclerosis, diabetes and related sequelae, macular degeneration, heart disease (e.g., from ischemia), emphysema, chronic obstructive pulmonary disease, myocarditis, pulmonary and systemic hypertension and their sequelae, infection, and other conditions relating to expression of inflammatory-, angiogenesis- or neovasculature-related proteins, such as those described herein.
  • Expression of angiogenesis-related proteins is a contributor to a variety of malignant, ischemic, inflammatory, infectious and immune disorders.
  • the methods are similarly applicable to such conditions, which are collectively referred to herein as“pathology”.
  • the targeted drug composition comprises an antibody that specifically binds a targeted protein, as described herein (e.g., Annexin A1, Aminopeptidase 2).
  • An antibody is an immunoglobulin molecule obtained by in vitro or in vivo generation of the humoral response, and includes both polyclonal and monoclonal antibodies.
  • the term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies), and recombinant single chain Fv fragments (scFv).
  • antibody also includes multivalent antibodies as well as antigen binding fragments of antibodies, such as Fab', F(ab')2, Fab, Fv, rIgG, and, inverted IgG, as well as the variable heavy and variable light chain domains.
  • An antibody immunologically reactive with a targeted protein can be generated in vivo or by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors. See, e.g., Huse et al. (1989) Science 246:1275-1281; and Ward, et al. (1989) Nature 341:544- 546; and Vaughan et al. (1996) Nature Biotechnology, 14:309-314.
  • an antigen-binding fragment includes any portion of an antibody that binds to the targeted protein.
  • An antigen-binding fragment may be, for example, a polypeptide including a CDR region, or other fragment of an immunoglobulin molecule that retains the affinity and specificity for the targeted protein.
  • the targeting agent comprises a member of a high-affinity binding pair high-affinity binding pair, optionally a molecule selected from the group consisting of an antibody, an antigen-binding antibody fragment, a receptor, a ligand-binding receptor fragment, a receptor ligand, a small molecule, and an aptamer.
  • a multivalent antibody is used.
  • One moiety of the multivalent antibody can serve as the targeting agent component, and a second moiety of the multivalent antibody can serve as the active agent component.
  • the targeting agent component is linked to the active (or therapeutic) agent component.
  • they can be covalently bonded directly to one another.
  • the bond may be formed by forming a suitable covalent linkage through an active group on each moiety.
  • an acid group on one compound may be condensed with an amine, an acid or an alcohol on the other to form the corresponding amide, anhydride or ester, respectively.
  • Suitable active groups for forming linkages between a targeting agent component and an active agent component include sulfonyl groups, sulfhydryl groups, and the haloic acid and acid anhydride derivatives of carboxylic acids.
  • the targeting agent component and the therapeutic agent component may be covalently linked to one another through an intermediate linker.
  • the linker advantageously possesses two active groups, one of which is complementary to an active group on the targeting agent component, and the other of which is complementary to an active group on the active agent component.
  • the linker may suitably be a diacid, which will react with both compounds to form a diether linkage between the two residues.
  • other suitable active groups for forming linkages between pharmaceutically active moieties include sulfonyl groups, sulfhydryl groups, and the haloic acid and acid anhydride derivatives of carboxylic acids.
  • Suitable diacid linkers include oxalic, malonic, succinic, glutaric, adipic, pimelic, suberic, azelaic, sebacic, maleic, fumaric, tartaric, phthalic, isophthalic, and terephthalic acids. While diacids are named, the skilled artisan will recognize that in certain circumstances the corresponding acid halides or acid anhydrides (either unilateral or bilateral) are preferred as linker reprodrugs.
  • a preferred anhydride is succinic anhydride.
  • Another preferred anhydride is maleic anhydride.
  • Other anhydrides and/or acid halides may be employed by the skilled artisan to good effect.
  • Suitable amino acids include butyric acid, 2-aminoacetic acid, 3-aminopropanoic acid, 4- aminobutanoic acid, 5-aminopentanoic acid, 6-aminohexanoic acid, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
  • the acid group of the suitable amino acids may be converted to the anhydride or acid halide form prior to their use as linker groups.
  • Suitable diamines include 1, 2-diaminoethane, 1,3-diaminopropane, 1,4-diaminobutane, 1,5- diaminopentane, 1,6-diaminohexane.
  • Suitable aminoalcohols include 2-hydroxy-1-aminoethane, 3- hydroxy-1-aminoethane, 4-hydroxy-1-aminobutane, 5-hydroxy-1-aminopentane, 6-hydroxy-1- aminohexane.
  • Suitable hydroxyalkyl acids include 2-hydroxyacetic acid, 3-hydroxypropanoic acid, 4- hydroxybutanoic acid, 5-hydroxypentanoic acid, 5-hydroxyhexanoic acid.
  • the various linker groups can be designated either “weak” or “strong” based on the stability of the covalent bond which the linker functional group will form between the spacer and either the polar lipid carrier or the biologically active compound.
  • the weak functionalities include, but are not limited to phosphoramide, phosphoester, carbonate, amide, carboxyl-phosphoryl anhydride, ester and thioester.
  • the strong functionalities include, but are not limited to ether, thioether, amine, sterically hindered amides and esters.
  • Enzymatic release is also possible, but such enzyme-mediated modes of release will not necessarily be correlated with bond strength in such embodiments of the invention.
  • Spacer moieties comprising enzyme active site recognition groups, such as spacer groups comprising peptides having proteolytic cleavage sites therein, are envisioned as being within the scope of the present invention.
  • the linker moiety includes a spacer molecule that facilitated hydrolytic or enzymatic release of the active agent component from the targeting agent component.
  • the spacer functional group is hydrolyzed by an enzymatic activity found in the target vascular tissue, preferably an esterase.
  • the active therapeutic agent component which is linked to the targeting agent component, can be or comprise any agent that achieves the desired therapeutic result, including agents such as but not limited to the following, which can be used as an active agent component for a targeted therapeutic agent, as appropriate: a radionuclide (e.g., I 125 , I 123 , I 124 , I 131 or other radioactive agent, including but not limited to Lu 177 , Pb 212 , Tc 99 , Zr 89 , Y 90 , Ac 225 ); a chemotherapeutic agent (e.g., an antibiotic, antiviral or antifungal, including but not limited to toxins such as the epipolythiodioxopiperazine (ETP) class of fungal toxins); inhibitors, such as but not limited to chemotherapeutic inhibitors (e.g.
  • a radionuclide e.g., I 125 , I 123 , I 124 , I 131 or other radioactive agent, including but not
  • alkylating agents anthracyclines, cytoskeletal disruptors (taxanes), epothilones, histone deacetylase inhibitors, histone methyltransferase inhibitors, inhibitors of topoisomerase I, inhibitors of topoisomerase II, kinase inhibitors, nucleotide analogs and precursor analogs, peptide antibiotics , and platinum-based agents; an immune stimulatory agent (e.g., a cytokine); an anti-neoplastic agent: an anti-inflammatory agent; a pro-inflammatory agent; a pro- apoptotic agent (e.g., peptides or other agents to attract immune cells and/or stimulate the immune system); a pro-coagulant; a toxin (e.g., ricin, enterotoxin, LPS); an antibiotic; a hormone; a protein (e.g., a recombinant protein or a recombinant modified protein); an immune stimulatory agent,
  • chemotherapeutic agent is a chemical compound useful in the treatment of cancer.
  • examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), C
  • celecoxib or etoricoxib proteosome inhibitor
  • proteosome inhibitor e.g. PS341
  • bortezomib VELCADE®
  • CCI-779 tipifamib (R11577); orafenib, ABT510
  • Bcl-2 inhibitor such as oblimersen sodium (GENASENSE®)
  • pixantrone EGFR inhibitors
  • tyrosine kinase inhibitors such as gefitinib, imatinib, vemurafenib, and vismodegib
  • serine-threonine kinase inhibitors such as rapamycin (sirolimus, RAPAMUNE®)
  • anthracyclines famesyltransferase inhibitors such as lonafamib (SCH 6636, SARASAR®)
  • histone deacetylase inhibitors such as vorinostat and romidepsin
  • hydrazines such as
  • Chemotherapeutic agents as defined herein include “anti-hormonal agents” or “endocrine therapeutics” which act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer. They may be hormones themselves, including, but not limited to: anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON.cndot.toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole
  • a radionuclide or other radioactive agent can be used as the active agent component.
  • the targeting agent component delivers the radioactive agent in a tissue- specific manner, allowing local radiation damage and resulting in radiation-induced apoptosis and necrosis throughout the solid tumor or diseased tissue including in tumor cells, stromal calls, and endothelial cells of the tumor or throughout the diseased area.
  • an active agent that exhibits anti-fibrotic activity can be used to treat pulmonary fibrosis.
  • the active agent is effective to treat acute lung injury.
  • the targeting agent component delivers the agent in a highly precise manner directly into diseased tissue and therapeutic activity is enhanced because the ultra low dose that is administered is concentrated at the desired site where it can be most effective.
  • antisense oligonucleotides or other agents can be used as the active agent component, to alter, and particularly to inhibit, production of a gene in a targeted tissue, such as a gene that is overexpressed in a tumor tissue (e.g., an oncogene or a gene associated with neoplasm, such as c-Jun, c-Fos, HER-2, E2F-1, RAS, FAS, NF, BRCA), or a gene that is overexpressed in angiogenesis.
  • a tumor tissue e.g., an oncogene or a gene associated with neoplasm, such as c-Jun, c-Fos, HER-2, E2F-1, RAS, FAS, NF, BRCA
  • a gene that is overexpressed in angiogenesis e.g., an oncogene or a gene associated with neoplasm, such as c-Jun, c-Fos, HER-2, E2F-1
  • oligonucleotides or genes can be used to alter, and particularly to enhance, production of a protein in the targeted tissue, such as a gene that controls apoptosis or regulates cell growth; oligonucleotides or genes can also be used to produce a protein that is under- expressed or deleted in the targeted tissue, or to express a gene product that is directly or indirectly destructive to the neoplasm.
  • an anti-fibrotic agent can be used as the active agent.
  • Representative agents include rapamycin, troglitazone, therapeutic peptides such as Thy-1, and therapeutic antibodies such as those again TGF- ⁇ .
  • an anti-inflammatory agent can be used as the active agent.
  • Representative agents include a non-steroidal anti- inflammatory agent; a steroidal or corticosteroidal anti-inflammatory agent; or other anti- inflammatory agent (e.g., histamine).
  • the active agent can be an agent to alter blood pressure (e.g., a diuretic, a vasopressin agonist or antagonist, angiotensin).
  • pro- inflammatory agents can be used as active agents (e.g., to enhance angiogenesis or increase development of neovasculature, as described herein).
  • chemotherapeutic agents for neoplastic diseases can be used as the active agent component.
  • Representative agents include alkylating agents (nitrogen mustards, ethylenimines, alkyl sulfonates, nitrosoureas, and triazenes), antimetabolites (folic acid analogs such as methotrexate, pyrimidine analogs, and purine analogs), natural products and their derivatives (antibiotics, alkaloids, enzymes), hormones and antagonists (corticosteroids; adrenocorticosteroids, progestins, estrogens), and other similar agents.
  • the chemotherapeutic agent can be acytotoxic or cytostatic drugs.
  • Chemotherapeutics may also include those that have other effects on cells such as reversal of the transformed state to a differentiated state or those which inhibit cell replication.
  • Examples of known cytotoxic agents useful in the present invention are listed, for example, in Goodman et al., "The Pharmacological Basis of Therapeutics," Sixth Edition, A. G. Gilman et a.l, eds./Macmillan Publishing Co. New York, 1980.
  • taxol nitrogen mustards, such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard and chlorambucil; ethylenimine derivatives, such as thiotepa; alkyl sulfonates, such as busulfan; nitrosoureas, such as carmustine, lomustine, semustine and streptozocin; triazenes, such as dacarbazine; folic acid analogs, such as methotrexate; pyrimidine analogs, such as fluorouracil, cytarabine and azaribine; purine analogs, such as mercaptopurine and thioguanine; vinca alkaloids, such as vinblastine and vincristine; antibiotics, such as dactinomycin, daunorubicin, doxorubicin, bleomycin, mithramycin and mitomycin; enzymes, such as L-asparagina
  • Drugs that interfere with intracellular protein synthesis can also be used; such drugs are known to these skilled in the art and include puromycin, cycloheximide, and ribonuclease.
  • chemotherapeutic agents currently in use in treating cancer possess functional groups that are amenable to chemical crosslinking directly with an amine or carboxyl group of a targeting agent component.
  • functional groups that are amenable to chemical crosslinking directly with an amine or carboxyl group of a targeting agent component.
  • free amino groups are available on methotrexate, doxorubicin, daunorubicin, cytosinarabinoside, cis-platin, vindesine, mitomycin and bleomycin while free carboxylic acid groups are available on methotrexate, melphalan, and chlorambucil.
  • These functional groups, that is free amino and carboxylic acids are targets for a variety of homobifunctional and heterobifunctional chemical crosslinking agents which can crosslink these drugs directly to a free amino group.
  • Peptide and polypeptide toxins are also useful as active agent components, and the present invention specifically contemplates embodiments wherein the active agent component is a toxin.
  • Toxins are generally complex toxic products of various organisms including bacteria, plants, etc.
  • Examples of toxins include but are not limited to: ricin, ricin A chain (ricin toxin), Pseudomonas exotoxin (PE), diphtheria toxin (DT), Clostridium perfringens phospholipase C (PLC), bovine pancreatic ribonuclease (BPR), pokeweed antiviral protein (PAP), abrin, abrin A chain (abrin toxin), cobra venom factor (CVF), gelonin (GEL), saporin (SAP), modeccin, viscumin and volkensin.
  • ricin ricin A chain
  • PE Pseudomonas exotoxin
  • DT diphtheria toxin
  • an anti-inflammatory agent can be used as the active agent.
  • Representative agents include a non-steroidal anti-inflammatory agent; a steroidal or corticosteroidal anti-inflammatory agent; or other anti-inflammatory agent (e.g., histamine).
  • pro-inflammatory agents can be used as active agents (e.g., to enhance angiogenesis or increase development of neovasculature, as described herein).
  • Prodrugs or promolecules can also be used as the active agent.
  • a prodrug that is used as an active agent can subsequently be activated (converted) by administration of an appropriate enzyme, or by endogenous enzyme in the targeted tissue.
  • the activating enzyme can be co-administered or subsequently administered as another active agent as part of a therapeutic agent as described herein; or the prodrug or promolecule can be activated by a change in pH to a physiological pH upon administration.
  • prodrugs include Herpes simplex virus thymidine kinase (HSV TK) with the nucleotide analog GCV; cytosine deaminase ans t-fluorocytosine; alkaline phosphatase/etoposidephosphate; and other prodrugs (e.g., those described in Greco et al., J. Cell. Phys. 187:22-36, 2001; and Konstantinos et al., Anticancer Research 19:605-614, 1999; see also Connors, T.A., Stem Cells 13(5): 501-511, 1995; Knox, R.J., Baldwin, A. et al., Arch. Biochem.
  • HSV TK Herpes simplex virus thymidine kinase
  • the targeting agent component and/or the active agent component comprises a chelate moiety for chelating a metal, e.g., a chelator for a radiometal or paramagnetic ion.
  • the a chelator is a chelator for a radionuclide.
  • Radionuclides useful within the present invention include gamma-emitters, positron-emitters, Auger electron-emitters, X-ray emitters and fluorescence-emitters, with beta- or alpha-emitters preferred for therapeutic use.
  • radionuclides useful as toxins in radiation therapy include: 225 Ac, 89 Zr, 32 P, 33 P, 43 K, 47 Sc, 52 Fe, 57 Co, 64 Cu, 67 Ga, 67 Cu, 68 Ga, 71 Ge, 75 Br, 76 Br, 77 Br, 77 As, 77 Br, 81 Rb/ 81M Kr, 87M Sr, 90 Y, 97 Ru, 99 Tc, 100 Pd, 101 Rh, 103 Pb, 105 Rh, 109 Pd, 111 Ag, 111 In, 113 In, 119 Sb 121 Sn, 123 I, 125 I, 127 Cs, 128 Ba, 129 Cs, 131 I, 131 Cs, 143 Pr, 153 Sm, 161 Tb, 166 Ho, 169 Eu, 177 Lu, 186 Re, 188 Re, 189 Re, 191 Os, 193 Pt, 194 Ir, 197 Hg, 199 Au, 203 Pb, 211 At,
  • Preferred therapeutic radionuclides include 188 Re, 186 Re, 203 Pb, 212 Pb, 212 Bi, 109 Pd, 64 Cu, 67 Cu, 90 Y, 125 I, 131 I, 77 Br, 211 At, 97 Ru, 105 Rh, 198 Au and 199 Ag, 166 Ho, 225 Ac, 89 Zr, or 177 Lu.
  • Conditions under which a chelator will coordinate a metal are described, for example, by Gansow et al., U.S. Pat. Nos.4,831,175, 4,454,106 and 4,472,509.
  • 99m Tc can be used as a radioisotope for therapeutic and diagnostic applications (as described below), as it is readily available to all nuclear medicine departments, is inexpensive, gives minimal patient radiation doses, and has ideal nuclear imaging properties. It has a half-life of six hours which means that rapid targeting of a technetium-labeled antibody is desirable.
  • the therapeutic targeting agent includes a chelating agents for technium.
  • the therapeutic targeting agent can also comprise radiosensitizing agents, e.g., a moiety that increase the sensitivity of cells to radiation.
  • radiosensitizing agents include nitroimidazoles, metronidazole and misonidazole (see: DeVita, V. T. Jr. in Harrison's Principles of Internal Medicine, p.68, McGraw-Hill Book Co., N.Y. 1983, which is incorporated herein by reference).
  • the therapeutic targeting agent that comprises a radiosensitizing agent as the active moiety is administered and localizes in the endothelial call and/or in any other cells of the neoplasm. Upon exposure of the individual to radiation, the radiosensitizing agent is "excited" and causes the death of the cell.
  • the chelating ligand can be a derivative of 1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA) and 1-p-Isothiocyanato-benzyl-methyl- diethylenetriaminepentaacetic acid (ITC-MX).
  • DOTA 1,4,7,10-tetraazacyclododecanetetraacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • DTPA diethylenetriaminepentaacetic acid
  • ITC-MX 1-p-Isothiocyanato-benzyl-methyl- diethylenetriaminepentaacetic acid
  • Such groups include, e.g., benzylisothiocyanate, by which the DOTA, DTPA or EDTA can be coupled to, e.g., an amine group of the inhibitor.
  • the agent is an“N x S y ” chelate moiety.
  • N x S y chelates includes bifunctional chelators that are capable of coordinately binding a metal or radiometal and, preferably, have N2S2 or N3S cores. Exemplary NxSy chelates are described, e.g., in Fritzberg et al. (1988) PNAS 85:4024-29; and Weber et al.
  • a problem frequently encountered with the use of conjugated proteins in radiotherapeutic and radiodiagnostic applications is a potentially dangerous accumulation of the radiolabeled moiety fragments in the kidney.
  • the conjugate is formed using a acid-or base-labile linker, cleavage of the radioactive chelate from the protein can advantageously occur. If the chelate is of relatively low molecular weight, it is not retained in the kidney and is excreted in the urine, thereby reducing the exposure of the kidney to radioactivity.
  • active agents include agents that induce intravascular coagulation, or which damage the endothelium, thereby causing coagulation and effectively infracting a neoplasm or other targeted pathology.
  • enzymes activated by other agents e.g., biotin, activated by avidin
  • active agents can be used as active agents or as part of the therapeutic targeting agent.
  • the therapeutic targeting agents can be synthesized, by standard methods known in the art (e.g., by recombinant DNA technology or other means), to provide reactive functional groups that can form acid-labile linkages with, e.g., a carbonyl group of the ligand.
  • suitable acid-labile linkages include hydrazone and thiosemicarbazone functions. These are formed by reacting the oxidized carbohydrate with chelates bearing hydrazide, thiosemicarbazide, and thiocarbazide functions, respectively.
  • base-cleavable linkers that have been used for the enhanced clearance of the radiolabel from the kidneys, can be used. See, for example, Weber et al. 1990 Bioconjug.
  • the coupling of a bifunctional chelate via a hydrazide linkage can incorporate base-sensitive ester moieties in a linker spacer arm.
  • ester-containing linker unit is exemplified by ethylene glycolbis(succinimidyl succinate), (EGS, available from Pierce Chemical Co., Rockford, Ill.), which has two terminal N-hydroxysuccinimide (NHS) ester derivatives of two 1,4-dibutyric acid units, each of which are linked to a single ethylene glycol moiety by two alkyl esters.
  • One NHS ester may be replaced with a suitable amine-containing BFC (for example 2- aminobenzyl DTPA), while the other NHS ester is reacted with a limiting amount of hydrazine.
  • the resulting hyrazide is used for coupling to the targeting agent component, forming an ligand-BFC linkage containing two alkyl ester functions.
  • Such a conjugate is stable at physiological pH, but readily cleaved at basic pH.
  • Therapeutic targeting agents labeled by chelation are subject to radiation-induced scission of the chelator and to loss of radioisotope by dissociation of the coordination complex.
  • metal dissociated from the complex can be re-complexed, providing more rapid clearance of non-specifically localized isotope and therefore less toxicity to non-target tissues.
  • chelator compounds such as EDTA or DTPA can be infused into patients to provide a pool of chelator to bind released radiometal and facilitate excretion of free radioisotope in the urine.
  • a Boron addend such as a carborane
  • carboranes can be prepared with carboxyl functions on pendant side chains, as is well known in the art. Attachment of such carboranes to an amine functionality, e.g., as may be provided on the targeting agent component can be achieved by activation of the carboxyl groups of the carboranes and condensation with the amine group to produce the conjugate.
  • Such therapeutic agents can be used for neutron capture therapy.
  • RNAi is used.
  • "RNAi construct” is a generic term used throughout the specification to include small interfering RNAs (siRNAs), hairpin RNAs, and other RNA species which can be delivered ectopically to a cell, cleaved by the enzyme dicer and cause gene silencing in the cell.
  • small interfering RNAs or “siRNAs” refers to nucleic acids around 19-30 nucleotides in length, and more preferably 21-23 nucleotides in length.
  • siRNAs can be chemically synthesized, or derive by enzymatic digestion from a longer double-stranded RNA or hairpin RNA molecule.
  • an siRNA will generally have significant sequence similarity to a target gene sequence.
  • the siRNA molecules includes a 3' hydroxyl group, though that group may be modified with a fatty acid moiety as described herein.
  • the phrase "mediates RNAi” refers to (indicates) the ability of an RNA molecule capable of directing sequence-specific gene silencing, e.g., rather than a consequence of induction of a sequence-independent double stranded RNA response, e.g., a PKR response.
  • the RNAi construct used for the active agent component is a small- interfering RNA (siRNA), preferably being 19-30 base pairs in length.
  • the RNAi construct is a hairpin RNA which can be processed by cells (e.g., is a dicer substrate) to produce metabolic products in vivo in common with siRNA treated cells, e.g., a processed to short (19-22 mer) guide sequences that induce sequence specific gene silencing.
  • the treated animal is a human.
  • RNAi constructs contain a nucleotide sequence that hybridizes under physiologic conditions of the cell to the nucleotide sequence of at least a portion of the mRNA transcript for the gene to be inhibited (i.e., the”target” gene).
  • the double-stranded RNA need only be sufficiently similar to natural RNA that it has the ability to mediate RNAi.
  • the invention has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism or evolutionary divergence.
  • the number of tolerated nucleotide mismatches between the target sequence and the RNAi construct sequence is no more than 1 in 5 basepairs, or 1 in 10 basepairs, or 1 in 20 basepairs, or 1 in 50 basepairs. Mismatches in the center of the siRNA duplex are most critical and may essentially abolish cleavage of the target RNA. In contrast, nucleotides at the 3' end of the siRNA strand that is complementary to the target RNA do not significantly contribute to specificity of the target recognition.
  • Sequence identity may be optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Greater than 90% sequence identity, or even 100% sequence identity, between the inhibitory RNA and the portion of the target gene is preferred.
  • the duplex region of the RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 501C or 701C hybridization for 12-16 hours; followed by washing).
  • a portion of the target gene transcript e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 501C or 701C hybridization for 12-16 hours; followed by washing).
  • RNAi constructs can be carried out by chemical synthetic methods or by recombinant nucleic acid techniques. Endogenous RNA polymerase of the treated cell may mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vitro.
  • RNAi constructs may include other modifications, such as to the phosphate-sugar backbone or the nucleoside, e.g., to reduce susceptibility to cellular nucleases, improve bioavailability, improve formulation characteristics, and/or change other pharmacokinetic properties.
  • the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom.
  • Modifications in RNA structure may be tailored to allow specific genetic inhibition while avoiding a general cellular response to dsRNA (a APKR-mediated response@).
  • bases may be modified to block the activity of adenosine deaminase.
  • the RNAi construct may be produced enzymatically or by partial/total organic synthesis, any modified ribonucleotide can be introduced by in vitro enzymatic or organic synthesis.
  • RNAi constructs see, for example, Heidenreich et al. (1997) Nucleic Acids Res, 25:776-780; Wilson et al. (1994) J Mol Recog 7:89-98; Chen et al. (1995) Nucleic Acids Res 23:2661-2668; Hirschbein et al. (1997) Antisense Nucleic Acid Drug Dev 7:55-61).
  • MMI methylene(methylimin
  • the double-stranded structure may be formed by a single self-complementary RNA strand or two complementary RNA strands. RNA duplex formation may be initiated either inside or outside the cell.
  • the RNAi construct is designed so as not to include unmodified cytosines occurring 5' to guanines, e.g., to avoid stimulation of B cell mediated immunosurveillance.
  • the backbone linkages can be chosen so as titrate the nuclease sensitivity to make the RNAi sufficiently nuclease resistant to be effective in the tissue of interest (e.g., the neoplasm), but not so nuclease resistant that significant amounts of the construct could escape the tissue undegraded.
  • tissue of interest e.g., the neoplasm
  • RNAi constructs are available for gene silencing in the tissue of interest, but are degraded before they can enter the wider circulation.
  • the RNA may be introduced in an amount that allows delivery of at least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies per cell) of double-stranded material may yield more effective inhibition, while lower doses may also be useful for specific applications. Inhibition is sequence-specific in that nucleotide sequences corresponding to the duplex region of the RNA are targeted for genetic inhibition.
  • the subject RNAi constructs are siRNAs. These nucleic acids are around 19-30 nucleotides in length, and even more preferably 21-23 nucleotides in length, e.g., corresponding in length to the fragments generated by nuclease“dicing” of longer double-stranded RNAs.
  • the siRNAs are understood to recruit nuclease complexes and guide the complexes to the target mRNA by pairing to the specific sequences. As a result, the target mRNA is degraded by the nucleases in the protein complex.
  • the 21-23 nucleotides siRNA molecules comprise a 3' hydroxyl group.
  • siRNA molecules of the present invention can be obtained using a number of techniques known to those of skill in the art.
  • the siRNA can be chemically synthesized or recombinantly produced using methods known in the art.
  • short sense and antisense RNA oligomers can be synthesized and annealed to form double-stranded RNA structures with 2- nucleotide overhangs at each end (Caplen, et al. (2001) Proc Natl Acad Sci USA, 98:9742-9747; Elbashir, et al. (2001) EMBO J, 20:6877-88).
  • These double-stranded siRNA structures can then be directly introduced to cells, either by passive uptake or a delivery system of choice, such as described below.
  • the siRNA constructs can be generated by processing of longer double-stranded RNAs, for example, in the presence of the enzyme dicer.
  • the Drosophila in vitro system is used.
  • dsRNA is combined with a soluble extract derived from Drosophila embryo, thereby producing a combination. The combination is maintained under conditions in which the dsRNA is processed to RNA molecules of about 21 to about 23 nucleotides.
  • the siRNA molecules can be purified using a number of techniques known to those of skill in the art. For example, gel electrophoresis can be used to purify siRNAs. Alternatively, non-denaturing methods, such as non-denaturing column chromatography, can be used to purify the siRNA. In addition, chromatography (e.g., size exclusion chromatography), glycerol gradient centrifugation, affinity purification with antibody can be used to purify siRNAs.
  • gel electrophoresis can be used to purify siRNAs.
  • non-denaturing methods such as non-denaturing column chromatography
  • chromatography e.g., size exclusion chromatography
  • glycerol gradient centrifugation glycerol gradient centrifugation
  • affinity purification with antibody can be used to purify siRNAs.
  • Modification of siRNA molecules with fatty acids can be carried out at the level of the precursors, or, perhaps more practically, after the RNA has been synthesized. The latter may be accomplished in certain instances using nucleoside precursors in the synthesis of the polymer that include functional groups for formation of the linker-fatty acid moiety.
  • At least one strand of the siRNA molecules has a 3' overhang from about 1 to about 6 nucleotides in length, though may be from 2 to 4 nucleotides in length. More preferably, the 3’ overhangs are 1-3 nucleotides in length. In certain embodiments, one strand having a 3' overhang and the other strand being blunt-ended or also having an overhang. The length of the overhangs may be the same or different for each strand. In order to further enhance the stability of the siRNA, the 3' overhangs can be stabilized against degradation. In one embodiment, the RNA is stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides.
  • substitution of pyrimidine nucleotides by modified analogues e.g., substitution of uridine nucleotide 3' overhangs by 2'-deoxythyinidine is tolerated and does not affect the efficiency of RNAi.
  • the absence of a 2' hydroxyl significantly enhances the nuclease resistance of the overhang in tissue culture medium and may be beneficial in vivo.
  • the RNAi construct is in the form of a long double-stranded RNA.
  • the RNAi construct is at least 25, 50, 100, 200, 300 or 400 bases.
  • the RNAi construct is 400-800 bases in length.
  • the double-stranded RNAs are digested intracellularly, e.g., to produce siRNA sequences in the cell.
  • use of long double-stranded RNAs in vivo is not always practical, presumably because of deleterious effects which may be caused by the sequence-independent dsRNA response.
  • the use of local delivery systems and/or agents which reduce the effects of interferon or PKR are preferred.
  • the RNAi construct is in the form of a hairpin structure (named as hairpin RNA).
  • hairpin RNAs can be synthesized exogenously or can be formed by transcribing from RNA polymerase III promoters in vivo. Examples of making and using such hairpin RNAs for gene silencing in mammalian cells are described in, for example, Paddison et al., Genes Dev, 2002, 16:948-58; McCaffrey et al., Nature, 2002, 418:38-9; McManus et al., RNA, 2002, 8:842-50; Yu et al., Proc Natl Acad Sci U S A, 2002, 99:6047-52).
  • hairpin RNAs are engineered in cells or in an animal to ensure continuous and stable suppression of a desired gene. It is known in the art that siRNAs can be produced by processing a hairpin RNA in the cell.
  • the therapeutic targeting agent alone or in a composition, is administered in a therapeutically effective amount, which is the amount used to treat the neoplasm or to treat angiogenesis or unwanted development of neovasculature.
  • a therapeutically effective amount which is the amount used to treat the neoplasm or to treat angiogenesis or unwanted development of neovasculature.
  • the amount that will be therapeutically effective will depend on the nature of the neoplasm, neovasculature or angiogenesis, the extent of disease and/or metastasis, and other factors, and can be determined by standard clinical techniques.
  • in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges.
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • TISSUE ENGINEERING Because certain proteins have been identified as being prevalent on tumor endothelium, as described herein, methods are now available to create cell types in culture that are more similar to those in vivo. (See, e.g., Engelmann, K. Et al., Exp Ehye Res (2004) 78(3):573-8; Kirkpatrick, C.J. et al., biomol. Eng. (2002):19(2-6):211-7; Nugent, H.M. and Edelman, E.R., Circ. Res. (2003) 92(10):1068-780).
  • Tumor cells in vitro that are more similar to those in vivo, by virtue of producing similar panels of proteins on the endothelial surface, provide a better tool for assessing agents that may be useful in therapies such as the therapies described herein.
  • Cells can be modified, for example, by incorporation of nucleic acids or vectors expressing proteins that are produced in excess in neoplasms, compared to expression in normal cells. Such modified cells allow more accurate assessment of effects of a potential therapeutic agent on neoplasm cells.
  • the invention provides antibodies to certain targeted proteins, that can be used, for example, in the methods of the invention.
  • Aantibody,@ is described above.
  • the invention provides polyclonal and monoclonal antibodies that bind to a targeted protein.
  • Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a desired immunogen, e.g., the targeted protein or a fragment or derivative thereof.
  • the antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules directed against the targeted protein can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature, 256:495-497, the human B cell hybridoma technique (Kozbor et al. (1983) Immunol. Today, 4:72), the EBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques.
  • standard techniques such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature, 256:495-497, the human B cell hybridoma technique (Kozbor et al. (1983) Immunol. Today, 4:72), the EBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or triom
  • hybridomas The technology for producing hybridomas is well known (see generally Current Protocols in Immunology (1994) Coligan et al. (eds.) John Wiley & Sons, Inc., New York, NY). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with an immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds a polypeptide of the invention.
  • lymphocytes typically splenocytes
  • a monoclonal antibody to a targeted protein can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the targeted protein, to thereby isolate immunoglobulin library members that bind to the targeted protein.
  • Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAPJ Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Patent No.
  • recombinant antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.
  • antibodies of the invention can be used in the methods of the invention.
  • an antibody specific for a targeted protein can be used in the methods of the invention to image a neoplasm, in order to evaluate the abundance and location of the neoplasm.
  • Antibodies can thus be used diagnostically to, for example, determine the efficacy of a given treatment regimen, by imaging before and after the treatment regimen.
  • EXAMPLES The proteomic mapping of vascular endothelium and its caveolae, reveals tissue-specific delivery targets that enable precision drug delivery to the site of desired pharmacologic activity. Specific targeted agents can now penetrate rapidly and actively into a single diseased tissue to increase therapeutic potency at ultra-low doses as the direct result of improved precision drug delivery. Targeting vascular endothelial cell proteins expressed on the outer surface of the cellular membrane permits precision delivery to, treatment of, and imaging of diseased tissue in vivo. EXAMPLE 1
  • VEGF Mouse VEGF (B20-4.1.1, G6-31) were provided by Dr. Orlandoe Ferrara (Genentech, San Francisco, CA). Herceptin (trastuzumab), doxorubicin, and Taxotere (doxetaxel) were obtained via UCSD pharmacy. Mouse IgGs were obtained from Southern Biotech (Birmingham, AL). VEGF antibodies (B20-4.1.1 and G6-31) were a gift from Genentech (South San Francisco, CA). mAnnA1 and mAPP2 antibodies were made as described previously (Oh et al., 2007; Oh et al., 2004; Oh et al., 2014). Cisplatin was obtained from Sigma-Aldrich (St. Louis, MO), as were other chemicals and reagents unless otherwise noted.
  • mice Female nu/nu athymic nude, C57b6 and FVB mice from either Charles River Laboratories (Wilmington, MA) or Jackson Laboratories (Bar Harbor, ME) were used for the dorsal skinfold implantations and for donor tissues. We used mice that were >25g for both the chambered mice and the donor tissues.
  • BT-474 (Cat# HTB-20- ATCC, Manassas, VA) were maintained in Hybridoma-SFM supplemented with L- Glutamine (2mM), Penicillin (100 U/ml), Streptomycin (100 U/ml), Sodium Pyruvate (1 mM) (Invitrogen, Carlsbad, CA) and 10% heat inactivated FBS (Omega Scientific, Tarzana, CA).
  • TRAMPC2 (Cat#CRL-2731– ATCC, Manassas, VA) cells were maintained in Dulbecco's modified Eagle's medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose supplemented with 0.005 mg/ml bovine insulin and 10 nM dehydroisoandrosterone, 90%; fetal bovine serum, 5%; Nu-Serum IV, 5%.
  • the histone H2B-GFP was subcloned into the SalI/HpaI sites in the LXRN vector (Clontech, Palo Alto, CA) using SalI and blunted NotI sites from the BOSH2BGFPN1 vector (Kanda, Sullivan, & Wahl, 1998).
  • the monovalent cherry (mCherry) vector was created from the H2B-GFP vector by cloning the mCherry gene (a kind gift from Dr. Roger Tsien, UCSD) to replace the GFP gene.
  • GP2-293 cells were infected with VSV and the H2B-GFP or H2B-mCherry containing virus to produce viable virus.
  • N202, BT-474, TRAMPC2 and LLC1 cells were transduced with the viable virus to stably incorporate the H2B-GFP or H2B-mCherry gene.
  • the transduced cells were FACs sorted twice to ensure 100% of the cells stably expressed the H2B-GFP or H2B-mCherry protein.
  • mice usually athymic nude mice (25-30 g body weight), were anesthetized (7.3 mg ketamine hydrochloride and 2.3 mg xylazine per 100 g body weight, intraperitoneal injection) and placed on a heating pad. A titanium frame was placed onto the dorsal skinfold of mice to sandwich the extended double layer of skin. A 15 mm diameter full-thickness circular layer of skin was then excised.
  • Tumor spheroids were formed by plating 50,000 cells onto 1% agar-coated 96-well non-tissue culture treated flat bottom dishes (20 ⁇ l cells in 100 ⁇ l medium) and centrifuging 4 times at 2000 rpm for 15 min, rotating the dish after every centrifugation. The cells were incubated an additional 3-7 days (depending on cell type) at 37 o C in 5% CO2 in air to form tight spheroids.
  • BT-474 cells required 500,000 cells in the presence of Matrigel (BD Bioscience, San Diego) (1:1 cell volume dilution) to form spheroids in culture.
  • the tumor spheroids were implanted in the window chamber directly onto the exposed dorsal skin either alone or with lung (for LLC1) or mammary (lactating female mammary fat pad for N2O2 and BT-474) tissue which was excised from a donor mouse and minced into small pieces in Penicillin (10,000 ⁇ g/ml– Streptomycin (10,000 ⁇ g/ml) solution. Tumors were allowed to vascularize over 7- 14 days depending on model before being tested for vascular leakiness and response to therapy. Mice with BT-474 tumors were supplemented with intramuscular injection of estrogen (20 ⁇ g, twice weekly).
  • mice were injected intravenously via tail vein with 1 x 10 4 13762 Mat B III mammary adenocarcinoma cells.
  • Rat body weights were monitored over time. Body weights of healthy non-tumor bearing rats were also monitored for comparison.
  • female Fisher rats were injected intravenously via tail vein with 1 x 10 6 13762 Mat B III mammary adenocarcinoma cells.
  • Tumor Growth Tumors were imaged using intravital fluorescence microscopy, as described (Borgstrom et al., 2013; Oh et al., 2007; Oh et al., 2014). Tumor growth was analyzed off-line from the recorded, digital, grayscale 0-to-256 images using Image-Pro Plus (Media Cybernetics, Bethesda, MD). Tumor growth was determined in 2 ways, by measuring the area with fluorescence signal from the GFP or CFP expressing tumor cells or by quantifying the cumulative fluorescence signal for the tumor over time. Tumor area is measured by counting the number of pixels with a grayscale intensity above 75, thereby making it easier to reliably follow irregularly shaped tumors. The cumulative tumor fluorescence signal was measured by signal summation of all pixels over 75. All growth curves are normalized to the tumor on day 0. In all cases, growth measured by area or aggregate fluorescence signal were found to be very similar so only one of the results is usually shown.
  • VEGF antibody and Herceptin were fluorescently labelled using AlexaFluor 488 or 568 as per manufacturer’s instruction (Invitrogen, Carlsbad, CA).
  • mAnnA1 and mAPP2 antibodies were labelled as previously described (Oh et al., 2007; Oh et al., 2004; Oh et al., 2014). Size exclusion chromatography was used to separate free dye from labelled antibody (EconoPak 10 DG, Bio Rad, Hercules, CA).
  • Targeting antibodies mAnna1 or mAPP2
  • doxorubicin, docetaxel, and antibody-conjugatable maytansinoids (DM1), and cisplatin were conjugated to mAnnA1 to create antibody-drug conjugates, and both drug and antibody binding activity were maintained.
  • Cisplatin was conjugated as in (Deng et al., 2013; Shen et al., 2005). Briefly, carboxylic acid groups have been introduced onto dextran molecules by chemical modification (Shen et al., 2005).
  • Standard acid activation chemistry is then used to attach the modified dextran to free amines on the antibody (Shen et al., 2005).
  • the free acid groups remaining on the dextran molecule are used to chelate and carry cisplatin, the amount of which (Wakankar et al., 2010) is determined spectroscopically as in (Deng et al., 2013).
  • Doxorubicin was immunoconjugated and quantified as per (Griffiths et al., 2003).
  • DM1 was conjugated using standard linkage chemistry (see for example (Burris, Tibbitts, Holden, Sliwkowski, & Lewis Phillips, 2011; Chari et al., 1992; Chari, Miller, & Widdison, 2014; Shao et al., 2018; Wakankar et al., 2010).
  • Radiolabelling of antibodies with 125 I was performed as described previously (D. P. McIntosh et al., 2002). Briefly, affinity purified antibodies were conjugated to 125 I using Iodogen as described (D.P.
  • dendrimers Prior to antibody attachment, dendrimers were modified up to 30-40% with particular moiety/chelator for coupling of each radioisotope. Remaining primary amines on dendrimer surfaces were blocked by acetic anhydride.
  • PAMAM dendrimers were labeled with fluorophore (AlexaFluor-488 or AlexaFluor-568) using corresponding N-hydroxysuccinimide (NHS)-activated esters reactive toward primary amines of dendrimers. Unmodified primary amines on dendrimer surface were then shielded by reaction with acetic anhydride. Fluorophore-labeled dendrimers were purified by size exclusion chromatography before antibody functionalization.
  • the surface of PAMAM dendrimers were modified up to 30-40% with N- succinimidyl-3-(4-hydroxyphenyl)propionate.
  • Iodine was introduced by electrophilic substitution onto aromatic ring of p-hydroxyphenyl residues.
  • the degree of 125 I incorporation was estimated by mass spectrometry using nonradioactive“cold” iodine.
  • Conditions were optimized for maximum iodine incorporation of 125 I. We achieved specific activities ranging from 65 ⁇ Ci/ ⁇ g to 2 mCi/ ⁇ g ( ⁇ 30 atoms of 125 I per nanoparticle).
  • fluorophore-labeled and/or radionuclide-loaded PAMAM dendrimers were conjugated to mAnnA1 or control IgG antibodies through maleimide/thiol chemistry.
  • Formulations with a molecular substitution ratio (MSR) of dendrimer to antibody at 1.2:1, and 4:1 were evaluated. All formulations were characterized for immunoreactivity using saturation binding assay on recombinant AnnA1 protein, where immunoreactive fraction was determined by extrapolation of binding to infinite antigen excess.
  • the desired stoichiometry of the conjugates was confirmed by high-performance liquid chromatography (HPLC) on a size exclusion column and subjected to purification, if necessary.
  • HPLC high-performance liquid chromatography
  • Fluorescence Confocal Microscopy Confocal microscopy was used to acquire dual fluorescence images via a Nikon E2000 microscope (20x and 60x objective lens) equipped with a Perkin Elmer UltraView 5ERS confocal system with an Orca ER camera. To construct movies, dual color images were taken every second; exposures for a single fluorophore were kept under 400 msec.
  • IVM Tumor uptake of antibody.
  • IVM is a powerful imaging modality that complements gamma- scintigraphy and CT- SPECT imaging, by providing greater detail to permit live, dynamic imaging of antibody binding to the EC surface as well as direct visualization of transport across the EC barrier and accumulation in the tissue interstitium and parenchyma. IVM enables real time imaging to visualize tumor penetration of different therapies.
  • Her2/neu, 13762 metastatic rat mammary adenocarcinoma, TRAMPC2 transgenic model were anaesthetized and injected via the tail vein with 125 I-labeled monoclonal antibody (1-5 ⁇ g IgG; 10 ⁇ Ci/ ⁇ g) before being subjected to planar gamma scintigraphic imaging and SPECT-CT. After whole body imaging, in some cases, normal organs and target tissue (i.e. tumors or lung) were excised for planar imaging ex vivo.
  • ADC caveolae-targeting antibody-drug conjugates
  • Fig. 1 shows a dose-dependent impact with minimal benefit at 0.2 ⁇ g/kg of doxorubicin in the ADC, clear tumor regression at 2 ⁇ g/kg and eradication at 20 ⁇ g/kg. Any fluorescence signal observed after 5 days was not in intact nuclei, but as remnants of cellular debris.
  • Cisplatin is a widely used chemotherapy for treating solid malignancies. It has been used to treat various types of cancers, including sarcomas, some carcinomas (e.g. small cell lung cancer, squamous cell carcinoma of the head and neck, and ovarian cancer), lymphomas, bladder cancer, cervical cancer, germ cell tumors, prostate and breast cancer. Unfortunately, cisplatin has several serious side effects that can limit its use. We conjugated cisplatin to targeting mAnnA1 antibodies to reduce toxicity and boost therapeutic potency through improved tumor delivery and assessed the ability to eradicate tumors in IVM models.
  • Fig. 3 shows IVM images of a dose response study of the cisplatin-CMD-mAnnA1 in a mammary tumor model system. Results show mammary tumor regression and complete destruction after a single injection at a low cisplatin dose (2.2 and 7.2 ⁇ g/kg cisplatin) in the conjugate. The control non-specific IgG-immunoconjugate at 7.2 ⁇ g/kg cisplatin had no effect. Both 10-fold lower doses (0.22 and 0.72 ⁇ g/kg) stopped tumor growth more effectively than 1000-fold greater dose of 5 mg/kg cisplatin alone.
  • cisplatin-CMD-mAnnA1 also eradicated TRAMP C2 prostate tumor spheroids, exhibiting a dramatic increase in therapeutic potency at low doses even when compared to docetaxel (row 2) or doxorubicin (row 3).
  • Significant retardation of growth was observed with the mAnnA1-doxorubin conjugates (row 3) with less robust tumor eradication compared to the mAnnA1-docetaxel conjugate (row 2).
  • Rats were treated approximately 2 weeks after cell inoculation with 100 ⁇ g/kg mAnnA1- DM1.
  • the percent body weight on day of treatment for no-tumor control and tumor-bearing animals are shown in Fig. 5.
  • a cohort of rats received one dose of 100 ⁇ g/kg mAnnA1-DM1 while another cohort received two doses one week apart (indicated by *).
  • Non-treated tumor-bearing animals died within 7 days. Tumor-bearing animals dosed once with the ADC did not survive beyond 20 days (0 of 3 survived), while animals dosed twice survived to 100 days (3 of 3 survived) when the experiment reached endpoint.
  • Figs. 7a and b confirm mAnnA1 binds tumor ECs but not normal lung blood vessels in situ as expected in this model; using a biotinylated form of mAnnA1, paraffin sections of neoplastic and normal lung tissues further show the perfused antibody preferentially localizes to tumor blood vessels (Fig.7 a-b).
  • Fig. 7j shows the rapid time course of tumor uptake and blood clearance.
  • overall tumor accumulation of the radiotracer quickly exceeded peak blood levels measured immediately after injection ( ⁇ 7 %ID/gblood). Indeed, more than 50% of the entire injected dose can accumulate in tumors with a total weight > 1.5g.
  • the tissue-targeting index for tumors exceeded 1 at each time point measured, while remaining ⁇ 1 for all other organs tested.
  • TTI tumor indicated tumor concentrations of 125 I were 6.0 ⁇ 2.1 times greater than concurrent blood levels (Fig. 7k).
  • Fig. 7k As additional tumor uptake further reduced blood levels of 125 I-mAnnA1, this value increased steadily reaching 53 ⁇ 4.2 by 24 hrs (Fig. 7k).
  • TTI tissue selectivity index
  • mAnnA1 To more directly quantify the targeting and retention efficiency as well as the concentrating power of the caveolae pump via mAnnA1, we also determined the standardized uptake value (SUV) (Fig. 7m) and the Tissue Concentration Power Index (TCPI) (Fig. 7n).
  • the SUV specifically calculates how much more concentrated mAnnA1 is in specific tissues such as tumors relative to its equal distribution throughout the whole body. It is a useful way to: i) quantify the efficiency with which a probe concentrates in a tissue; and ii) the degree of a probe’s specific uptake and retention.
  • the TCPI quantifies the ability of different tissues, each with their unique uptake mechanisms, to concentrate a circulating test probe beyond its maximum concentration in the blood immediately after intravenous injection. It is calculated as the ratio of tissue uptake normalized to the maximum blood concentration (%ID/g tissue divided by max %ID/g blood). Peak blood level is a guiding parameter for normalizing delivery, because it defines the initial and maximum blood-to-tissue concentration gradient, which drives the probe passively across the vascular wall.
  • the TCPI for mAnnA1 in tumors shows probe accumulation in tumors at 1 and 24 hrs is 3-fold and 5-fold greater, respectively, than maximum levels in the blood. In contrast, TCPIs for other organs and antibodies remained well under 1 at all time points (Fig. 7n and data not shown).
  • tissue were harvested after 2 hrs circulation and results shows overwhelming accumulation of signal in tumor- bearing lungs as compared to normal lung with no tumors (Fig.8). Moreover, a significant amount of signal remained in the blood of normal animals at 2 hrs ( ⁇ 60%), as compared to tumor-bearing animals ( ⁇ 10%).
  • mAnnA1 125 I- mAnnA1 (3 ⁇ g) was injected i.v. into female Her2/Neu mice, a well-established mouse mammary tumor model (Quaglino, Mastini, Forni, & Cavallo, 2008). Expressing the Neu (Erbb2) gene under the transcriptional control of a mouse mammary tumor virus promoter/enhancer, these mice develop spontaneous mammary tumors (Guy, Edinburgh, & Muller, 1992; Guy, Webster, et al., 1992; Reilly et al., 2000).
  • Tumor accumulation of 125 I-mAnnA1 produced a robust and highly localized tumor signal in SPECT/CT images captured as early as 1 h post-injection (Fig. 9).
  • Region of interest analysis shows the tumor-specific signal persists even 24 hrs post-injection with little to no apparent accumulation in off-target organs such as the liver (Fig. 9 a-d, i), unlike as isotype matched 125 I-IgG control (Fig. 9 f-h), note the different imaging scales to enhance detection of the control IgG signal).
  • the data shows very specific, robust and rapid targeting of multiple solid tumors.
  • the results from the IVM and non-IVM mammary tumor models show that caveolae pumping is useful to delivery precisely payloads across the EC barrier, effectively concentrating them in the tumor interstitium and parenchyma where a tumor cell killing agent could be most effective. It does so in small drug-resistant IVM tumors, spontaneous tumors in immunocompetent transgenic mice, and also in large tumor burden hard-to-treat metastatic models of disease.
  • AnnA1 is specifically exposed to the blood only on tumor endothelium and readily accessible to i.v. injected antibodies.
  • the Annexin A1 protein is known to be expressed intracellulary in other cell types (e.g. neuronal, endocrine, some leukocyotes, but not normal endothelium (Gerke & Moss, 2002)). However, it is not externalized on the cell surface, nor is it in contact with the blood.
  • mAnnA1 vascular endothelial growth factor
  • Anti-VEGF antibodies suppresses the growth of new blood vessels by inhibiting cell signaling pathways that promote angiogenesis.
  • Anti-angiogenic drugs here exemplified by anti-VEGF antibodies, neutralize the function of VEGF primarily expressed by tumor and immune (macrophage) cells inside the tumors, and importantly, are located within the tumor interstitium and extracellular matrix surrounding all cells within the tumor.
  • this bifunctional antibody is comprised of a precision delivery moiety (AnnA1 antibody) and a therapeutic moiety (anti-VEGF antibody).
  • Bevacizumab The humanized form of the anti-VEGF monoclonal antibody is known as bevacizumab (marketed as Avastin).
  • Bevacizumab was used as a first-line of therapy for metastatic colorectal cancer, thus validating the idea that VEGF is a key mediator of tumor angiogenesis and that blocking the formation of new blood vessels is an effective strategy to treat solid tumors.
  • Administration of bevacizumab frequently leads to adverse side effects because of systemic exposure.
  • Main effects include hypertension and a heightened risk of bleeding. Bowel perforation has been reported, and fatigue and infection are also common. These adverse events are largely avoided in opthalmological use since the drug is introduced directly into the eye, thus directly accessing its target and minimizing any effects on the rest of the body.
  • VEGF vascular endothelial growth factor
  • N202 mammary tumor cells
  • Fig.11A and B we measured the uptake of mAnnA1-mVEGF as compared to non-specific IgG-mVEGF control dual-antibody conjugate.
  • Antibody conjugates were labeled with GFP and injected into the tail vein of mice that were then imaged over the course of hours and days to assess accumulation of fluorescent signal within the tumor (Fig. 11A). Results indicate 100-fold more mVEGF uptake by the tumor (based on fluorescent intensity) when conjugated to mAnnA1 as opposed to the non-specific IgG isotype matched control (Fib.11B).
  • Fig. 11C shows that the mAnnA1-mVEGF conjugate had little effect on halting tumor growth at the equivalent of 10 ⁇ g/kg mVEGF, but significantly retarded growth at 30 ⁇ g/kg compared to 5 mg/kg of mVEGF alone. Indeed, tumor growth was not affected with administration of mVEGF alone, even at this high dosage, when compared to the untreated tumor control (top 2 rows). In contrast, comparison of tumor size from Day 0 to 14 indicates that administration of 0.1 or 0.3 mg/kg of mAnnA1-mVEGF essentially stops tumor growth (bottom 2 rows; Fig. 11C). The boost in potency of a therapeutic antibody with anti-angiogenic activity was significant and unexpected.
  • Herceptin binds to HER2 growth factor receptors on the surface of breast cancer cells effectively inhibiting their functions, including cell signaling, ultimately, causing cancer regression. Herceptin is frequently used in the clinic and considered a minilified by traszutumab, which functions through specific recognition and inhibition of the critical tumor cell surface growth factor receptor Her2/neu (also called ErbB2) which turns off one or more key signaling cascades (e.g.
  • AKT map kinase
  • Traszutumab when bound at the tumor cell surface, induces immune cells to kill that cell, and thereby, initiates inherently antibody-dependent cell-mediated cytotoxicity. It also induces and suppresses specific gene expression that has affects outside the tumor cell and inhibits tumor growth by several mechanisms, including inhibiting angiogenesis.
  • the dual antibody conjugate we generated (mAnnA1-Her2) is comprised of a therapeutic moiety and a precision delivery moiety, both of which bind tumor-specific antigens, albeit at different locations– inherently accessible EC surfaces (AnnA1) versus actual breast cancer cells (Her2 receptors).
  • mAnnA1-Herceptin conjugates were compared in a IVM model with spheroids of GFP-tagged human BT474 (HER2 positive) cells implanted into human donor mammary tissue.
  • Fig. 12 shows that the mAnnA1-Herceptin conjugate had little effect at 30 ⁇ g/kg equivalent dose of Herceptin, stopped BT474 tumor growth at 100 ⁇ g/kg, and induced substantial regression at 300 ⁇ g/kg.
  • the boost in Herceptin potency was dramatic and surprising (>100- fold as per molar equivalence).
  • the above studies represent a direct side-by-side comparison of tumor uptake and efficacy resulting from two very different targeting strategies; namely, targeting the caveolae pumping system to improve precision delivery to enhance therapeutic potency versus targeting a cell surface tumor antigen. It thus appears that caveolae-targeting can enhance the therapeutic potency of other antibodies as a direct consequence of improved delivery that concentrates low doses inside tumors where it can be most effective.
  • the mAnnA1-mVEGF and mAnnA1-Herceptin conjugates represent a unique class of bifunctional antibodies optimized and targeted for transvascular transport to enhance the potency of low doses of the therapeutic antibody as a result of achieving precision delivery.
  • APP2 targeting antibodies mAPP2
  • ALI acute lung injury
  • idiopathic pulmonary fibrosis a staggering number of biological molecules have been implicated as therapeutic targets for pulmonary fibrosis.
  • TGF- ⁇ transforming growth factor-beta
  • TGF- ⁇ is a multifunctional cytokine that has been implicated as a‘master switch’ in induction of fibrosis in many organs including the lung (Sime & O'Reilly, 2001).
  • the TGF- ⁇ 1 isoform is thought to play the most significant role in wound healing and subsequent fibrosis (Flanders, 2004).
  • TGF- ⁇ and TGF- ⁇ -responsive genes are upregulated in lungs of patients with IPF (Kaminski et al., 2000; Konigshoff et al., 2009; Lazenby, Crouch, McDonald, & Kuhn, 1990; Tager et al., 2004; Vyalov, Gabbiani, & Kapanci, 1993; H. Y.
  • TGF- ⁇ 1 Overexpression of active TGF- ⁇ 1 in rat lung induces a dramatic fibrotic response (Sime, Xing, Graham, Csaky, & Gauldie, 1997), whereas inhibiting TGF- ⁇ signaling pathways can prevent bleomycin-induced pulmonary fibrosis (Anscher, Thrasher, Rabbani, Teicher, & Vujaskovic, 2006; Ask et al., 2006; du Bois, 2010; Giri, Hyde, & Hollinger, 1993; Kim et al., 2005; Pittet et al., 2001; Wilson et al., 2010; K.
  • TGF- ⁇ is chemotactic for fibroblasts and myofibroblasts (Postlethwaite, Keski-Oja, Moses, & Kang, 1987), stimulates differentiation of fibroblasts into myofibroblasts while suppressing myofibroblast apoptosis (H. Y.
  • TGF- ⁇ neutralizing antibodies have entered clinical trials for treatment of surgical scarring, sclerosis, and IPF (Ask et al., 2006; Denton et al., 2007; Khaw et al., 2007; Siriwardena et al., 2002).
  • TGF- ⁇ is clearly an excellent therapeutic candidate, inhibiting its function ubiquitously in the body present many problems. TGF- ⁇ is ubiquitously expressed, plays important roles in immune function, wound and cartilage repair (Blaney Davidson, van der Kraan, & van den Berg, 2007) and blood vessel stability (Sounni et al.), and can act as both a tumor suppressor and a tumor promoter (Yang & Moses, 2008).
  • TGF- ⁇ inhibitors are perinatally lethal due to severe inflammatory responses (Kulkarni et al., 1993) and a wide range of defects in normal organ development (Kaartinen et al., 1995; Sanford et al., 1997) and vasculogenesis (Martin et al., 1995). Though manipulating levels of TGF- ⁇ may have significant therapeutic value, there is much concern about the danger of off-target toxic effects (Blaney Davidson et al., 2007; Maher, Wells, & Laurent, 2007; Prud'Neill, 2007). Precision delivery of TGF- ⁇ inhibitors inside lungs could be a powerful new therapeutic approach because higher concentrations inside lungs could be achieved and broad effects on normal physiological functions of TGF- ⁇ in other parts of the body would be limited.
  • Fig. 13A Targeted delivery of mAPP2-mTGF- ⁇ prevented activation of pSMAD2 (Fig. 13A), a key mediator of TGF- ⁇ signaling.
  • Fig. 13B The anti-fibrotic effect of this targeted bifunctional immunoconjugate was also evident as shown through IHC analysis (Fig. 13B) and measurement of lung collagen content (Fig. 13C).
  • Fig. 13B Comparison of collagen deposition using Trichrome staining
  • Fig. 13C morphometric analysis revealed that the response to treatment was improved for the caveolae-targeted mAPP2-mTGF- ⁇ immunoconjugate than to mTGF- ⁇ alone.
  • Thy-1 is a fibrosis suppressor which modulates critical aspects of the fibrogenic phenotype, including proliferation, cytokine and growth factor expression and responsiveness, migration, myofibroblastic differentiation, and cell survival (Hagood et al., 2002; Hagood et al., 1999; Hagood et al., 2005; Sanders, Kumbla, & Hagood, 2007).
  • the effects of Thy-1 on the myofibroblast phenotype are broad, including: decreased expression of a number of muscle-specific proteins and myogenic transcription factors, inhibition of contractility, and promotion of apoptosis.
  • Thy-1 because key functions of Thy-1 are recapitulated by administering soluble Thy-1, Thy-1 itself may have therapeutic benefit (Zhou, Hagood, Lu, Merryman, & Murphy-Ullrich, 2010). There is significant evidence that harnessing the anti-myofibroblastic effects of Thy-1 may allow for a therapeutic“phenotype switch” in lung myofibroblasts, and thus either halt the progression or speed the resolution of lung fibrosis. Soluble Thy-1 (sThy-1) has been shown to mediate similar effects to endogenous expression (Zhou et al., 2010), it may be possible to administer sThy-1, or its derivative, as a therapeutic agent.
  • Nanotechnology has the potential to offer paradigm-shifting solutions to improve the outcome of diagnosis and therapy for patients suffering from cancer and other diseases.
  • Nanomedicine, or the use of nanoscale (10– 200 nm) constructs for therapeutic delivery is emerging as a powerful tool in cancer care.
  • Significant advances in nanomaterials and nanotechnology have paved the way for several carriers, such as dendrimers, liposomes and polymeric micelles, for clinical use.
  • the goal is to enhance the safety and efficacy of therapeutic agents through encapsulation or other attachment (covalent or non-covalent) with carriers to form nanoparticles (NP).
  • Some advantages afforded for drug delivery using NP include prolonged blood circulation time, increased loading capacity, improved stability and slower release time of the drug or active agent.
  • NP have been designed as nanocarriers to improve the delivery of therapeutic and imaging agents, but have thus far met with limited success (see for example, (Bae & Park, 2011; Chen, Ehlerding, & Cai, 2014; Lazarovits et al., 2015; Min, Caster, Eblan, & Wang, 2015; Park, 2013; Wang et al., 2012; Wilhelm et al., 2016; Wolfbeis, 2015; J. Xu et al., 2017).
  • Multi-functional NP for multi-modality imaging and therapy are especially suited for image-guided drug delivery (Cavalieri, Zhou, & Ashokkumar, 2010; Foy et al., 2010; Homan et al., 2010; Koning & Krijger, 2007; Myhr, 2007; Peng et al., 2011; L. Zhang et al., 2010).
  • NP may be particularly useful for small, rapidly excreted agents by increasing their residence time in the circulation and thus, their opportunity to reach target tissue (Farokhzad & Langer, 2009). Although NP size helps reduce unwanted rapid clearance from the blood, its size greatly hinders transport across biological interfaces.
  • NP neuropeptides
  • NP-based targeting and delivery strategies to overcome in vivo barriers to improve efficiency, efficacy and reduce side effects (Anchordoquy et al., 2017; Dawidczyk, Kim, et al., 2014; Dawidczyk, Russell, & Searson, 2014; Wilhelm et al., 2016).
  • nanostructures such as gold particles and nanostreptabodies (biotin-engineered antibody fragments on a streptavidin scaffold with a defined capacity for additional biotinylated payloads).
  • nanostreptabodies biotin-engineered antibody fragments on a streptavidin scaffold with a defined capacity for additional biotinylated payloads.
  • PAMAM poly(amidoamine)
  • Poly(amidoamine) (PAMAM) dendrimers are the most extensively studied in their class (Tomalia, 1991; Tomalia, Reyna, & Svenson, 2007; Wei et al., 2007) of hyper-branched, well- defined, monodisperse polymers with a highly uniform size and molecular weight.
  • the abundance of terminal groups that exponentially increases with each generation (Fig.16A) provides a large capacity for attachment of imaging agents and radiopharmaceuticals (Tomalia et al., 2007) (Kobayashi & Brechbiel, 2004).
  • PAMAM capable of flexible derivatization
  • various metallic imaging agents Gd(3+)
  • PAMAM scaffold was also used in VEGF-targeted boron neutron capture therapy (Backer et al., 2005).
  • PAMAM dendrimers can be excellent carriers for radionuclides, toxic metals and other therapeutic agents.
  • mAPP2 aminopeptidase 2
  • mAPP2 a target protein concentrated in lung vascular EC caveolae
  • two different radiolabeled and uncloaked (non- PEGylated) dendrimers assessed the ability of each caveolae-targeted immunoconjugate to target lung tissue after i.v. injection.
  • Both ⁇ -scintigraphy and SPECT-CT imaging showed robust lung uptake of both mAPP2-G5 and mAPP2-G4 PAMAM dendrimers (Fig. 16, panels B, D and E), whereas the control untargeted NP, as expected, accumulated rapidly in the liver and spleen (see panels A, C and F).
  • the caveolae pumping system in the lung and the RES of the liver and spleen were very robust, efficient and complete in accumulating their respective NPs. Most to nearly all of the uptake occurred for both in the first 30 min. The natural tropism of these uncloaked NP to the RES was largely avoided with conjugation to mAPP2.
  • the caveolae pumping system can compete favorably with the RES and be robust enough to obviate RES uptake of NP and re-target them specifically to a single tissue (i.e. lung). Further optimization can reduce RES uptake by cloaking (PEGylation) of the NP.
  • the antibody-drug conjugate as described in Example 3 that we generated using carboxymethyl dextran (CMD) as a carrier for cisplatin was able to successfully eradicate tumors at ultra low doses as the result of improved targeted drug delivery.
  • CMD carboxymethyl dextran
  • the free carboxylic acid groups of the dextran molecule make it possible to chelate cisplatin, as well as other toxic metals with therapeutic benefit (e.g. 177 Lu, 225 Ac, 221 Fr and 213 Bi) to this polymeric linker.
  • These loaded CMD polymeric structures are small enough to be considered as nanocarriers, like the PAMAM dendrimers described above, to enhance delivery and potency of low doses of therapeutic agents when targeted for transvascular transport via the caveolae pumping system.
  • osteoarthritis Osteoarthritis Cartilage, 15(6), 597-604.
  • vascular permeability factor vascular endothelial growth factor
  • Vascular permeability factor vascular endothelial growth factor
  • T-DM1 Trastuzumab emtansine: a novel agent for targeting HER2+ breast cancer. Clin Breast Cancer, 11(5), 275-282. doi:10.1016/j.clbc.2011.03.018
  • Vascular permeability factor/vascular endothelial growth factor a critical cytokine in tumor angiogenesis and a potential target for diagnosis and therapy. J Clin Oncol, 20(21), 4368-4380.
  • vascular permeability factor vascular endothelial growth factor
  • Transforming growth factor beta modulates the expression of collagenase and metalloproteinase inhibitor. Embo J, 6(7), 1899-1904.
  • Histone-GFP fusion protein enables sensitive
  • WNT1-inducible signaling protein-1 mediates pulmonary fibrosis in mice and is upregulated in humans with idiopathic pulmonary fibrosis. J Clin Invest, 119(4), 772-787. doi:33950 [pii]
  • TGF-beta is a critical mediator of acute lung injury. J Clin Invest, 107(12), 1537- 1544. doi:10.1172/jci11963
  • ErbB2 transgenic mice a tool for
  • Thy-1 as a regulator of cell-cell and cell-matrix interactions in axon regeneration, apoptosis, adhesion, migration, cancer, and fibrosis.
  • HER-2/neu is a tumor rejection target in tolerized HER-2/neu transgenic mice. Cancer Res, 60(13), 3569-3576.
  • TGFbeta2 knockout mice have multiple developmental defects that are non-overlapping with other TGFbeta knockout phenotypes. Development, 124(13), 2659- 2670.
  • Endothelial caveolae have the molecular transport machinery for vesicle budding, docking, and fusion including VAMP, NSF, SNAP, annexins, and GTPases. J Biol Chem, 270(24), 14399-14404.
  • plasmalemma of rat lung endothelium microdomains enriched in caveolin, Ca(2+)-ATPase, and inositol trisphosphate receptor. Proc Natl Acad Sci U S A, 92(5), 1759-1763.
  • Nanoparticles for the delivery of therapeutic antibodies Dogma or promising strategy? Expert Opin Drug Deliv, 1-14. doi:10.1080/17425247.2017.1273345
  • Tager A. M., Kradin, R. L., LaCamera, P., Bercury, S. D., Campanella, G. S., Leary, C. P., ...
  • Rat alveolar myofibroblasts acquire alpha-smooth muscle actin expression during bleomycin-induced pulmonary fibrosis.
  • Wakankar, A. A. Feeney, M. B., Rivera, J., Chen, Y., Kim, M., Sharma, V. K., & Wang, Y. J. (2010).
  • Thy-1-integrin alphav beta5 interactions inhibit lung fibroblast contraction-induced latent transforming growth factor-beta1 activation and myofibroblast differentiation. J Biol Chem, 285(29), 22382-22393. doi:10.1074/jbc.M110.126227

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Abstract

L'invention concerne des procédés et des compositions pour améliorer l'activité thérapeutique d'agents à faibles doses suite à une administration de médicament ciblée améliorée, à savoir, une administration de précision. Les procédés et les compositions peuvent être utilisés pour diminuer les quantités administrées efficaces d'agents thérapeutiques actifs par l'administration de précision à des cibles susceptibles de médier le transport actif à travers la barrière biologique formée par le système vasculaire qui conduit les vaisseaux sanguins. L'invention concerne également des anticorps (et autres espèces d'agents de ciblage) pour administrer précisément un principe actif (c'est-à-dire, un agent thérapeutique) à des cellules exprimant, par exemple, une protéine cible sur leurs membranes cellulaires, ainsi que des méthodes de traitement d'une maladie.
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EP3886913A4 (fr) * 2018-11-30 2023-01-11 Prism | Proteogenomics Research Institute for Systems Medicine Administration ciblée améliorée d'agents thérapeutiques

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US7429563B2 (en) * 2004-03-05 2008-09-30 The Board Of Trustees Of The University Of Illinois Peptide carrier for drug delivery
US20100260676A1 (en) * 2007-02-09 2010-10-14 Northeastern University Precision-guided nanoparticle systems for drug delivery
US20160279248A1 (en) * 2009-09-09 2016-09-29 Centrose, Llc Extracellular targeted drug conjugates

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7429563B2 (en) * 2004-03-05 2008-09-30 The Board Of Trustees Of The University Of Illinois Peptide carrier for drug delivery
US20100260676A1 (en) * 2007-02-09 2010-10-14 Northeastern University Precision-guided nanoparticle systems for drug delivery
US20160279248A1 (en) * 2009-09-09 2016-09-29 Centrose, Llc Extracellular targeted drug conjugates

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3886913A4 (fr) * 2018-11-30 2023-01-11 Prism | Proteogenomics Research Institute for Systems Medicine Administration ciblée améliorée d'agents thérapeutiques

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