WO2011133658A1 - Compositions et procédés de ciblage et d'administration d'agents thérapeutiques dans des cellules - Google Patents

Compositions et procédés de ciblage et d'administration d'agents thérapeutiques dans des cellules Download PDF

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WO2011133658A1
WO2011133658A1 PCT/US2011/033229 US2011033229W WO2011133658A1 WO 2011133658 A1 WO2011133658 A1 WO 2011133658A1 US 2011033229 W US2011033229 W US 2011033229W WO 2011133658 A1 WO2011133658 A1 WO 2011133658A1
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receptor
growth factor
compound
cell
copi
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PCT/US2011/033229
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English (en)
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John R. Murphy
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Boston Medical Center Corporation
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • 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
    • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
    • 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
    • A61K47/66—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 the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3513—Protein; Peptide

Definitions

  • X is selected from a cytotoxic agent, a therapeutic agent, and a diagnostic agent and comprises at least one thiol-containing group capable of forming a disulfide bond with Y;
  • Z is a polypeptide targeting moiety that is bound to Y at its carboxy-terminal end.
  • X is selected from a siRNA, dsRNA, an RNAi molecule, a protein nucleic acid (PNA) molecule, and a polypeptide (e.g., a transcription factor or growth factor).
  • the lysine-rich domain includes one or more lysine rich motifs having a dibasic signature selected from KKXX and KXKXX, or aromatic amino acid sequences selected from FFXXBB(X) repeat,.
  • the methods of the invention include the administration of this composition to treat cytomegalovirus infections in the eye, e.g., in patients with HIV.
  • a detectable label when coupled to a conjuage compound of the invention emits a signal that can be detected by a signal transducing machine.
  • the detectable label can emit a signal spontaneously, such as when the detectable label is a radionuclide.
  • the detectable label emits a signal as a result of being stimulated by an external field such as when the detectable label is a relaxivity metal.
  • signals include, without limitation, gamma rays, X-rays, visible light, infrared energy, and radio waves.
  • Examples of signal transducing machines include, without limitation, gamma cameras including SPECT/CT devices, PET scanners, fluorimeters, and Magnetic Resonance Imaging (MRI) machines.
  • a “pharmaceutically acceptable excipient” is meant a carrier that is physiologically acceptable to the treated mammal while retaining the therapeutic properties of the compound with which it is administered.
  • One exemplary pharmaceutically acceptable excipient is physiological saline.
  • Other physiologically acceptable excipients and their formulations are known to one skilled in the art and described, for example, in “Remington: The Science and Practice of Pharmacy” (20th ed., ed. A.R. Gennaro AR., 2000, Lippincott Williams & Wilkins).
  • Figure 6 In silico prediction of 25 amino acid fragment of transmembrane domain helices 1 and 2 (DWDVIRDKTKTKISSLKEHGH) of diphtheria toxin.
  • the three dimensional model was constructed using the PEP-Fold algorithm (Maupetit et al, 2009) which is based on the structural alphabet SA letters to describe the conformation of four consecutive residues, and then couples the predicted series of SA letter to a greedy algorithm and course-grained force field.
  • the resulting structure was the visualized using the Accelrys DS Visualizer (v 2.0.1.7347).
  • EF, and botulinum neurotoxins serotypes A, C, and D on the cytoplasmic side of endosomal vesicle membranes can mimic cargo motifs and/or the KKXX and FFXX of adaptor proteins which are known to bind COPI complex proteins.
  • replacement of the native transmembrane helix 1 with the 13 amino acid COPI binding domain from the cytoplasmic tail of p23 results in a domain swap mutant whose cytotoxic potency is identical to that of the wild type toxin.
  • COPI complex binding to the N-terminal lysine- rich portion of the transmembrane domain of these toxins is an essential feature for C- domain delivery to the eukaryotic cell cytosol.
  • the COPI coatomer complex plays an essential role in the efficient delivery of toxins, such as DT and other toxins, e.g., anthrax LF and EF and botulinum neurotoxins serotypes A, C, and D, across the endosomal vesicle membrane and into the eukaryotic cell cytosol.
  • toxins such as DT and other toxins, e.g., anthrax LF and EF and botulinum neurotoxins serotypes A, C, and D
  • the requirements for COPI complex protein for both the DT C-domain and LF entry into the cytosol confirm that there is a common mechanism of entry for these highly divergent bacterial protein toxins.
  • Y is a polypeptide containing a lysine-rich domain that is capable of interacting with cellular COPI complex proteins and that includes at least one bond that is cleavable by an intracytosolic enzyme (e.g., the polypeptide may include a cysteine residue that forms a disulfide bond with the at least one thiol group (e.g., a cysteine residue) of the X moiety that is cleavable by an intracytosolic enzyme (e.g., thioredoxin reductase)), in which the lysine-rich domain includes one or more lysine rich motifs having a dibasic signature (e.g., KKXX, XKXX) and/or an aromatic amino acid sequences (e.g,.
  • the X moiety may also include a linker that connects the therapeutic agent (e.g., a siRNA, dsRNA, RNAi, a protein nucleic acid (PNA) molecule, or a protein agent (e.g., a transcription factors and other polypeptides)) to a thiol-containing group that can be used to form a disulfide bond with the thiol-containing group of the Y moiety.
  • the therapeutic agent e.g., a siRNA, dsRNA, RNAi, a protein nucleic acid (PNA) molecule, or a protein agent (e.g., a transcription factors and other polypeptides)
  • the transmembrane and receptor binding domains of the non- toxic DT mutant CRM 197 and the DT-related fusion protein toxins are used as structural platforms for the development of non-viral, cell receptor-specific siRNA cytosolic delivery systems. These systems employ their respective cell receptor- specific targeting and endosomal vesicle membrane pore forming ability and COPI binding motifs for enhanced delivery of peptide-siR As to the target cell cytosol.
  • the X moiety is prepared so that it is suitable for thiol-specific attachment via a free cysteine to the Y moiety of the conjugate compound.
  • Thiol-specific drug attachment to a peptide analog can be direct or indirect, i.e. via a chelator (e.g., MX-DTPA, which is useful in preparing the peptide analogs of the invention; the maleimido derivatives of MX-DTPA chelator is reactive with thiol groups of a peptide portion of the X moeity (i.e., SH groups of one or more free cysteines) to form a thioether linkage).
  • the thiol attachment methods of the present invention are generally applicable to the attachment of drugs/chelators to the Y/Z portion of the conjugate compound.
  • the thiol linkage can be a stable linkage, for example as a thioether linkage.
  • a drug or chelator is functionalized with a thiol reactive group (e.g., a maleimido group) that provides a stable thioether linkage.
  • a drug can comprise a cleavable site, such that the X moiety can be released from Y moiety (e.g., by reducing the disulfide bond).
  • representative cleavable sites include acid-labile and enzyme-labile sites.
  • Therapeutic agents that can be used as the "cargo" of the compounds of the invention include cytotoxic polypeptides, such as cytochrome c, caspase 1-10, granzyme A or B, tumor necrosis factor-alpha (TNF-a), TNF- ⁇ , Fas, Fas ligand, Fas-associated death doman-like IL- ⁇ ⁇ converting enzyme (FLICE), TRAIL/AP02L, TWEAK/AP03L, Bax, Bid, Bik, Bad, Bak, RICK, vascular apoptosis inducing proteins 1 and 2 (VAP1 and VAP2), pierisin, apoptosis-inducing protein (AIP), IL-l propiece polypeptide, apoptin, apoptin-associated protein 1 (AAP-1), endostatin, angiostatin, and biologically-active fragments thereof.
  • cytotoxic polypeptides such as cytochrome c, caspase 1-10, granzyme A
  • the X moiety can also be selected from nucleic acid molecules, e.g., siR As, dsRNAs, and other nucleic acid molecules that are known in the art to silence gene expression.
  • the nucleic acid molecules are those that silence genes that express polypeptides that are known to be involved in disease.
  • siRNA molecules for use in the treatment of diseases are known in the art (see, e.g., U.S. Patent Nos. 7,056,704; 7,678,896; 7,678,897; and 7691998; and U.S. Patent Application Publication Nos. 20100062051 (entitled Composition for Treatment of Cervix Cancer); 20100062436; 20100062951; 20100062967; 20100063131;
  • 20100063132 (entitled Small Interfering RNA and Pharmaceutical Composition for Treatment of Hepatitis B Comprising the Same); 20100063134 (entitled Treatment of Neurodegenerative Disease Through Intracranial Delivery of siRNA); and 20100063308; 20080249046; 20080260854; 20090318536; and 20100098664; each of which is incorporated by reference herein in their entirety).
  • Any of the siRNA molecules described in these publications can be used as the X moiety in the conjugate compounds of the invention for use in the treatment of diseases for which the siRNA molecules are known to treat or were developed to treat.
  • the X moiety may also be a transcription factor or a nucleic acid molecule that encodes a transcription factor.
  • the transcription factor can be selected from helix-turn- helix motif proteins, homeodomain proteins, zinc finger motif proteins, steroid receptor proteins, leucine zipper motif proteins, helix-loop-helix motif proteins, and ⁇ -sheet motif proteins.
  • the X moiety is a nucleic acid binding compound that binds nonspecifically to nucleic acids and is selected from the group consisting of poly- L-lysine, protamine, histone and spermine.
  • the X moiety is a nucleic acid binding domain that binds the coding region of a ribosome inactivating protein such as saporin.
  • Transcription factors for use as the X moiety in the preparation of conjugate compounds of the invention are known in the art (see, e.g., U.S. Patent No. 6,037,329, incorporated by reference herein in its entirety).
  • the X moiety may also be a growth factor polypeptide or a nucleic acid molecule that encodes the growth factor selected from leptin receptor (LPTR), granulocyte colony stimulating factor receptor (GCSFR), LIF/OSM/CNTF common beta chain (GPI30), leukemia inhibiting factor receptor (LIFR), oncostatin-M receptor beta chain (OSMR), interleukin-12 receptor beta-1 chain (IL12RB1), and interleukin-12 receptor beta-2 chain (IL 12RB2).
  • LPTR leptin receptor
  • GCSFR granulocyte colony stimulating factor receptor
  • LIF/OSM/CNTF common beta chain GPI30
  • LIF/OSM/CNTF common beta chain GPI30
  • LIF/OSM/CNTF common beta chain GPI30
  • OSMR leukemia inhibiting factor receptor
  • IL12RB1 interleukin-12 receptor beta-1 chain
  • IL 12RB2 interleukin-12 receptor beta-2 chain
  • IL12RB1 with IL12RB2 such as G-CSF, GM-CSF or M-CSF
  • SCF stem cell factor
  • SCPF stem cell proliferation factor
  • IL1 , IL4, IL5, IL6, IL11, IL12 various Interleukins
  • TGF- ⁇ , MIP-1- ⁇ , TNF-a and also many other low molecular weight factors.
  • the X moiety may also be a growth factor polypeptide or a nucleic acid molecule that encodes the growth factor (e.g., a growth factor selected from stem cell factor (SCF), FLT3, IL-3, IL-6, GSF, GM-CSF, and erythropoietin).
  • a growth factor selected from stem cell factor (SCF), FLT3, IL-3, IL-6, GSF, GM-CSF, and erythropoietin.
  • the conjugate compound can include a targeting moiety (Z) that targets the conjugate compound to a stem cell (e.g., a hemotopoeitic stem cell or a mesenchymal stem cell) and can include an X moiety, such as one of the growth factors described above, that promotes the differentiation of stem cells into a desired lineage (e.g., neuronal, hepatic, osteogenic, chondrogenic, tendonogenic, ligamentogenic, myogenic, marrow stromagenic, adipogenic or dermogenic lineage). Growth factors that stimulate the differentiation of stem cells are known in the art (see, e.g,. U.S. Patent No. 5,942,225, incorporated by reference herein).
  • Stem cells may also be differentiated using a conjugate compound of the invention into pancreatic islet cells (or primary cells of the islets of Langerhans), which may then be transplanted into a subject suffering from diabetes (e.g., diabetes mellitus, type 1), see e.g., Burns et al., (2006) Curr. Stem Cell Res. Ther., 2:255-266.
  • pancreatic beta cells derived from induced cells and differentiated using a conjugate compound of the invention may be transplanted into a subject suffering from diabetes (e.g., diabetes mellitus, type 1).
  • Coronaviridae family which includes the severe acute respiratory syndrome (SARS) virus
  • SARS severe acute respiratory syndrome
  • Rhabdoviridae family which includes the rabies virus and vesicular stomatitis virus (VSV)
  • VSV vesicular stomatitis virus
  • RSV human respiratory syncytial virus
  • RV human respiratory syncytial virus
  • NSV vesicular stomatitis virus
  • RV human respiratory syncytial virus
  • Newcastle disease virus hendravirus
  • nipahvirus measles virus
  • rindeipest virus canine distemper virus
  • Sendai virus human parainfluenza virus (e.g., 1, 2, 3, and 4)
  • rhinovirus and mumps virus
  • Picornaviridae family which includes the poliovirus, human enterovirus (A, B, C, and D), hepatitis A virus, and the coxsackievirus
  • Hepadnaviridae family which includes the hepatitis B virus; a member of the
  • Dezaguanine Dezaguanine Mesylate; Diaziquone; Docetaxel; Dolasatins; Doxorubicin;
  • Fluorouracil 5-FdUMP; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Gold Au 198; Homocamptothecin; Hydroxyurea; Idarubicin
  • Mitogillin Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin;Ormaplatin; Oxisuran; Paclitaxel;
  • Testolactone Thiamiprine; Thioguanine; Thiotepa; Thymitaq; Tiazofurin; Tirapazamine; Tomudex; TOP53; Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine;
  • cyclophosphamide nielphaian; chlorambucil; ifosfamide; busulfan; N-methyl- Nnitrosourea (MNU); N, N'-Bis (2-chloroethyl)-N-nitrosourea (BCNU); N- (2- chloroethyl)-N' cyclohexyl-N-nitrosourea (CCNU); N- (2-chloroethyl)-N - (trans-4- methylcyclohexyl-N-nitrosourea (MeCCNU); N- (2-chloroethyl)-N'- (diethyl) ethylphosphonate-N-nitrosourea (fotemustine); streptozotocin; diacarbazine (DTIC); mitozolomide; temozolomide; thiotepa; mitomycin C; AZQ; adozelesin; Cisplatin;
  • Dactinomycin (Actinomycin D); amsacrine; pyrazoloacridine; all-trans retinol; 14- hydroxy-retro-retinol; all-trans retinoic acid; N- (4- Hydroxyphenyl) retinamide; 13-cis retinoic acid; 3-Methyl TTNEB; 9-cis retinoic acid; fludarabine (2-F-ara-AMP); or 2- chlorodeoxyadenosine (2-Cda).
  • aclarubicin acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors;
  • antagonist D antagonist G
  • antarelix anti-dorsalizing morphogenetic protein- 1 ; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; asulacrine;
  • camptothecin C calphostin C
  • camptothecin derivatives e.g., 10-hydroxy-camptothecin
  • IL-2 capecitabine
  • carboxamide-amino-triazole carboxyamidotriazole
  • CaRest M3 CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A
  • collismycin B combretastatin A4; combretastatin analogue; conagenin;
  • crambescidin 816 crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanihraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; 2'deoxycoformycin
  • didox diethylnorsperrnine; dihydro-5-azacytidine; dihydrotaxol, 9- ; dioxamycin; diphenyl spiromustine; discodermolide; docosanol; dolasetron; doxifluridine;
  • ganirelix gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;
  • heregulin hexamethylene bisacetamide; homoharringtonine (HHT); hypericin;
  • ibandronic acid idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;
  • imidazoacridones imiquimod; immunostimulant peptides; insulin-like growth factor- 1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane;
  • iododoxorubicin ipomeanol, 4- ; irinotecan; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin: letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide + estrogen + progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;
  • masoprocol maspin
  • matrilysin inhibitors maspin
  • matrix metalloproteinase inhibitors masoprocol inhibitors
  • mycobacterial cell wall extract myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone + pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase;
  • nilutamide nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; 06- benzyl guanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin;
  • palmitoylrhizoxin pamidronic acid; panaxytriol; panomifene; parabactin;
  • pazelliptine pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin;
  • phenylacetate phosphatase inhibitors
  • picibanil pilocarpine hydrochloride
  • podophyllotoxin porfimer sodium; porfiromycin; propyl bis-acridone: prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex;
  • glycosaminoglycans tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;
  • thalidomide thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene dichloride; topotecan; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors;
  • tyrphostins UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.
  • the X moiety may also be a lytic peptide.
  • Such lytic peptides induce cell death and include, but are not limited to, streptolysin O; stoichactis toxin; phallolysin; staphylococcus alpha toxin; holothurin A; digitonin; melittin; lysolecithin;
  • the X moiety may also be a synthetic peptide that shares some sequence homology or chemical characteristics with any of the naturally occurring peptide lysins; such characteristics include, but are not limited to, linearity, positive charge, amphipathicity, and formation of alpha-helical structures in a hydrophobic environment (Leuschner et al, Biology of Reproduction 73:860-865, 2005).
  • Agents of the invention can also be coupled to an agent that induces complement-mediated cell lysis such as, for example, the immunoglobulin F c subunit.
  • Iodopyracet 125 I Iodopyracet 131 I; lofetamine Hydrochloride 12 I; Iomethin 125 I;
  • daunorubicine chlorhydrate doxorubicine chlorhydrate, epirubicine chlorhydrate, idarubicine chlorhydrate, pirarubicine, or zorubicine chlorhydrate; a camptothecin, or its derivatives or related compounds, such as 10, 11 methylenedioxycamptothecin; or a member of the maytansinoid family of compounds, which includes a variety of structurally-related compounds, e.g., ansamitocin P3, maytansine, 2'-N- demethylmaytanbutine, and maytanbicyclinol.
  • ansamitocin P3 maytansine, 2'-N- demethylmaytanbutine, and maytanbicyclinol.
  • Detectable labels can be used as the X moiety to prepare conjugate compounds of the invention for use as diagnostic agents.
  • a detectable label is used as the "cargo" of the compounds of the invention.
  • Detectable labels can be selected from a radioactive, bioluminescent, fluorescent, or heavy metal label, or an epitope tag.
  • Detectable labels of the conjugate compounds can include radioactive metals for use in radiographic imaging or radiotherapy.
  • Preferred radioisotopes also include 99m Tc, 5 I Cr, 67 Ga, 68 Ga, m In, 168 Yb, 14C La, 90 Y, 88 Y, 153 Sm, 156 Ho, 165 Dy, 64 Cu, 97 Ru, 103 Ru, 186 Re, 188 Re, 203 Pb, 2n Bi, 212 Bi, 2I3 Bi, and 214 Bi.
  • the choice of metal is determined based on the desired therapeutic or diagnostic application.
  • the metal complexes of the invention are useful as diagnostic and/or
  • a detectable label may be a metal ion from heavy elements or rare earth ions, such as Gd , Fe , Mn , or Cr .
  • Conjugates that include paramagnetic or superparamagnetic metals are useful as diagnostic agents in MRI imaging
  • Paramagnetic metals that may be used in the conjugates include, but are not limited to, chromium (III), manganese (II), iron (II), iron (III), cobalt (II), nickel
  • Fluorescent molecules that can also serve as detectable labels include green fluorescent protein (GFP), enhanced GFP (eGFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), and dsRed.
  • GFP green fluorescent protein
  • eGFP enhanced GFP
  • YFP yellow fluorescent protein
  • CFP cyan fluorescent protein
  • RFP red fluorescent protein
  • dsRed red fluorescent protein
  • the bioluminescent molecule is luciferase.
  • the epitope tag is c- myc, hemagglutinin, or a histidine tag.
  • the lysine rich motif can be selected from the transmembrane helix 1 of DT, the Tl motif, or an amino acid sequence having at least 80% or more (e.g., 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to a contiguous amino acid sequence corresponding to at least amino acids 201 to 222 of DT (e.g., the lysine rich region has substantial sequence identity (80% or more, as described above) to a contiguous amino acid sequence corresponding to at least amino acids 201-235, amino acids 195 to 222, amino acids 195 to 235, amino acids 201 to 300, amino acids 195 to 300, amino acids 201 to 389, or amino acids 195 to 389, of DT).
  • the lysine rich motif has substantial sequence identity (80% or more, as described above) to a contiguous amino acid sequence corresponding to at least amino acids 201-235, amino acids 195 to 222, amino acids 195 to 235, amino acids 201 to 300, amino
  • amino acid sequences for use as the Y moiety can be selected from the transmembrane domains of other toxins (see, e.g., Table 1). These sequences can be modified at their amino-terminal end to include a cysteine residue in order to establish the formation of a disulfide bond between the Y/Z moieties and the X moiety of the conjugate compound.
  • polypeptides for use as the Y moiety include the consensus peptide sequence of CTF-binding moiety: RDKTKTKIESLKEHGPIKNS, the consensus peptide sequence of CTF-binding moiety including KXKXX sequences in bold:
  • polypeptides for use as the Y moiety are described in, e.g., US 2008- 0306003, incorporated by reference herein in its entirety, and below.
  • the compounds of the invention can be targeted to a specific cell or cells by using a targeting moiety (Z) that directs the compounds to a desired target cell.
  • Z targeting moiety is selected based on its ability to target conjugate compounds of the invention to a desired or selected cell population that expresses the corresponding binding partner (e.g., either the corresponding receptor or ligand) for the selected Z targeting moiety.
  • a conjugate compound of the invention could be targeted to cells expressing epidermal growth factor receptor (EGFR) by selected epidermal growth factor (EGF) as the Z targeting moiety.
  • EGFR epidermal growth factor receptor
  • EGF epidermal growth factor
  • the targeting moiety, Z is erythroblastic leukemia viral oncogene homolog (ErbB) receptor (e.g., ErbBl receptor; ErbB2 receptor; ErbB3 receptor; and ErbB4 receptor).
  • ErbB erythroblastic leukemia viral oncogene homolog
  • the Z targeting moiety can also be selected from bombesin, gastrin, gastrin- releasing peptide, tumor growth factors (TGF), such as TGF-a and TGF- ⁇ , and vaccinia virus growth factor (VVGF).
  • TGF tumor growth factors
  • VVGF vaccinia virus growth factor
  • Non-peptidyl ligands can also be used as the Z targeting moiety and may include, for example, steroids, carbohydrates, vitamins, and lectins.
  • the Z targeting moiety may also be selected from a peptide, such as somatostatin (e.g., a somatostatin having the core sequence cyclo[Cys-Phe-D-Trp-Lys-Thr-Cys], and in which, preferably, the C-terminus of the somatostatin analog is: Thr-NFL), a somatostatin analog (e.g., octreotide and lanreotide), bombesin, a bombesin analog, or an antibody, such as a monoclonal antibody.
  • somatostatin e.g., a somatostatin having the core sequence cyclo[Cys-Phe-D-Trp-Lys-Thr-Cys]
  • a somatostatin analog e.g., octreotide and lanreotide
  • bombesin e.g., octreotide and lan
  • peptides for use as the Z targeting moiety in the conjugate compounds of the invention can be selected from iSS peptides and analogs, urotensin II peptides and analogs, GnRH I and II peptides and analogs, octreotide, depreotide, vapreotide, vasoactive intestinal peptide (VIP), cholecystokinin (CCK), RGD-containing
  • ITIPP(psi) annexin-V
  • endothelin endothelin
  • leukotriene B4 LLB4
  • chemotactic peptides e.g., N-formyl-methionyl-leucyl-phenylalanine-lysine (fMLFK)
  • GP Iib/IIIa e.g., N-formyl-methionyl-leucyl-phenylalanine-lysine (fMLFK)
  • Immunoreactive ligands for use as the targeting moiety Z in the invention include an antigen-recognizing immunoglobulin (also referred to as "antibody"), or antigen-recognizing fragment thereof.
  • immunoglobulin refers to any recognized class or subclass of immunoglobulins such as IgG, IgA, IgM, IgD, or IgE. Preferred are those immunoglobulins which fall within the IgG class of immunoglobulins.
  • the immunoglobulin can be derived from any species. Preferably, however, the immunoglobulin is of human, murine, or rabbit origin. In addition, the immunoglobulin may be polyclonal or monoclonal, but is preferably monoclonal.
  • the immunoglobulin used in conjugates of the invention may be a "chimeric antibody” as that term is recognized in the art.
  • the immunoglobulin may be a "bifunctional” or “hybrid” antibody, that is, an antibody which may have one arm having a specificity for one antigenic site, such as a tumor associated antigen while the other arm recognizes a different target, for example, a hapten which is, or to which is bound, an agent lethal to the antigen-bearing tumor cell.
  • the bifunctional antibody may be one in which each arm has specificity for a different epitope of a tumor associated antigen of the cell to be therapeutically or biologically modified.
  • Hybrid antibodies thus have a dual specificity, preferably with one or more binding sites specific for the hapten of choice or one or more binding sites specific for a target antigen, for example, an antigen associated with a tumor, an infectious organism, or other disease state.
  • Biological bifunctional antibodies can also be used as the Z targeting moiety in the conjugate compounds of the invention (such antibodies are described in, for example, European Patent Publication, EPA 0 105 360, which is hereby incorporated by reference in its entirety).
  • Such hybrid or bifunctional antibodies may be derived either biologically, by cell fusion techniques, or chemically, such as with cross- linking agents or disulfide bridge-forming reagents, and may be comprised of whole antibodies and/or fragments thereof.
  • bifunctional antibodies are those biologically prepared from a "polydoma” or “quadroma” or which are synthetically prepared with cross-linking agents such as bis- (maleimido)-methyl ether (“BMME”), or with other cross-linking agents familiar to those skilled in the art.
  • BMME bis- (maleimido)-methyl ether
  • the immunoglobin may be a single chain antibody ("SCA").
  • SCA may consist of single chain Fv fragments ("scFv”) in which the variable light (“V L ”) and variable heavy (“V H ”) domains are linked by a peptide bridge or by disulfide bonds.
  • the immunoglobulin may consist of single VH domains (dAbs) that possess antigen-binding activity. See G. Winter and C. Milstein, Nature 349:295, 1991; R. Glockshuber et al., Biochemistry 29:1362, 1990; and, E. S. Ward et al., Nature 341 :544, 1989.
  • chimeric monoclonal antibodies preferably those chimeric antibodies having specificity toward a tumor associated antigen.
  • the term "chimeric antibody” refers to a monoclonal antibody comprising a variable region, i.e. a binding region, from one source or species and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques.
  • Chimeric antibodies having a murine variable region and a human constant region are especially preferred in certain applications of the invention, particularly human therapy, because such antibodies are readily prepared and may be less immunogenic than purely murine monoclonal antibodies.
  • Such murine/human chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding murine immunoglobulin variable regions and DNA segments encoding human immunoglobulin constant regions.
  • Other forms of chimeric antibodies for use in conjugates of the invention are those in which the class or subclass has been modified or changed from that of the original antibody.
  • Such "chimeric" antibodies are also referred to as "class-switched antibodies.”
  • Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques now well known in the art. See Morrison, S. L, et al., Proc. Nat'l Acad. Sci., 81 :6851, 1984.
  • chimeric antibody also includes a "humanized antibody,” namely, those antibodies in which the framework or “complementarity determining regions” (“CDR") have been modified to include the CDR of an immunoglobulin of different specificity, as compared to that of the parent immunoglobulin.
  • CDR framework or complementarity determining regions
  • a murine CDR is grafted into the framework region of a human antibody to prepare the "humanized antibody.” See, e.g., L. Riechmann et al., Nature 332:323, 1988; M. S. Neuberger et al, Nature 314:268, 1985.
  • Particularly preferred CDRs correspond to those representing sequences recognizing the antigens noted above for the chimeric and bifunctional antibodies. See, e.g., EPA 0 239 400 (published Sep. 30, 1987), which is hereby incorporated by reference in its entirety.
  • the noted constructions can be prepared with immunoglobulin fragments used as the starting materials; or, if recombinant techniques are used, the DNA sequences, themselves, can be tailored to encode the desired "fragment” which, when expressed, can be combined in vivo or in vitro, by chemical or biological means, to prepare the final desired intact immunoglobulin "fragment.” It is in this context that the term "fragment" is used herein.
  • the immunoglobulin (antibody), or fragment thereof, used as the Z targeting moiety in conjugate compounds of the present invention may be polyclonal or monoclonal in nature. Monoclonal antibodies are the preferred immunoglobulins.
  • the preparation of polyclonal or monoclonal antibodies is well known to those skilled in the art. See, e.g., G. Kohler and C. Milstein, Nature 256:495, 1975.
  • hybridomas and/or monoclonal antibodies which are produced by such hybridomas and which are useful in the practice of the present invention are publicly available. Linkers for Attaching the Cytotoxic, Therapeutic, or Diagnostic Agents
  • conjugate compounds of the invention can include cytotoxic, therapeutic, or diagnostic agents as "cargo” (e.g., nucleic acids molecules, PNAs, peptides, polypeptides, proteins, small molecules, antibodies, or antibody fragments). These agents can be associated with or bonded to the Y and Z moieties of the conjugate compound, which faciliate the delivery and targeting of the conjugate compound, respectfully, using, e.g., a linker or linking component.
  • cytotoxic, therapeutic, or diagnostic agents as "cargo” (e.g., nucleic acids molecules, PNAs, peptides, polypeptides, proteins, small molecules, antibodies, or antibody fragments).
  • conjugate compounds of the invention are prepared by incorporating a peptidic linking group into the the X moiety (e.g., nucleic acid molecules, such as siRNA and dsRNA), PNA molecules, peptides, polypeptides, proteins, small molecules, antibodies, or antibody fragments).
  • the peptide linking group can include, e.g., a cysteine residue that is used to form a disulfide bond between the X moiety and the Y moiety or it can be another peptide sequence that forms a bond that is cleavable by a known cytosolic enzyme.
  • linker can also couple the X moiety (i.e., the "cargo") to the Y moiety by reacting, e.g., a free amino group of a Thr residue of a peptide portion of X moiety to the conjugate compound (e.g., a portion of the Y moiety) with an appropriate functional group of a chelator, such as a carboxyl group or activated ester.
  • a conjugate may
  • EDTA ethylenediaminetetraacetic acid
  • Conjugate compounds of the invention may be administed to a mammalian subject, such as a human, directly or in combination with any pharmaceutically acceptable carrier or salt known in the art for use in the treatment or detection of disease.
  • Pharmaceutically acceptable salts may include non-toxic acid addition salts or metal complexes that are commonly used in the pharmaceutical industry.
  • acid addition salts include organic acids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethyl cellulose, or the like; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, or the like.
  • Metal complexes include zinc, iron, and the like.
  • One exemplary pharmaceutically acceptable carrier is physiological saline.
  • physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington's Pharmaceutical Sciences. (18 th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, PA.
  • compositions of a therapeutically effective amount of a conjugate compound of the invention, or pharmaceutically acceptable salt-thereof can be administered orally, parenterally (e.g., by intramuscular, intraperitoneal, intravenous, or subcutaneous injection, by inhalation, intradermally, using optical drops, or by implant), nasally, vaginally, rectally, sublingually, or topically, in admixture with a pharmaceutically acceptable carrier adapted for the route of administration.
  • parenterally e.g., by intramuscular, intraperitoneal, intravenous, or subcutaneous injection, by inhalation, intradermally, using optical drops, or by implant
  • nasally, vaginally, rectally, sublingually, or topically in admixture with a pharmaceutically acceptable carrier adapted for the route of administration.
  • compositions intended for oral use may be prepared in solid or liquid forms according to any method known to the art for the manufacture of pharmaceutical compositions.
  • the compositions may optionally contain sweetening, flavoring, coloring, perfuming, and/or preserving agents in order to provide a more palatable preparation.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound is admixed with at least one inert pharmaceutically acceptable carrier or excipient.
  • inert pharmaceutically acceptable carrier or excipient may include, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, sucrose, starch, calcium phosphate, sodium phosphate, or kaolin. Binding agents, buffering agents, and/or lubricating agents (e.g., magnesium stearate) may also be used. Tablets and pills can additionally be prepared with enteric coatings.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and soft gelatin capsules. These forms contain inert diluents commonly used in the art, such as water or an oil medium. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying agents, and suspending agents.
  • Liquid formulations can be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, or by irradiating or heating the compositions. Alternatively, they can also be manufactured in the form of sterile, solid compositions which can be dissolved in sterile water or some other sterile injectable medium immediately before use.
  • compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to active substances, excipients such as coca butter or a suppository wax.
  • Compositions for nasal or sublingual administration are also prepared with standard excipients known in the art.
  • Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops or spray, or as a gel.
  • the amount of active ingredient in the compositions of the invention can be varied.
  • One skilled in the art will appreciate that the exact individual dosages may be adjusted somewhat depending upon a variety of factors, including the type of conjugate compound being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the nature of the subject's conditions, and the age, weight, health, and gender of the patient.
  • the severity of the condition targeted by the conjugate compounds of the invention may also have an impact on the dosage level.
  • dosage levels of between 0.1 ⁇ g/kg to 100 mg/kg of body weight are administered daily as a single dose or divided into multiple doses.
  • the general dosage range is between 250 ⁇ g/kg to 5.0 mg/kg of body weight per day.
  • compositions of the invention may be as frequent as necessary to obtain the desired therapeutic effect. Some patients may respond rapidly to a higher or lower dose and may find much weaker maintenance doses adequate. Other patients, however, receive long-term treatments at the rate of 1 to 4 doses per day, in accordance with the physiological requirements of each patient.
  • the active product may be administered, e.g., intravenously, 1 to 4 times daily or via continuous infusion.
  • sustained released composition will generally be preferred.
  • Toxins as a Platform to Deliver Cytotoxic, Therapeutic, Diagnostic Agents as Cargo
  • Toxins e.g., the transmembrane and receptor binding domains of the DT
  • cargo e.g., nucleic acid molecules, such as siRNA, polypeptides, or chemical compounds
  • the "cargo" affixed to the toxins can be easily substituted to yield multiple different delivery constructs.
  • the toxins can be engineered to include a cysteine residue at its amino-terminal end or an existing cysteine residue within the toxin (e.g., the cysteine residue located at amino acid position 201 of DT (see, e.g., Figure 1 A) can be used to "load” the cargo onto the toxin construct via the formation of disulfide bonds between the cargo (referred to herein as the "X" moiety) and the toxin platform (referred to herein as the
  • diphtheria toxin structural platform as a nano-machine in the
  • the evolution of bacterial protein toxins can be viewed as the natural assembly of highly efficient nano-machines capable of cell surface receptor specific delivery of their respective catalytic domains, "cargo", to the cytosol.
  • the DT structural platform can be exploited to develop a system for the facilitated delivery of, e.g., nucleic acid molecules (e.g., siRNA), polypeptide therapeutics, or small molecules across the endosomal vesicle membrane and their subsequent release into the cytosol of target cells and tissues.
  • the conjugate compounds of the invention deliver their "cargo” using the same mechanism by which native DT delivers its C- domain cargo to the eukaryotic cell cytosol.
  • the intoxication of sensitive eukaryotic cells by DT follows an ordered series of events.
  • the first step in the intoxication process is the binding of toxin to its cell surface receptor, the heparin-binding (hb)-EGF-like precursor (Naglich el ah, 1992).
  • This binding is further enhanced by the DT receptor associated protein DTRAP27, which is the primate homologue of human CD9 (Iwamoto et al., 1994).
  • Receptor bound toxin is concentrated in clathrin coated pits and internalized into clathrin coated vesicles (CCVs), which are then converted into early endosomal vesicles (EEVs) (Moya et al., 1985).
  • CCVs clathrin coated vesicles
  • EEVs early endosomal vesicles
  • the activity of the vacuolar ATPase lowers the luminal pH within the EEV. Acidification of the vesicle lumen is required to trigger the unfolding of the DT transmembrane domain (Boquet et al, 1976).
  • the transmembrane domain undergoes a dynamic reorganization which results in the spontaneous insertion of two a-helical hairpins into the vesicle membrane resulting in the formation a transmembrane 18-25 A pore (Donovan et ah, 1981; Kagan et al., 1981).
  • Pore formation is a critical step in the intoxication process since it provides the conduit for C-domain translocation from the cis to trans side of the endosomal vesicle membrane with subsequent events effecting C-domain release into the target cell cytosol.
  • CTF Cytosolic Translocation Factor
  • the DT C-domain is an ADP-ribosyltransferase that catalyzes the NAD+ -dependent ADP-ribosylation of eukaryotic elongation factor 2 (EF-2) thereby inhibiting cellular protein synthesis (Honjo et ah, 1968; Collier and Kandel, 1971 ; Gill and Pappenheimer, 1971).
  • EF-2 eukaryotic elongation factor 2
  • Tl motif is highly conserved in anthrax LF, anthrax EF, and the botulinum neurotoxins. I have also demonstrated that the Tl motif and its closely associated XKXX sequences mediate the facilitated delivery of anthrax LF into the eukaryotic cell cytosol (Tamayo et al., 2008).
  • the Tl motif and its closely positioned KXKXX sequences closely resemble the dilysine (KKXX) and interrupted dilysine motifs (KXKXX) in the cytoplasmic tails of both p23/24 adaptor and cargo proteins (Cosson & Letourneue, 1994) that bind COPI coatomer complex proteins and direct vesicle trafficking (Fiedler et al., 1966).
  • endosomal vesicle membrane to form a pore through which the "cargo " is threaded by the chaperone-like properties of the transmembrane domain: and, upon the emergence of transmembrane helix 1 sequences on the cytosolic surface of the endosomal vesicle membrane,
  • transmembrane domain Tl motif and/or KXKXX sequences e.g., dibasic signature (e.g., KKXX, KXKXX) and/or an aromatic amino acid sequences (e.g. FFXXBB(X) horr) that function as p23/24 adaptor mimetics and mediate the binding of COPI complex proteins then facilitate the delivery of the catalytic domain "cargo " into the cytosol; the cargo, which is bound to the transmembrane domain and receptor binding domain via a disulfide bond is then reduced by thioredoxin reductase, thereby releasing the cargo into the cytosol.
  • KXKXX sequences e.g., dibasic signature (e.g., KKXX, KXKXX) and/or an aromatic amino acid sequences (e.g. FFXXBB(X) press) that function as p23/24 adaptor mimetics and mediate the binding of COPI complex proteins then facilitate
  • “nicked" toxin may be separated into a 21.1 kDa N-terminal polypeptide (residues 1-193), or Fragment A, and a 41.2 kDa C-terminal Fragment B (residues 194 to 535), which carries both the transmembrane and receptor binding domains (Uchida et al, 1971; Choe et al, 1992; Bennett et al, 1994).
  • DAB 389 lL-2 was the first, and to date only, targeted toxin to be approved by the FDA for human clinical use (Foss, 2000).
  • the DT structural platform is remarkably amenable to receptor binding domain substitution, thereby allowing this domain of DT to be substituted with a range of desired binding domain (e.g., a receptor, the receptor ligand, and other binding molecules, such as antibodies or antibody fragments, that can be used to target the conjugate compound to a desired cell or tissue).
  • desired binding domain e.g., a receptor, the receptor ligand, and other binding molecules, such as antibodies or antibody fragments, that can be used to target the conjugate compound to a desired cell or tissue.
  • transmembrane domain that are required for COPI complex facilitated delivery of "cargo" from the lumen of acidified endosomal vesicles to the cytosol allows for the use of the DT structural platform for the development of a system to efficiently deliver cytotoxic, therapeutic, or diagnostic agents (e.g., nucleic acid molecules, such as siRNA and PNA molecules, polypeptides, such as growth and transcription factors, cytotoxic agents, such as paclitaxel, and anti-viral agents) to the cytosol of target cells.
  • cytotoxic, therapeutic, or diagnostic agents e.g., nucleic acid molecules, such as siRNA and PNA molecules, polypeptides, such as growth and transcription factors, cytotoxic agents, such as paclitaxel, and anti-viral agents
  • This system envisions the use of the DT B-fragment transmembrane and receptor binding domains (and substitutions thereof) for (i) the targeting of specific cell surface receptors or ligands for cell-specific and/or tissue specific delivery, and (ii) the transmembrane domain for endosomal vesicle membrane pore formation and COPI complex binding for the facilitated delivery of siRNA "cargo" to the target cell cytosol. Delivery of siRNA molecules using Conjugate Compounds of the Invention
  • the antisense RISC complex Upon degradation of the sense strand, the antisense RISC complex then seeks out and degrades the messenger (m)RNA that is complementary to the antisense strand (Ameres et al., 2007). Since the antisense siRNA-RISC complex is stable, mRNA that is targeted continues to be degraded over time. In fact, siRNA mediated knockdown of mRNA is known to last for 3-7 days in rapidly dividing cells, and for weeks in non-dividing cells (Bartlett & Davis, 2006).
  • siRNAs have been developed for hepatitis B virus (Morrissey et al., 2005); human papilloma virus (Niu et al., 2006), liver cirrhosis (Sato et al., 2008), ovarian cancer (Haider et al., 2006), and bone cancer (Takeshita et al., 2005); the siRNA molecules described in the publications and others can be incorporated into the conjugate compounds of the invention to treat these and other diseases.
  • cytoplasmic delivery of siRNAs in vitro have been shown to require one of a variety of transfection methods: linkage to cell penetrating peptides (Turner et al., 2007; Deshayes et al., 2008; Lebleu et al., 2008); conjugation to cholesterol
  • RNAi is a cytoplasmic process
  • efficient delivery to the cytosol following either adsorption to the cell surface or from the lumen of an endosomal vesicle after fluid phase endocytosis is absolutely essential.
  • a diphtheria toxin-based system can be used to facilitate cytosolic delivery of oligonucleotide antisense cargo.
  • peptide-PNA can be conjugated with Fragment B from the non-toxic mutant of diphtheria toxin, CRM 197.
  • the approach is based upon methods that were developed for the reconstitution of "native" diphtheria toxin from two non-toxic mutants, CRM 197 and CRM45 (Uchida et al., 1973).
  • Fragment B197 can be purified from CRM197 according to the method of Uchida et al. (1973). Briefly, purified CRM197 (List Biological Laboratories, Campbell, CA) is treated with immobilized trypsin for 10 min at 37°C in the presence of 10 mM dithiothreitol in 0.02M Tris-HCl buffer at pH 8.0. The reaction is stopped by centrifuging the reaction mix through a spin column. This method allows the separation of the cleaved ppolypeptides from the immobilized trypsin which does not pass through the column frit. Under denaturing conditions the trypsin treated CRM 197 is then be purified by HPLC sizing in order to separate fragment A197 from B197.
  • CHO- Kl cells express the hb-EGF-like precursor receptor for diphtheria toxin, the peptide- PNA-Fragment B197 conjugate should readily bind to the cell surface and be internalized into an early endosomal compartment.
  • peptide-PNA alone should not, by itself, be delivered and therefore should not give rise to luciferase expression
  • CRM 197 can be used as a competitive inhibitor of peptide-PNA-Fragment B197 binding to the hb-EGF-like precursor receptor
  • Bafilomycin Al can be used as an inhibitor of the vesicular ATPase in order to establish that vesicle acidification (required for Fragment B transmembrane domain insertion and pore formation in the endosomal membrane) is required for delivery of the peptide-PNA into the target cell cytosol.
  • Trizol extracted RNA can be used for RT-PCR to confirm the expression of full length luc mRNA.
  • RNA extracted from Luc-IVS2 CHO-Kl cells can be used as the positive control for RT-PCR.
  • primers which hybridize to sequences flanking Luc-IVS2 introns can be used: TTGATATGTGGATTTCGAGTCGTC and
  • Diphtheria fragment B be used to successfully deliver a "chemically unique" PNA cargo.
  • the DT platform described herein can be used to deliver to the cytosol of a target cell a chemically unique PNA cargo. It is known that N-terminal extension fusion proteins of fragment A which include a duplicate A fragment and some of apolipoprotein Al can be delivered into the eukaryotic cell cytosol (Madhus et al., 1992). Thus, other cargo, such as a peptide-PNA (see Fig.l 1) disulfide cross-linked to fragment B of diphtheria toxin, can be delivered into the eukaryotic cell cytosol.
  • a peptide-PNA see Fig.l 1
  • An alternative construct may be to cross link a PNA-Cys to a modified Fragment A (A197).
  • A197 modified Fragment A
  • a Cys residue can be inserted at the N-terminal end of fragment A 197 in order to form a construct that is analogous to those described by Madhus et al. (1992).
  • the recombinant protein can be expressed, purified and used to make the conjugate PNA-Cys-S-S-CRM197.
  • the sense or antisense strand can be modified with a 3'-thiopropyl moiety that can be used for peptide coupling (this same modification can be used to couple any of the nucleic acid molecules described herein as "cargo" to the Y moiety for formation of the conjugate compounds of the invention).
  • this same modification can be used to couple any of the nucleic acid molecules described herein as "cargo" to the Y moiety for formation of the conjugate compounds of the invention.
  • peptide-PNAs and peptide-siRNAs can be synthesized chemically using techniques known in the art.
  • Peptide-siRN A/Fragment B 197 mediated silencing of Luc gene expression in CHO-Kl cells: As described above, protective antigen PA63 mediated the effective delivery of peptide-PNA and correction of aberrantly spliced
  • GCCTGAAGTCTCTGATTAAGT-3 ' reverse 5'- ACACCTGCGTCGAAGT-3 '
  • mRNA isolated from siRNA treated and control samples and correlated to direct measurements of luciferase activity using the Promega luciferase assay system with RLU determined by Wallac Microbeta, Victor2 (Perkin Elmer) counter or Turner Designs luminometer.
  • the A-form of RNA can be thread through the pore formed by the fragment B197 transmembrane domain
  • transmembrane domain helices 5-9 of diphtheria toxin has been measured by Kagan et al. (1981) to be >18A and by Zalman & Wisnieski (1984) to be 24A in diameter. Based upon these reports the pore formed in the endosomal vesicle by the diphtheria toxin transmembrane domain is of sufficient size to allow the passage of peptide- siRNA and peptide PNA from the vesicle lumen to the cytosol.
  • the disulfide bond between the cargo (peptide-siRNA and peptide-PNA) and fragment B197 can be reduced in the endosomal vesicle lumen
  • native diphtheria toxin may be "nicked” by proteases either during its purification from culture filtrates of Corynebacterium diphtherias, in serum, or on the cell surface by the endoproteinase furin.
  • both native diphtheria toxin and the diphtheria toxin-related fusion protein toxins must be nicked in the protease sensitive loop between the A and B fragments into or to deliver the A fragment to the target cell cytosol.
  • the A and B fragment of nicked diphtheria toxin are known to remain disulfide bond cross-linked until the fragments emerge from the transmembrane pore and are presented on the cytosolic surface of the endosomal vesicle and into the reducing environment of the cytosol.
  • Ratts et al., (2003) demonstrated that the disulfide bond linking the A and B fragment of the toxin are reduced in the cytosol by thioredoxin reductase.
  • the peptide-siRNA fragment B197 conjugate and the peptide-PNA fragment B197 conjugate are expected to remain disulfide bond cross-linked until the peptide-siRNA is delivered into the cytosol.
  • a bacterial protein toxin can facilitate the cytosolic delivery of antisense oligon ucleotide.
  • anthrax protective antigen binds to its cell surface receptors capillary morphogenesis gene 2 (CMG2) and tumor endothelial cell marker 8 (TEM8) (Bradley et al., 2001 ; Scobie et al., 2003). Once bound to its receptor, PA83 is cleaved to PA63 by the endoprotease furin (Klimpel et al., 1992; Gordon et al., 1995) and then reorganizes into a homo-heptamer (Milne et al., 1994).
  • CMG2 capillary morphogenesis gene 2
  • TEM8 tumor endothelial cell marker 8
  • Each heptamer is then able to bind up to three molecules of Lethal Factor and/or Edema Factor.
  • Anthrax toxin is then internalized in clathrin coated pits which become early endosomal vesicles. Upon acidification of the endosomal lumen, PA63 undergoes a dynamic change and spontaneously inserts into the vesicle membrane forming a pore.
  • the mechanism of Lethal Factor entry into cytosol parallels that of diphtheria toxin and requires COPI complex binding to one or more of multiple KXKXX sequences and a Tl-like motif in the N-terminal region of the protein (Tamayo et al, 2008).
  • PA63 would facilitate both the delivery of PNA-Lys 8 through the PA63 pore and its release into the eukaryotic cell cytosol.
  • CHO-K1 Luc-VIS-654 cells were exposed to PNA-Lys 8 in the absence or presence of either PA83 or PA63 and measured cytosolic delivery by induction of functional luciferase activity by measuring both chcmiluminescence and luc mRNA.
  • Figure 1 A shows a partial amino acid sequence of diphtheria toxin fragment B from Serine] 95 to Asparagine 235 showing transmembrane helices 1 and 2, the Tl motif and the multiple lysine residues that were anticipated to be most likely to mediate binding to COPI complex proteins and thereby facilitate catalytic domain entry.
  • DAB 389 IL-2 is composed of native DT catalytic and transmembrane domain sequences, amino acids 1 -389, to which human interleukin 2 is genetically fused in the correct translational reading frame (Williams et ah, 1990).
  • transmembrane domain helix 1 a combination of at least three lysine residues is most likely to be required for binding to COPI complex proteins and subsequent delivery of the C-domain. Furthermore, the spacing between these residues appears to affect the efficiency of C-domain entry into the cytosol as reflected by the 2-log range in cytotoxic potency of this group of single K ⁇ A mutants. If this were the case, then either the introduction of any pair of K ⁇ A mutations or the quadruple K ⁇ A mutation in transmembrane helix 1 should lead to a complete loss of cytotoxic activity.
  • double mutants DAB(K213A, K215A) 389 IL-2 and DAB(K215A, K217A) 389 IL-2, and the quadruple mutant DAB(K213A, K215A, K217A, K222A) 389 IL-2 were constructed. Following expression and purification, each mutant fusion protein was then assayed for cytotoxic activity. As anticipated, both of the double K ⁇ A mutants as well as the quadruple K ⁇ A mutant were found to be non-toxic (IC 50 > 5 x 10 "7 M) (Figs.lC & ID).
  • the structural gene encoding DAB 389 IL2 was modified such that amino acids 212 - 223 which encompasses transmembrane helix 1 was deleted and replaced with the 13 amino acid sequence encoding the COPI binding portion of the p23 cytoplasmic tail (REILKKAKFFRRL). Following its genetic construction, the plasmid encoding the mutant toxin DAB(212p23) 3 8 9 IL-2 was cloned, sequenced to verify correct insertion and reading frame of the COPI binding segment, and the recombinant mutant protein was expressed and purified as described in Experimental procedures below. As shown in Figure 2, dose response analysis of
  • DAB(212p23) 389 IL-2 on Hutl02 cells is identical to that of the wild type DAB 389 IL-2 (IC50 3 ⁇ 4 10 pM).
  • the functional equivalence between the lysine-rich transmembrane helix 1 and the COPI binding segment from the cytoplasmic tail of the p23 adaptor protein suggests maintenance of the COPI complex binding function of this region is an essential feature in the catalytic domain entry process.
  • CBM 1,3- cyclohexanebis(methylamine)
  • Lemichez et al. (1997) were the first to describe an in vitro assay to investigate the requirements for translocation of the diphtheria toxin catalytic domain from the lumen of acidified endosomal vesicles to the external medium.
  • endosomal vesicles were pre-loaded with native diphtheria toxin in the presence of Bafilomycin Al, and the early endosomal vesicle enriched fraction was isolated by sucrose density gradient ultracentrifugation. Upon removal of
  • TKIESLKEHG transmembrane helix 1 of diphtheria toxin. Since the cytosolic expression of a peptide which carried the Tl motif in HuT102 cells was found to confer resistance to the toxin and knock down of peptide expression restored toxin sensitivity, it was apparent that either the Tl motif or transmembrane helix 1 of diphtheria toxin is likely to play an essential role in the catalytic domain entry process. In addition, pull down experiments using GST-DT 140-271 demonstrated that at least the ⁇ -COP subunit of the COPI complex specifically bound to at least a portion of the Tl motif, and that this association was also essential for catalytic domain entry process.
  • the Tl motif includes two lysine residues which may play a role in COPI binding and catalytic domain entry; however, in anthrax lethal factor and anthrax edema factor the separation of their respective Tl- like motifs from the multiple upstream KXKXX COPI binding sequences raises addition questions as to the role that the Tl motif per se may play in the entry process.
  • a site-directed mutational analysis of the multiple lysine residues in the N-terminal end of lethal factor was begun in order to determine the minimal sequence necessary for both COPI complex binding and delivery of
  • transmembrane helix 1 is dependent upon the presence and spacing of at least three of the four lysine residues, two of which are positioned immediately upstream of the consensus Tl motif ( TKTKIESLKEHG).
  • a site- directed mutational analysis of transmembrane helix 1 was first conducted in order to determine the minimal number of lysine residues that were necessary to facilitate catalytic domain delivery to the eukaryotic cell cytosol. Following site-directed mutagenesis and DNA sequence analysis to ensure the introduction of each mutation, individual mutant recombinant proteins were expressed, purified, and tested for cytotoxic activity by dose response analysis on HuT102 cells.
  • K215A With exception of a single mutation (K215A), all of the single K ⁇ A mutant forms of DAI1 ⁇ 2 ⁇ )IL-2 displayed only a modest 1 - 2-log reduction in their respective cytotoxic potency. In marked contrast, the introduction of double K ⁇ A mutations ⁇ e.g., K213A, K215A or K215A, K217A) resulted in over a 5-log reduction in cytotoxic potency in their respective mutant fusion protein toxins.
  • double K ⁇ A mutations ⁇ e.g., K213A, K215A or K215A, K217A
  • transmembrane helix 1 of diphtheria toxin and COPI components appears to be unlike the interactions mediated by the canonical di- lysine signature KKXX with a-COP and ⁇ '-COP (Eugster et ah, 2004; Letourneur et ah, 1994).
  • cargo and adaptor protein interactions with ⁇ -COP have been reported to exhibit more diversity in their binding profile including KKXX and KXKXX motifs (Eugster et al., 2004; Zerangue et al. 2001), and results presented here suggest this diversity of binding may be extended to transmenbrane helices 1 and 2 of diphtheria toxin as well.
  • the molecular process by which the catalytic domain from DAB 38 9iL-2 is delivered to the eukaryotic cell cytosol appears to follow the following steps: (i) binding of the toxin to its respective cell surface receptor (Naglich et al, 1992), (ii) the internalization of the toxin: :receptor complex by receptor mediated endocytosis into an early endosomal compartment (Moya et al., 1985), (iii) upon acidification of the vesicle lumen by the (v)ATPase, the transmembrane domain spontaneously inserts into the endosomal vesicle membrane forming an 18 - 22A pore or channel (Donovan et al, 1981 ; Kagan et al., 1981 ).
  • transmembrane helices 1-4 of the toxin with its disulfide bond linked catalytic domain appear to be un-tethered and readily “pulled” through the transmembrane vesicle pore formed by helices 5 - 9.
  • Bovine liver COPI purification COPI enriched fractions were prepared from bovine liver cytosol as described by Waters et al. (1991) with some modifications described by Tamayo et al. (2008).
  • the 13S fraction containing intact COPI was further purified by DE52 (Whatman) column following manufacture's specifications in a Biologic LP (Bio-rad). Briefly, the DE52 cellulose was equilibrated with 25 mM Tris-HCl (pH 7.4)/ 100 mM KCl, 1 mM DTT, 10% Glycerol.
  • the column was eluted with a step gradient of 150, 500, 750 and 1000 mM KCl in 25 mM Tris-HCl (pH 7.4)/ 10% Glycerol/ 1 mM DTT.
  • the elution corresponding to 500 mM KCl containing intact COPI complex and associated material was dialyzed against 25 mM Tris.HCl (pH 7.4), 10% Glycerol and used as input material for the precipitation assays.
  • COPI subunits In Vitro Synthesis of COPI subunits.
  • Vector pCMV6-XL5 carrying the structural human gene encoding ⁇ ' COP (NM_004766.1), ⁇ -COP (NM 016128.3) and ⁇ -COP (NM_007263.3) were purchased from OriGene Technologies (Rockville, MD).
  • Full length COPI subunits were synthesized in vitro by using the TNT Quick Coupled Transcription/Translation System (Promega) in the presence of [ 35 S] -methionine following the manufacturer's instructions (2 ⁇ g of plasmid DNA/ 100 ⁇ of reaction volume).
  • reaction mixture was diluted to 300 ⁇ with binding buffer (50 mM Tris HC1, pH 7.4/150 mM NaCl/1 mM EDTA/1% Nonidet P- 40/ IX protease inhibitor cocktail (Roche)). Pull-down experiments with GST and GST-DT140-271 were performed as described above. Elutions were then analyzed by SDS/PAGE and autoradiographed according to standard methods.
  • binding buffer 50 mM Tris HC1, pH 7.4/150 mM NaCl/1 mM EDTA/1% Nonidet P- 40/ IX protease inhibitor cocktail (Roche)
  • Bacterial strains, plasmids and fusion toxin products Bacterial strains, plasmids and fusion toxin products.
  • the parental plasmid pET- JV127 (vanderSpek et al., 1993) encoding for the fusion toxin DAB 389 IL-2 (AAA72359) was used for cloning and purification of the mutant toxins.
  • the introduction of the alanine exchange mutations and the p23 adaptor COPI binding sequence swap was performed by site-directed mutagenesis between the Nsil and Rsill restriction sites (Table 4).
  • Table 4 Plasmids and IL-2 receptor-targeted fusion toxins used in this study.
  • Plasmid tox gene product amino acid sequence (212-225) pETJV127 DAB 3 89lL-2 DKTKTKIESLKEHG pETCT20 DAB(K213A) 389 IL-2 DATKTKIESLKEHG pETCT30 DAB(K215A)389IL-2 D TATKIESLKEHG pETCT40 DAB(K217A)3 89 IL-2 DKTKTAIESLKEHG pETCT50 DAB(K222A) 389 IL-2 DKTKTKIESLAEHG pETCT80 DAB(K213 A,K215 A) 389 IL-2 DATATKIESLKEHG pETCT90 DAB(K215 A,K217A) 389 lL-2 DKTATAIESLKEHG pETCT60 DAB(K213,K215,K217,K222- >A) 389 IL-2 DATATAIESLAEHG pETCT70 DAB(212p23) 389 IL-2
  • Cytotoxicity Assay Cytotoxicity assays were performed as described by vanderSpek et al. (1994). Figures were created in GraphPad Prism version 5.01 for Windows, GraphPad Software, San Diego California USA. References
  • ADP-ribosylation factor a small GTP-binding protein, is required for binding of coatomer protein beta- COP to Golgi membranes. Proc Natl Acad Sci, USA 89: 6408-6412.
  • Coatomer is essential for retrieval of dilysine- tagged proteins to the endoplasmic reticulum. Cell 79: 1199-1207.
  • Nickel, W., and Wieland, F.T. 2001 Receptor-dependent formation of COPI-coated vesicles from chemically defined donor liposomes. Methods Enzymol 329: 388-404.
  • Boquet P Silverman MS, Pappenheimer AM Jr, & Vernon WB. (1976) Binding of triton X-100 to diphtheria toxin, crossreacting material 45, and their fragments. Proc Natl Acad Sci, USA, 73: 4449- 4453.
  • RNAi is related to intracellular release of SIRNA via a covalently attached signal peptide.
  • RNA [Epub ahead of print].
  • RNA interference-mediated gene silencing of pleiotrophin through poyethylenimine-complexed small interfering RNAs in vivo exerts antitumoral effects in glioblastoma xenografts.
  • RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med, 9: 347-351.
  • COPI coatomer complex proteins facilitate the translocation of Anthrax Lethal Factor across vesicular membranes in vitro. Proc. Natl. Acad. Sci., USA, 105: 5254-5259.

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Abstract

L'invention concerne des compositions pour l'administration d'agents cytotoxiques, thérapeutiques, diagnostiques à une cellule cible et des procédés d'utilisation de ces compositions pour le traitement de maladies.
PCT/US2011/033229 2010-04-22 2011-04-20 Compositions et procédés de ciblage et d'administration d'agents thérapeutiques dans des cellules WO2011133658A1 (fr)

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WO2016191869A1 (fr) * 2015-06-01 2016-12-08 The Hospital For Sick Children Administration de cargo polypeptidique de diverses structures dans des cellules mammaliennes par une toxine bactérienne
WO2020206375A1 (fr) * 2019-04-03 2020-10-08 Technical University Of Denmark Conjugués de protéine de facteur neurotrophique et modes de réalisation associés
US11045546B1 (en) 2020-03-30 2021-06-29 Cytodyn Inc. Methods of treating coronavirus infection

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