WO2009131435A1 - Lieur contenant de la bungarotoxine et un peptide de liaison - Google Patents

Lieur contenant de la bungarotoxine et un peptide de liaison Download PDF

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Publication number
WO2009131435A1
WO2009131435A1 PCT/NL2008/050236 NL2008050236W WO2009131435A1 WO 2009131435 A1 WO2009131435 A1 WO 2009131435A1 NL 2008050236 W NL2008050236 W NL 2008050236W WO 2009131435 A1 WO2009131435 A1 WO 2009131435A1
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Prior art keywords
binding pair
peptide
btx
bungarotoxin
active compound
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PCT/NL2008/050236
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English (en)
Inventor
Ralph Alexander Willemsen
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Erasmus University Medical Center Rotterdam
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Priority to PCT/NL2008/050236 priority Critical patent/WO2009131435A1/fr
Publication of WO2009131435A1 publication Critical patent/WO2009131435A1/fr

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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention is in the field of drug targeting.
  • the present invention is in the field of the design and development of drugs that can be targeted and internalized into target cells in order to exert their preventive or curative effect.
  • the invention provides methods of producing targeted drugs as well as drug conjugates comprising a pharmaceutical active and a targeting moiety, linked via a specific peptide linker.
  • Drug targeting is generally aimed at altering the distribution of a drug in the body of the recipient such that the accumulation at the desired site of action is enhanced or favoured.
  • the site of action is usually remote from the site of administration.
  • Non-targeted drug compounds typically reach their intended target cells via whole-body diffusion and passive diffusion or receptor mediated uptake over the cell membrane.
  • targeted drugs home-in and concentrate mainly at the targeted tissues. Consequently, targeted drugs require smaller dosages while still allowing the drug to reach therapeutically effective levels inside the target cells.
  • the preferred lipophilic character of non -targeted drugs which facilitates their easy passage over the cell membrane and which feature is not always in agreement with other requirements of the drug, is less relevant to targeted drugs.
  • the targeting of drugs to specific cells is an attractive method to enhance specificity, to decrease systemic toxicity and to allow for the therapeutic use of compounds that are in principle less suitable or unsuitable as systemic drugs.
  • drug delivery technologies are aimed at altering the interaction of the drug with the in vivo environment and achieve that objective by conjugation of the drug with other molecules, entrapment of the drug within matrices or particles or simply by co-administration with other agents.
  • the net result is either drug targeting or enhanced drug transport across biological barriers such that its bioavailability is improved with a reduction of the incidence of clinical side effects in subjects.
  • Cell-selective delivery of drugs can, in principle, be obtained by coupling drug molecules to targeting moieties (macromolecular carriers that contain a chemical moiety that is specifically recognised by target cells in the diseased tissue).
  • targeting moieties macromolecular carriers that contain a chemical moiety that is specifically recognised by target cells in the diseased tissue.
  • cell-specific drug targeting preparations are only scarcely available due to major technological difficulties.
  • the linkage between the therapeutic agent and the targeting device often poses significant problems. For instance, chemically reactive groups for conventional conjugation chemistry may be absent, or chemically reactive groups may be (abundantly) present, but covalent linkage may (irreversibly) inhibit the bioactivity of the coupled therapeutic agent.
  • BTX bungarotoxin
  • BTX-tag a 13 amino acid polypeptide
  • the present invention provides a method for preparing a targetable drug comprising an active compound and a targeting moiety, comprising:
  • said active compound acts intracellular Iy.
  • said active compound is a virus, a non-viral particle, cytostatic agent or a radionuclide.
  • the targeting moiety is preferably a receptor ligand, an antibody or an antibody fragment, a T-cell receptor or T- cell receptor fragment or a small molecule.
  • the present invention provides a targetable drug comprising an active compound and a targeting moiety linked via the members of a binding pair, said binding pair consisting of the bungarotoxin (BTX) protein having the amino acid sequence as provided in SEQ ID NO:2 or 4 as a first member of said binding pair and a peptide comprising the bungarotoxin- binding site having the amino acid sequence as provided in SEQ ID NO:7 as a second member of said binding pair.
  • the active compound acts intracellularly, that is, it acts by traversing the cell membrane of a target cell.
  • the active compound is a virus, a non-viral particle, a cytostatic agent or a radionuclide.
  • the targeting moiety is a receptor ligand, an antibody or an antibody fragment, or a small molecule.
  • the present invention provides the use of a binding pair consisting of bungarotoxin (BTX) and a peptide comprising the bungarotoxin-binding site as defined above as a linker for binding a targeting moiety to an active compound, to thereby provide a targetable drug complex for transport over the cytoplasmic membrane of a target cell.
  • BTX bungarotoxin
  • the present invention provides a linker for linking two biomolecules, said linker comprising the binding pair consisting of the bungarotoxin (BTX) protein having the amino acid sequence as provided in SEQ ID NO:2 or 4 as the first member of said binding pair and a peptide comprising the bungarotoxin-binding site having the amino acid sequence as provided in SEQ ID NO:7 as the second member of said binding pair.
  • BTX bungarotoxin
  • the present invention provides a conjugate of at least two biomolecules wherein said at least two biomolecules are linked via the linker of claim 10.
  • the biomolecules are preferably proteins, peptides or nucleic acids.
  • a conjugate according to the present invention preferably does not include the conjugate or complex that is described in WO2007/073147. Therefore, in a preferred embodiment of aspects of the present invention the first or second member of the binding pair of the present invention are not comprised in a linker peptide comprising a multimerisation motif, and the first or second member of the binding pair are not comprised in a polypeptide capable of recognizing and binding to a specific Major Histocompatibility Complex (MHC)- peptide complex, under conditions wherein said linker peptide and said polypeptide are part of a multivalent monospecific protein complex comprising at least six of said polypeptides and at least one linker peptide, as specified in WO2007/073147.
  • MHC Major Histocompatibility Complex
  • Figure Ia shows the result of specific transfection of polyplexes loaded with anti-PSMA scFv/BTX (J591). Preformed polyplexes containing the BTX peptide were loaded with scFv/BTX fusion protein, specific for PSMA and incubated with PSMA negative cells (MZ2-MEL3.0) and PSMA positive cells (LNCap). As a control, LNCap cells were also incubated with polyplexes that were not loaded with fusion protein.
  • Figure Ib shows the result of specific transfection of polyplexes loaded with anti-HCV-NS3 scTCR/BTX (J591).
  • Preformed polyplexes containing the BTX peptide were loaded with scTCR/BTX fusion protein, specific for HCV-NS3and incubated with HCV-NS3 negative B-cells andHCV- NS3 positive B-cells.
  • HCV-NS3 positive B-cells were also incubated with polyplexes that were not loaded with fusion protein.
  • Figure 2 shows the in vivo targeting of orthotopic prostate cancer.
  • Four Athymic nude mice containing the PC346Cxenograft were injected with pre-formed polyplexes loaded with the PSMA specific scFv/BTX (J591). Tumor, spleen, lung, liver, lymph-node and cecum were harvested after sacrifice and lysed. Luciferase was measured. Data represent mean of four samples.
  • Figure 3 shows the result of specific infection of adenovirus Type 5 redirected by the addition of BTX fusion proteins.
  • 3A Specific infection of Adenovirus type 5 mediated through the addition of scFv/BTX and scTCR/BTX fusion proteins.
  • Upper left infection of HLA-AlPOS/MAGE-AlPOS, CARNEG melanoma cells by Ad5 loaded with scFv Hyb/BTX.
  • Lower left control HLA- AlPOS/MAGE-Alneg, CARNEG melanoma cells that are not infected with scFv Hyb3/BTX loaded Ad5.
  • Figure 4 shows the result of specific retargeting of polymer coated Adenovirus type 5 by loading with anti-PSMA specific scFvBTX fusion protein.
  • Upper left infection of PSMAPOS LNCap cells with Ad (wildtype Ad5), polymer coated Ad5 and polymer coated Ad5 after adding the psma specific scFvBTX fusion protein. Polymer coating of Ad5 reduces infection to background levels, which is restored after addition of the scFv/BTX fusion protein.
  • Upper right Infection of PSMANEGPC-3 cells with Ad (wildtype Ad5), polymer coated Ad5 and polymer coated Ad5 after adding the psma specific scFvBTX fusion protein.
  • Adenoviral particle count determined by QPCR of infected PSMANEG PC- 3cells: no enhanced number of particles after addition of scFv/BTX to polymer coated virus.
  • active compound refers to biologically- active (therapeutic) agents or medicaments, preferably those that act intracellularly, including but not limited to small molecules, proteins, nucleic acids, polyplexes, and viruses.
  • active compound is a cytostatic agent.
  • targeting moiety refers to the moiety conjugated to the therapeutic agent by means of the linking system as disclosed herein, wherein said moiety causes the site selective delivery of the conjugate.
  • the targeting moiety may be any molecule that can provide delivery specificity to an active compound attached thereto and that is capable of binding specifically to a certain cell, cell surface molecule, or tissue.
  • targeting moieties include, but are not limited to, cell surface receptor ligands, antibodies, targeting sequences such as the protein transduction domain (PTD), small molecules, etc.
  • Cell surface receptors such as T-cell receptors can be reformatted into single chain TCR (scTCR) and when expressed as fusions with BTX be used to target cells expressing intracellular antigens such as the melanoma associated antigen gplOO or Hepatitis C Virus non-structural protein 3 (see examples).
  • bungarotoxin refers to ⁇ -bungarotoxin. ⁇ -
  • Bungarotoxin is one of the components of the venom of the snake species Bungarus multicinctus (also known as the Taiwanese banded krait).
  • the protein binds irreversibly and competitivly to the acetylcholine receptor found at the neuromuscular junction, causing paralysis, respiratory failure and death in the victim of the snake's bite.
  • ⁇ -Bungarotoxin is also a selective antagonist of the ⁇ 7 nicotinic acetylcholine receptor (AchR) in the brain, and as such has applications in neuroscience research.
  • ⁇ -bungarotoxin binds with great affinity to the ACh-binding sites of muscle-type nicotinic AChRs thereby inhibiting their function, and can be used as a histological label, affinity column ligand, and probe for the ACh-binding site of the purified AChRs.
  • Two isoforms exist of the 74 amino acid protein, which differ in an A to V mutation at position 31 (isoforms A31 and V31, respectively), which mutation is due to a single C— »T transition in the gene. Both isoforms are useful in aspects of the present invention.
  • small molecule refers to a non-peptidic, non-DNA, and non-polymeric organic compound either synthesized in the laboratory or found in nature typically containing several carbon-carbon bonds with a molecular weight of less than 2800 daltons, preferably less than 1500 daltons.
  • Known naturally-occurring small molecules include, but are not limited to, penicillin, erythromycin, taxol, cyclosporin, and rapamycin.
  • synthetic small molecules include, but are not limited to, ampicillin, methicillin, sulfamethoxazole, and sulfonamides.
  • peptide and protein are interchangeable, and each refers to a sequence of naturally occurring amino acids linking through a peptide bond.
  • peptide is intended to refer to a sequence of less than 50, preferably less than 20 amino acid residues, "protein” to a sequence of 20 or more, preferably 50 or more amino acid residues.
  • the terms include reference to naturally occurring peptides and proteins, as well as synthetic peptides and proteins.
  • peptide and “protein” are also inclusive of modifications including, but not limited to, alkylation, acylation, glycosylation, lipid attachment, sulfation, gamma- carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. Both full-length proteins and fragments thereof are encompassed by the terms “peptide” and “protein”.
  • the terms also include modifications, such as deletions, additions and substitutions (generally conservative in nature), to the native amino acid sequence.
  • nucleic acid includes reference to a deoxyribonucleotide or ribonucleotide polymer, i.e. a polynucleotide, in either single-or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e. g., peptide nucleic acids).
  • a polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof.
  • DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein.
  • DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.
  • polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including among other things, simple and complex cells.
  • antibody includes reference to antigen binding forms of antibodies (e. g., Fab, F(ab)2).
  • antibody frequently refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof which specifically bind and recognize an analyte (antigen).
  • antibody fragments can be defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology.
  • antibody fragments such as single chain Fv, chimeric antibodies (i. e., comprising constant and variable regions from different species), humanized antibodies (i. e., comprising a complementarity determining region (CDR) from a non-human source) and heteroconjugate antibodies (e. g., bispecific antibodies).
  • Non-viral particle refers to particles that are not intact viruses.
  • Non-viral paritcles include, but are not limited to, microp articles, nanoparticles, virosomes, virus-like particles, liposomes and encapsulated nucleoproteins, including wholly or partially assembled viral particles, in lipid bilayers.
  • Non-viral particles may also include, for example, cationic liposomes and polycations.
  • Virus-like particles are structures built in an organized and geometrically regular manner from many polypeptide molecules of one or more types. Being comprised of more than one molecule, VLPs can be referred to as being supramolecular. VLPs lack the viral genome and, therefore, are noninfectious.
  • VLPs can be produced in large quantities by heterologous expression and can be easily purified.
  • the geometry of a VLP typically resembles the geometry of the source virus particle, so for most cases discussed below, the geometry has icosahedral or pseudo-icosahedral symmetry. VLPs are being exploited in the area of vaccine production because of their structural properties, their ease in large scale preparation and purification, and their non-infectious nature.
  • VLPs include but are not limited to the self- assemblages of capsid or nucleocapsid polypeptides of hepatitis B virus [WO 92/11291], rotavirus [US 5,071,651 and US 5,374,426] retroviruses [WO 96/30523; US 6,602,705], the retrotransposons [e.g., the Ty polypeptide pi, in US 6,060,064], and human papilloma virus [WO 98/15631]. Plant-infecting virus capsid polypeptides also self- assemble into VLPs in vitro and in vivo.
  • radioactive isotope refers to an atom with an unstable nucleus, which undergoes radioactive decay, and emits a gamma ray(s) and/or subatomic particles.
  • the term is equivalent to the term radioactive isotope or radioisotope.
  • affinity tag refers to an amino acid sequence that has been engineered into or on a protein (for instance in the form of a fusion protein) to make its purification easier.
  • the tag could be another protein or a short amino acid sequence, allowing purification by affinity chromatography.
  • purification tag is equivalent to the term purification tag.
  • the targetable drug delivery system in aspects of the present invention formed by targeting-moiety-conjugated drug complex of the invention, is especially suitable for intracellular delivery, such as intra- organelle or intranuclear delivery.
  • the present invention thus provides for the intracytoplasmic and/or intranuclear delivery of active compounds, such as small molecules, proteins or DNA.
  • Therapeutic agents may be used interchangeably herein and include genetic therapeutic agents, non-genetic therapeutic agents and cells.
  • High-and low-molecular weight therapeutic agent can be selected from suitable members of the lists of therapeutic agents to follow.
  • non-genetic therapeutic agents for use in connection with the present invention include:
  • antithrombotic agents such as heparin, heparin derivatives, urokinase, and PPack (dextrophenylalanine proline arginine chloromethy Ike tone) ;
  • anti-inflammatory agents or immunosuppressants such as betamethasone, budesonide, corticosterone, dexamethasone, estrogen, everolimus, glucocorticoids, mesalamine, prednisolone, sirolimus, sulfasalazine and tacrolimus;
  • anti- neoplastic/antiproliferative/anti-miotic agents such as acetylsalicylic acid, actinomycin, adriamycin, amlodipine, angiopeptin, angiostatin, azathioprine, cisplatin, doxazosin, endostatin, enoxaprin, epothilones, 5-fluorouracil, halofuginone, hirudin, methotrexate, mutamycin, paclitaxel, vinblastine, vincristine, monoclonal antibodies capable of blocking cell proliferation, and thymidine kinase inhibitors;
  • anti- neoplastic/antiproliferative/anti-miotic agents such as acetylsalicylic acid, actinomycin, adriamycin, amlodipine, angiopeptin, angiostatin, azathioprine, cisplatin, doxazosin
  • anesthetic agents such as lidocaine, bupivacaine and ropivacaine
  • anti-coagulants such as D-Phe-Pro-Arg chloromethyl keton, an RGD peptide-containing compound, heparin, antithrombin compounds, platelet receptor antagonists, anti-thrombin anticodies, anti-platelet receptor antibodies, aspirin (aspirin is also classified as an analgesic, antipyretic and anti-inflammatory drug), dipyridamole, protamine, hirudin, prostaglandin inhibitors, platelet inhibitors and tick antiplatelet peptides
  • cell growth promoters such as growth factors (FEGF, all types including VEGF-2), growth factor receptors, transcriptional activators, and translational promoters
  • cell growth inhibitors such as growth factor inhibitors, growth factor receptor antagonists, transcriptional repressors, translational repressors, replication inhibitors, inhibitory antibodies, antibodies directed against growth factors, bifunctional molecules consisting of a growth factor and a cytotoxin, bifunctional molecules consisting of an antibody and a cytotoxin;
  • protein kinase and tyrosine kinase inhibitors such as tyrphostins, genistein, and quinoxalines;
  • prostacyclin analogs such as tyrphostins, genistein, and quinoxalines;
  • prostacyclin analogs such as tyrphostins, genistein, and quinoxalines;
  • j prostacyclin analogs;
  • cholesterol-lowering agents such as angiopoietins;
  • antimicrobial agents such as penicillin, cefoxitin, oxacillin, tobramycin, triclosan, cephalosporins, aminoglycosides and nitrofurantoin;
  • cytotoxic agents, cytostatic agents and cell proliferation affectors which cause cell death or inhibit cell proliferation primarily by interfering directly with the cell's functioning or inhibit or interfere with cell mytosis, including alkylating agents, tumor necrosis factors, intercalators, hypoxia activatable compounds, microtubule inhibitors/microtubule -stabilizing agents, inhibitors of mitotic kinesins, inhibitors of kinases involved in mitotic progression, antimetabolites; biological response modifiers; hormonal/anti- hormonal therapeutic agents, haematopoietic growth factors, monoclonal antibody targeted therapeutic agents, topoisomerase inhibitors, proteasome inhibitors and ubiquitin ligase inhibitors;
  • hormones such as probucol and alpha-tocopherol, and
  • drugs for heart failure such as dioxin, beta-blockers, angiotensin-converting enzyme (ACE) inhibitors including captopril and enalopril;
  • ACE angiotensin-converting enzyme
  • Toll-like receptor ligands such as poly (I, C).
  • exemplary genetic therapeutic agents for use in connection with the present invention include anti-sense DNA and RNA as well as DNA or genes coding for:
  • angiogenic factors including growth factors such as acidic and basic fibroblast growth factors, vascular endothelial growth factor, epidermal growth factor, transforming growth factor ⁇ and ⁇ , platelet-derived endothelial growth factor, platelet-derived growth factor, tumor necrosis factor ⁇ , hepatocyte growth factor and insulin-like growth factor;
  • cell cycle inhibitors including CD inhibitors, and
  • TK thymidine kinase
  • BMP's bone morphogenic proteins
  • BMP-7 bone morphogenic proteins
  • dimeric proteins can be provided as homodimers, heterodimers, or combinations thereof, alone or together with other molecules.
  • molecules capable of inducing an upstream or downstream effect of a BMP can be provided.
  • Such molecules include any of the "hedgehog" proteins, or the DNA's encoding them.
  • Genes are usually delivered into a patient's cells through a vector, for example, a retroviral vector whose DNA is genetically engineered to include a desired DNA sequence.
  • a vector for example, a retroviral vector whose DNA is genetically engineered to include a desired DNA sequence.
  • nonviral gene transfer methods can be used, for example, plasmid DNA vectors (which can be delivered, for example, along with polymeric carriers, DNA condensing agents, lipofection agents, receptor mediated delivery vectors, and so forth).
  • Vectors for delivery of genetic therapeutic agents include viral vectors such as adenoviruses, gutted adenoviruses, adeno-associated virus, reovirus, retroviruses, alpha virus (Semliki Forest, Sindbis, etc.), lentiviruses, herpesviruses and bacteriophages, replication competent viruses (e.g., attenuated adenovirus ONYX-Ol 5) and hybrid vectors; and non-viral vectors such as artificial chromosomes and mini-chromosomes, plasmid DNA vectors (e.g., pCOR plasmid), cationic polymers (e.g., polyethyleneimine, polyethyleneimine (PEI)), graft copolymers (e.g., polyether-PEI and polyethylene oxide-PEI), neutral polymer (poly-N-vinyl pyrrolidin-2-one, PVP), lipids such as cationic lipids, liposomes
  • Cells for use in connection with the present invention include cells of human origin (autologous or allogeneic), including whole bone marrow, bone marrow derived mono-nuclear cells, progenitor cells (e.g., endothelial progenitor cells), stem cells (e.g., mesenchymal, hematopoietic, neuronal), pluripotent stem cells, fibroblasts, myoblasts, satellite cells, pericytes, cardiomyocytes, skeletal myocytes or macrophage, or from an animal, bacterial or fungal source (xenogeneic), which can be genetically engineered, if desired, to deliver proteins of interest.
  • a therapeutic agent may be encapsulated in microcapsules or nano-capsules by known methods.
  • the present invention is useful in delivering high- molecular-weight therapeutic agents.
  • high-molecular -weight therapeutic agents include polysaccharide therapeutic agents having a molecular weight greater than 1,000; polypeptide therapeutic agents having a molecular weight greater than 10,000; polynucleotides, including antisense polynucleotides, having a molecular weight greater than 2,000, gene-encoding polynucleotides, including plasmids, having a molecular weight greater than 500,000; viral and non-viral particles having a diameter greater than about 50 nanometers, and cells.
  • Typical polynucleotide therapeutic agents include the genetic therapeutic agents specifically listed above, and more generally include DNA encoding for various polypeptide and protein products including those previously listed.
  • Some additional examples of polynucleotide therapeutic agents include DNA encoding for the following: cytokines such as colony stimulating factors (e.g., granulocyte-macrophage colony- stimulating factor), tumor necrosis factors (e.g., fas ligand) and interleukins (e.g., IL-10, an antiinflammatory interleukin), as well as protease inhibitors, particularly serine protease inhibitors (e.g., SERP-I ), tissue inhibiting metalloproteinases (e.g., TIMP-I through TIMP-4), monocyte chemoattractant proteins (e.g., MCP-I), protein kinase inhibitors including cyclin-dependent kinase inhibitors (e.g., p27, p21), endogenous and in
  • Some specific classes of therapeutic agents are anti -proliferative agents, anti-inflammatory agents, anti-thrombotic agents, lipid mediators, vasodilators, anti-spasm agents, remodeling agents, endothelial-cell specific mitogens, as well as nucleotide sequences (which may further include an associated delivery vector) encoding for therapeutic agents having any one or combination of these therapeutic effects.
  • examples include plasmids that encode an antiproliferative protein or anti-inflammatory protein.
  • the present invention is especially useful in delivering low- molecular -weight therapeutic agents, such as small molecules, or therapeutic agents which are sensitive to inactivation by conjugation to a targeting moiety, such as therapeutic peptides or proteins.
  • a wide range of therapeutic agents can be used in connection with the targeted delivery system of the present invention, with the therapeutically effective amount being readily determined by those of ordinary skill in the art and ultimately depending, for example, upon the condition to be treated, the age, sex and condition of the patient, the nature of the therapeutic agent, the nature of the release region, the nature of the medical article, and so forth.
  • Targeting moieties As defined herein, a targeting moiety is capable of binding to a target molecule associated with the surface of a target cell.
  • the target cells to which the genes of interest can be delivered are basically any target cells that have a target molecule associated with their surface with which it is possible to distinguish them from other cells using a targeting moiety. The ability to distinguish may lie in the abundance of a certain target molecule on a certain subset of cells. Furthermore, it is not necessary for the application of the invention that the nature of the target molecule is known.
  • Targeting moieties can be selected from combinatorial peptide libraries on the basis of differential binding to molecules expressed on the surface of different cell types.
  • Useful combinatorial peptide libraries for the invention include those in which a large variety of peptides is displayed on the surface of filamentous bacteriophages as is well known in the art. Screening for individual library members that interact with desired target cells allows the isolation of the nucleotide sequences encoding suitable peptide structures to be used as a targeting moiety in the invention. For this purpose, libraries displaying scFv variants are particularly useful. Moreover, methods that increase the combinatorial diversity of said libraries make the number of targeting moieties that can be generated for application in the invention almost limitless. Said methods include PCR-based random mutagenesis techniques known in the art.
  • the invention discloses methods for specific delivery of therapeutic agents into any target cell that can be phenotypically distinguished from other cells, also when the nature of this distinction has not been revealed.
  • Important target cells are tumor cells, virally infected cells, cells of the hematopoietic system, hepatocytes, endothelial cells, lung cells, cells of the central nervous system, muscle cells and cells of the gastrointestinal tract.
  • Usual target molecules are receptors, surface antigens and the like.
  • receptor targeting involves linking the therapeutic agent to a ligand which has an affinity for a receptor expressed on the cell surface of a desired target cell.
  • a drug is intended to adhere to the target cell following formation of a ligand-receptor complex on the cell surface. Entry into the cell could then follow as the result of internalization of ligand-receptor complex. Following internalization, the drug may exert its therapeutic effect on the cell.
  • any targeting moiety that can be fused or coupled to a member of the BTX-BBS binding pair is now usable with the targetable delivery system according to the present invention.
  • Any molecule associated with the surface of a target cell for which a specific binding partner is known or can be produced is in principle useful as a target for the presently invented gene delivery system, if in presence or abundance it differs from one subset of cells to another.
  • Antibodies of course are a good example of suitable targeting moieties of this embodiment.
  • a preferred embodiment of the present invention encompasses a targetable delivery system wherein the targeting moiety is an antibody or a fragment or a derivative thereof, recognizing the target molecule associated with the surface of the target cell.
  • Suitable targeting moieties are ligands wherein the target molecule is a receptor (for which the targeting moiety is a ligand) associated with the surface of the cell.
  • protein ligands For many cell type-specific antigens protein ligands have been identified which bind with high specificity and/or affinity, e.g. cytokines binding to their cellular receptors.
  • any protein ligand can be coupled or fused with a member of the BTX-BBS binding pair, using simple procedures. Suitable couplings can be achieved using succinimide esters of the member of the BTX-BBS binding pair. Binding to the protein ligand may proceed through free amino groups, normally of lysyl residues.
  • Another group of suitable targeting moieties, and one that is highly preferred, are small molecules.
  • fusion proteins may be produced between the protein ligand and the protein/peptide member of the BTX-BBS binding pair.
  • the fused proteins may be separated by a peptide spacer to allow for proper folding of the proteins and ensure that each maintains its desired function of specific binding.
  • the targeting moiety is be coupled or fused the first member of the BTX-BBS binding pair to produce a targeting part of the targetable delivery system of the present invention.
  • the other part of the targetable delivery system of the present invention, the therapeutic part is formed by the active compound coupled or fused to the counterpart of the member coupled or fused to the targeting moiety.
  • the linker The present invention also provides a linker for linking two biomolecules.
  • the linker of the present invention comprises the binding pair consisting of the bungarotoxin (BTX) protein having the amino acid sequence as provided in SEQ ID NO:2 or 4 as the first member of the binding pair and a peptide comprising the bungarotoxin-binding site (BBS) having the amino acid sequence as provided in SEQ ID NO:7 as the second member of the binding pair.
  • BTX bungarotoxin
  • BSS bungarotoxin-binding site
  • biomolecules that are mutually linked by the linker of the present invention can be synthetic molecules having biological activity or natural molecules, preferably organic molecules, most preferably proteins.
  • the two biomolecules linked by the linker of the present invention can be the same or different, preferably they are different, providing the complex formed upon mutual (or intermolecular) linking of the biomolecules a bifunctional character, such as a targeting activity and a therapeutic activity.
  • Multiple linkers can also applied in combination with multiple biomolecules to provide clusters of mutually linked biomolecules, or chains of biomolecules.
  • biomolecules as used herein includes reference to "active compounds" as defined herein.
  • the members of this binding pair are relatively small.
  • avidine in the avidine/biotine binding pair has a size of about 66,000 daltons, which is at least 10 times larger than the size of the largest member of the BTX-BBS binding pair. This small size allows for a very low level of interference with the desired activity of the biomolecules linked thereby.
  • the members of this binding pair will not be normally present in the envelope or the capsid of a virus and will thus also not be normally present on a viral gene delivery vehicles or other therapeutic agents, or on targeting moieties according to the invention.
  • these vehicles, therapeutic agents and targeting moieties have to be modified in a suitable manner, for instance chemically, to present the member of the binding pair, which may for instance be presented as fusion molecules or hybrid molecules linked to a component which ensures their presence in or on the capsid or envelope of the gene delivery vehicles their presence in or on the therapeutic agents and targeting moieties.
  • a suitable manner for instance chemically
  • the linker of the present invention may be provided as a kit of parts for "labeling" therapeutic agents and/or targeting moieties.
  • the present invention in one aspect thus provides a kit of parts for the production of a targetable drug, or for the provision of individual components for assembling such a targetable drug comprising the individual members of the BTX-BBS binding pair, optionally in a form wherein they can be coupled to a targeting moiety or therapeutic compound (i.e.
  • a reactive group such as for instance as a succinimide ester
  • an expression vector comprising nucleic acid sequences encoding the protein/peptide of the individual members of the BTX-BBS binding pair for the production of a fusion protein as described herein.
  • the alpha-bungarotoxin protein used in aspects of the present invention may be extracted from the venom of Bungarus multicinctus , or it may be produced by using recombinant techniques well known in the art.
  • recombinant techniques usually comprise the production of a nucleic acid construct wherein a nucleic acid sequence encoding the peptide or protein is operably linked to a transcription initiation sequence or promoter. This is usually accomplished by site specific introducing of coding sequence in an expression vector for expression in a host cell.
  • site specific introducing of coding sequence in an expression vector for expression in a host cell The skilled person is well aware of the various techniques that are available to express the protein in recombinant host cells and to purify the protein from said cells, and these are not limiting to the present invention.
  • Suitable nucleic acids encoding the alpha-bungarotoxin protein include those coding for the amino acid sequence as provided in SEQ ID NO. 2 or 3.
  • the protein is produced as precursor comprising a signal sequence (e.g. SEQ ID NO:5) and the sequence of the mature protein (e.g. SEQ ID NO:1).
  • the signal sequence may suitably be omitted.
  • Suitable nucleic acid sequences for the alpha-bungarotoxin protein are provided in SEQ ID NO: 1 and 3.
  • Suitable nucleic acids encoding the peptide the represents the alpha- bungarotoxin binding site include those sequences that encode the amino acid sequence as provided in SEQ ID NO. 7, An exemplary sequence that encodes a peptide that can be used as a counterpart to the alpha-bungarotoxin protein in the BTX-BBS binding pair is provided in SEQ ID NO.6. It will be appreciated that the member of the binding pair representing the bungarotoxin binding site of the present invention may consist of the amino acid sequence as provided in SEQ ID NO:7. However, derivatives, variants or mimetics of this peptide, capable of high-affinity binding to the alpha-bungarotoxin protein can easily be found by the skilled person.
  • the binding site on the niconinic acetylcholine receptor, to which the alpha-bungarotoxin protein specifically binds is larger than the 13 amino acid peptide of SEQ ID NO: 7.
  • the counterpart of the alpha-bungarotoxin protein in the BTX-BBS binding pair of the present invention may be larger, for instance comprising additional amino acids at the N-terminal or C-terminal part of the peptide without compromising the binding capacity.
  • the counterpart of the alpha- bungarotoxin protein in the BTX-BBS binding pair of the preset invention comprises the 13 amino acid peptide of SEQ ID NO:7.
  • the members of the BTX-BBS binding pair of the present invention may be coupled to the biomolecules via a spacer, which spacer may take any form, and be of any chemical nature.
  • the spacer is a peptide spacer.
  • the uses of the linker of the present invention include its use as an affinity tag for purification of proteins or cells.
  • Such embodiments include for instance chromatographic methods, in particular affinity chromatography, an Example of which is provided in Example 6 below.
  • the use includes such applications as nuclear imaging and therapy.
  • An example thereof is provided in Example 7 below, wherein the production of a product suitable for use in nuclear imaging and radiotherapy mediated by viral delivery is described.
  • the present invention also provides pharmaceutical compositions comprising the targetable drug of the present invention in combination with a pharmaceutically acceptable carrier, adjuvant or vehicle.
  • the targetable drug of the present invention is present in said pharmaceutical composition in a therapeutically effective amount.
  • the drug conjugates and pharmaceutical compositions of the present invention are useful in the treatment of transplant rejection (e.g., kidney, liver, heart, lung, pancreas (e.g., islet cells), bone marrow, cornea, small bowel and skin allografts, and heart valve xenografts) and autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, juvenile diabetes, asthma, inflammatory bowel disease (Crohn's disease, ulcerative colitus), lupus, diabetes, myasthenia gravis, psoriasis, dermatitis, eczema, seborrhoea, pulmonary inflammation, eye uveitis, hepatitis, Grave's disease, Hashimoto's thyroiditis, Behcet's or Sjorgen's syndrome (dry eyes/mouth), pernicious or immunohaemolytic anaemia, idiopathic adrenal insufficiency, polyglandular autoimmune syndrome, glomerul
  • compositions comprising at least one complex (drug conjugate according to the invention) comprising an active compound that is to be delivered to the cytoplasm and/or nucleus of a cell or cells.
  • the complex may be administered alone or with at least one additional active compound, and any pharmaceutically acceptable carrier, adjuvant or vehicle.
  • additional active compounds encompasses, but is not limited to, an agent or agents selected from the group consisting of an immunosuppressant, an anti-cancer agent, an anti-viral agent, an anti-inflammatory agent, an anti-fungal agent, an antibiotic, or an anti-vascular hyperproliferation compound.
  • pharmaceutically acceptable carrier, adjuvant or vehicle refers to a carrier, adjuvant or vehicle that may be administered to a subject, together with a complex of the present invention, and which does not destroy the pharmacological activity thereof.
  • Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of the present invention include, but are not limited to, the following: ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilieate, polyvin
  • Cyclodextrins such as ⁇ -, ⁇ - and y- cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl- ⁇ -cyclodextrins, or other solubilized derivatives may also be used to enhance delivery of the compounds of the present invention.
  • compositions of the present invention may contain other therapeutic agents, and may be formulated, for example, by employing conventional solid or liquid vehicles or diluents, as well as pharmaceutical additives of a type appropriate to the mode of desired administration (for example, excipients, binders, preservatives, stabilizers, flavors, etc.) according to techniques such as those well known in the art of pharmaceutical formulation.
  • compositions comprising at least one complex of the present invention may be administered by any suitable means, for example, orally, such as in the form of tablets, capsules, granules or powders; sublingually; buccally; parenterally, such as by subcutaneous, intravenous, intramuscular, or intrasternal injection or infusion techniques (e.g., as sterile injectable aqueous or non-aqueous solutions or suspensions), nasally such as by inhalation spray, topically, such as in the form of a cream or ointment; or rectally such as in the form of suppositories; in dosage unit formulations containing non-toxic, pharmaceutically acceptable vehicles or diluents.
  • suitable means for example, orally, such as in the form of tablets, capsules, granules or powders; sublingually; buccally; parenterally, such as by subcutaneous, intravenous, intramuscular, or intrasternal injection or infusion techniques (e.g., as sterile injectable
  • compositions of the present invention may, for example, be administered in a form suitable for immediate release or extended release. Immediate release or extended release may be achieved by the use of suitable pharmaceutical compositions comprising the present compounds, or, particularly in the case of extended release, by the use of devices such as subcutaneous implants or osmotic pumps.
  • the present complexes may also be administered liposomally.
  • a "therapeutically effective amount" of a complex of the present invention may be determined by one of ordinary skill in the art, and includes exemplary dosage amounts for an adult human of from about 0.1 to 100 mg/kg of body weight of active compound per day, which may be administered in a single dose or in the form of individual divided doses, such as from 1 to 3 times per day.
  • the specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the species, age, body weight, general health, sex and diet of the subject, the mode and time of administration, rate of excretion, drug combination, and severity of the particular condition.
  • Preferred subjects for treatment include animals, most preferably mammalian species such as humans.
  • terapéuticaally effective is meant an amount necessary to achieve a desired result, for example, alleviation of symptoms of a particular disorder in a patient, the improvement of an ascertainable measurement associated with a particular disorder, or the prevention of a particular immune response.
  • a desired result for example, alleviation of symptoms of a particular disorder in a patient, the improvement of an ascertainable measurement associated with a particular disorder, or the prevention of a particular immune response.
  • the specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the species, age, body weight, general health, sex and diet of the subject, the mode and time of administration, rate of excretion, drug combination, and severity of the particular condition.
  • Preferred subjects for treatment include animals, most preferably mammalian species such as humans.
  • the complexes of the present invention may be employed alone or in combination with each other and/or other suitable therapeutic agents, such as antiinflammatories, antiproliferatives, chemotherapeutic agents, and immunosuppressants.
  • suitable therapeutic agents such as antiinflammatories, antiproliferatives, chemotherapeutic agents, and immunosuppressants.
  • Diseases, disease states, disorders and conditions which may be treated by complexes of the present invention include transplant rejection and autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, juvenile diabetes, asthma, and inflammatory bowel disease, as well as inflammatory diseases, cancer, viral replication diseases and vascular diseases.
  • Example 1 Expression of ⁇ -Bungarotoxin (BTX) fusion proteins.
  • scFv Hyb3: A1/MAGE-A1 (SEQ ID NO: 8); J591: anti-PSMA (SEQ ID NO: 9)) and scTCR (MPD: A2/gpl00 (SEQ ID NO: 10); CL8: A2/MAGE-3 (SEQ ID NO: 11); and A2/HCV-NS3 (SEQ ID NO: 12)) were linked to the alpha-bungarotoxin (BTX) gene (sequence with restriction sites (italics) provided in SEQ ID NO: 13), that was generated as a synthetic gene (SEG ID NO: 14) by Baseclear b.v. (Leiden, the Netherlands) and cloned into the pGEMll vector.
  • BTX alpha-bungarotoxin
  • prokaryotic and eukaryotic systems were used for expression of the fusion proteins.
  • Prokaryotic (bacterial) expression of the fusion proteins was performed using the pCES 1, pStaby 1.2 vectors, both with the gp3 signal sequence for expression in the periplasm and media.
  • the pjl ⁇ expression vector was used with the pelB signal sequence for periplasmic expression.
  • Expression of the fusion proteins in bacteria (TGl, BL21, SEl) was induced upon addition of IPTG. After 4 hours or overnight culture, fusion proteins were purified using affinity chromatography (HIS-tag purification), followed by size exclusion, both on an AKTA-FPLC.
  • Retroviral expression in human 911 cells and insect cell expression using the DES expression system (invitrogen).
  • the BTX gene was cloned into the Not I and Xho I digested pBullet Hyb3-CD4/ ⁇ or J591 CD4/ ⁇ and pBullet MPD- CD4/ ⁇ or CL8-CD4/ ⁇ retroviral vectors removing the CD4/ ⁇ fragment and linking the BTX protein 3' to the scFv and scTCR. This resulted in the vectors pBullet-scFv /BTX and pBullet-scTCR/BTX.
  • 911 cells stably expressing the proteins were obtained by retroviral infection with the pBullet vectors.
  • Expression of single chain TCR was also performed in Drosophila S2 cells using the pMT/BiP/V5-His A vector.
  • scTCR/BTX constructs were cloned into the PMT/BiP/V5-His A vector and co-electroporated with the pCoHygro vector into S2 cells. After 5 to 8 days of selection with Hygromycin (300 ⁇ g/ml) expression was induced by adding copper sulphate (500 ⁇ g/ml) to the medium.
  • BTX fusion proteins were purified from the media of 911 and S2 cells by affinity chromatography and size exclusion.
  • Example 2 polyplex retargeting using anti-PSMA/BTX and anti-HCV NS3/BTX.
  • Polymer-based transfection particles named polyplexes and lipid- based systems named lipoplexes are being developed for target cell specific delivery of DNA, RNA, miRNA, siRNA, and synthetic RNA such as poly (I, C,) into human and non-human cells.
  • RNA Ribonucleic acid
  • synthetic RNA such as poly (I, C,) into human and non-human cells.
  • the surface charge of these complexes have to be masked and accomplished to avoid interactions with plasma components, erythrocytes, and the reticuloendothelial system.
  • polyplexes based on polyethylenimine (PEI), shielded with polyethylene glycol (PEG), and linked to the receptor binding ligands such as transferrin (Tf) or epidermal growth factor (EGF) have been developed in the art.
  • the BTX-ligand addition system may be used, which has the advantage that all scFv or scTCR are positioned in such a way that the binding site is preserved.
  • a BTX-peptide - PEG-PEI conjugate was synthesised:
  • the C-terminal cysteine of the BTX-peptide was coupled to a bifunctional maleimido-PEG-N-hydroxysuccinimide (NHS) ester via thioether bond formation between the cysteine mercapto group and the PEG maleimido group.
  • the PEG molecule was further linked via the amine -re active NHS-ester to amino groups of PEL
  • the BTX-peptide (13.2 mg, 6 ⁇ mol) with the sequence (N to C terminus): CGSGGSWRYYESSLEPYPD, with the N-terminus acetylated and the C-terminus amidated, was dissolved in 0.7 ml dimethylformamide and reacted with 6 ⁇ mol of an aqueous solution of maleimido-PEG (3400 Da)-NHS ester in 20 mM Hepes buffer pH 7.3 for 5 minutes at room temperature, before mixing and reacting with 75mg (3 ⁇ mol) PEI (25 kDa, branched) dissolved in 1 ml aqueous 20 mM HEPES buffer pH 7.3 .
  • the conjugate was purified by cation exchange chromatography on a MacroPrep High S column by elution with an 20 mM HEPES pH 7.3 buffered salt gradient starting at 0.5 M NaCl and reaching 3 M after 60 min.
  • the BTX-peptide-PEG-PEI conjugate was eluted at the end of the gradient and detected by quantifying the peptide by UV absorption at 280 nm, PEG using a PEG assay and PEI by applying a TNBS assay (both described below).
  • the conjugate containing BTX-peptide and PEI at a molar ratio of 2.5:1 (weight/weight ratio of 1:4) was dialyzed against 150 mM NaCl, 20 mM HEPES pH 7.3, snap-frozen and stored at -80 0 C.
  • PEG was determined analogously as described in C. E. Childs. The determination of polyethylene glycol in gamma globulin solutions, Microchemistry Journal, 20:190-192 (1975), via complex formation with Ba2+ and iodine, which can be measured at 590 nm.
  • Barium chloride solution was prepared by dissolving BaC12 in 0.1 M HCl to form a 5% (w/v) solution.
  • Iodine solution was prepared by dissolving 1.27 g iodine in 100 ml water containing 2 g of potassium iodide. The reaction is dependent on the concentration of the solutions. First a red charge transfer complex arises with triiodide ions that turns gradually into a nearly water insoluble iodine complex.
  • TNBS assay The concentration of PEI was measured by the trinitrobenzenesulfonic acid (TNBS) assay analogously as described in S. L. Snyder and P. Z. Sobocinski, An improved 2,4,6-trinitrobenzenesulfonic acid method for the determination of amines, Anal Biochem, 64:284-8 (1975).
  • TNBS trinitrobenzenesulfonic acid
  • Standard amine solutions with a known amount of reagent and the test solutions containing unknown amounts of the primary amine derivative were serially diluted in duplicates with 0.1 M sodium tetraborate to give a final volume of 100 ⁇ l in a 96 well plate.
  • Concentrations of approximately 0.0175 to 0.21 ⁇ mol/ml amine reagent were achieved and then 2.5 ⁇ l of TNBS (75 nmol) diluted in water were added to each well. After 5-20 minutes incubation time at room temperature (depending on the strength of the developed colouring) the absorption was measured at 405 nm using a microplate reader.
  • Plasmid DNA /cationic polymer complexes ('polyplexes') are prepared by mixing solutions of DNA (20 - 200 ⁇ g/ml) with equal volumes of modified PEI polycation solutions. In standard polyplexes, a molar ratio of PEI nitrogen atoms to DNA phosphates (N/P) of 6 was applied.
  • the modified PEI polycation solution is prepared by mixing first the BTX- peptide-PEG-PEI conjugate with other PEI polycations at a given molar ratio, for example with PEG(5kDa)-PEI and PEI (22kDa, linear) at a 20% : 10% : 70% molar ratio. Polyplexes were incubated for 20 min at RT before further use.
  • PEG (5kDa)-PEI was prepared from PEG (5kDa) SPA and PEI 25 kDa as described previously in M. Kursa, G. F. Walker, V. Roessler, M. Ogris, W. Roedl, R. Kircheis, and E. Wagner. Novel Shielded Transferrin- Polyethylene Glycol-Polyethylenimine/DNA Complexes for Systemic Tumor- Targeted Gene Transfer. Bioconjug. Chem. 14:222-231 (2003), and is optionally included into polyplexes for shielding against unspecific interactions.
  • Polyplexes can be formed in various buffers including HBS (HEPES buffered saline: 20 mM HEPES, 150 mM NaCl, pH 7.4) or HBG (HEPES buffered glucose: 20 mM HEPES, 5% glucose, pH 7.4).
  • HBS HPES buffered saline: 20 mM HEPES, 150 mM NaCl, pH 7.4
  • HBG HPES buffered glucose: 20 mM HEPES, 5% glucose, pH 7.4
  • Polyplexes made in HBS have usually large size of particle and the tendency to form aggregates.
  • Polyplexes made in HBG form particles of small size and are virtually free of aggregates.
  • they can be stored in frozen form at -80 0 C.
  • N/P ratios can be varied between 3 and 30 depending on the molecular weight and modification of PEI.
  • PEI also other cationic polymers can be used, see for example polymers described in V. Russ, H. Elfberg, C. Thoma, J. Kloeckner, M. Ogris, and E. Wagner. Novel degradable oligoethylenimine acrylate ester-based pseudodendrimers for in vitro and in vivo gene transfer. Gene Ther 15:18-29 (2008), and references therein.
  • PEG-conjugates and endosome-destabilizing peptide- polycation conjugates such as melittin-PEI (M. Ogris, R. C. Carlisle, T.
  • Example 2.3 Generation of targeted polyplexes 2.3.1.: in vitro targeting of
  • the BTX-peptide containing polyplexes described in example 2.2 can be incubated with scFv-BTX and scTCR/BTX fusion proteins for generating targeted polyplexes.
  • a concentration range of the PSMA specific scFv J591-BTX was mixed with polyplexes and incubated at room temperature for 1 hour.
  • Optimal transfection of psma positive cells was then determined by adding the polyplexes to the culture medium of LNCaP (PSMA-pos) and MZ2-MEL3.0 (PSMA-neg), followed by Luciferase activity assay and fluorescnence microscopy 24 hours post transfection.
  • the BTX-peptide-PEG-PEI conjugate can be first directly mixed with the scFv fusion protein and subsequently be used for polyplex formation with plasmid DNA and the other PEI conjugates.
  • a concentration range of, the BTX-peptide-PEG-PEI conjugate was mixed with the PSMA specific scFv/BTX fusion protein and incubated at room temperature for one hour.
  • HCV Hepatitis C virus
  • scTCR/BTX fusion protein was constructed as described in example 1, and loaded on pre formed polyplexes as described for the PSMA retargeted polyplexes.
  • target cells Epstein Barr Virus (EBV) transformed, HLA-A2 positive human B cells were used stably expressing the HCV NS3 gene.
  • Luciferase activity assays demonstrated specific transfection of HCV-NS3 cells only (Fig Ib). Human B cells that lacked the HCV- NS3 gene were not transfected.
  • the human prostate cancer cell line PC346C was derived from the PC346 xenograft.
  • PC346 originates from tissue of a non-progressive prostate cancer patient, which was obtained by transurethral resection of the prostate.
  • Both the xenograft and its related cell line are androgen responsive, secrete PSA and express a wild-type androgen receptor.
  • Luciferase expression was normalized for total protein content (Lowry assay; Bio-Rad Laboratories GmbH, M ⁇ nchen, Germany).
  • Fig 2 shows specific delivery of the Luciferase gene to PC346C cells in the prostate of mouse.
  • Example 3 Targeting Adenovirus type 5 via fiber-ligand addition.
  • Adenoviral proteins are expressed in the nucleus of infected cells were the environment does not favour the formation of disulphate bridges.
  • disulphate bridges are required for optimal folding of large molecules such as antibody -based scFv or TCR-based scTCR. As a consequence, these molecules are unable to acquire their proper three- dimensional structure and thus antigen binding capacity when expressed intracellular.
  • fusions of adenoviral capsid proteins and antibodies were poorly expressed and did not result in the generation of infectious particles.
  • a method was developed that is based on the separate routes of expression of scFv and scTCR on the one hand and adenoviral capsid proteins on the other hand.
  • Antibody and TCR fragments can be linked to adenoviral capsid proteins using the BTX ligand addition system. Expressing fusions of capsid proteins with the 13-aminoacid peptide allows binding of fusion proteins that comprise the BTX protein.
  • the 13 amino-acid peptide bound by BTX was introduced into the wildtype Adenovirus type 5 fiber at the 3' end by PCR.
  • a primer was designed that comprised the last 20 nucleotides of the 3' fiber, the 39 nucleotides corresponding to the 13 AA petide and a stop codon and XXX restriction site.
  • PCR was performed using PWO polymerase, a 5' fiber primer and the 3' primer on wildtype fiber DNA. The resulting fragment was then cloned into the lentiviral vector pLV.CMV.bc.neo resulting in LV.WTFBTXPEPT.
  • the 13 AA peptide was linked to a truncated Ad5 fiber comprising fiber tail, first three repeats, and lung surfactant D protein neck region peptide.
  • a primer was designed comprising 20 nt of the lung surfactant D protein neck region peptide, 39 nt encoding the 13 AA peptide and a stop codon and Xho IXX restriction site.
  • This chimeric fiber was then cloned into the lentiviral vector pLV.CMC.bc.neo resulting in LV.TR1- 3FBTX PEPT .
  • Cell lines stably expressing the chimeric fiber molecules were generated by lentiviral infection of 911 cells with LV.WTFBTX PEPT and LV.TR1-3FBTX PEPT . These cell lines were then infected with adenoviral particles that lack the fiber gene (Ad ⁇ EGFP ⁇ F). In this way, all particles that are produced by the stable chimeric fiber expressing 911 cells will contain the cimeric fibers. Supernatant from these cells was mixed (1:1) with supernatant from 911 cells stably expressing the fusion proteins scFv Hyb3/BTX or scTCR BLM/BTX to couple the fusion proteins to the viral particles.
  • HLA-A1 POS /MAGE A1 POS melanoma cells were infected with scFv Hyb3/BTX loaded adenoviral particles as follows: Adenoviral particles loaded with the scFv Hyb3/BTX fusion protein were incubated with 1) the HLA-A1 POS /MAGE A1 POS MZ2-MEL3.0 melanoma cells and 2) HLA-Al p o s /MAGE A1 NEG MZ2-MEL2.2 melanoma cells. Twenty- four hours later infectivity was analysed by fluorescence microscopy.
  • Example 4 retargeting polymer coated Viruses.
  • a major hurdle in the clinical use of viral vectors is the pre-existing immunity against most viruses.
  • adenoviral vectors but also other vectors, are rapidly cleared from the blood after systemic injection.
  • the viral vectors may be neutralised by antibodies, red blood cells and many other blood components. In practice this means that viral therapy is very inefficient as less than 1% of the injected dose will circulate in the bloodstream.
  • viral particles may be shielded from immune attacks by packaging the particles in inert materials.
  • Adenovirus, adeno-associated virus, and probably many other viruses can be packaged by polymers such as poly-[JV-(2- hydroxypropyl) methacrylamide] (PHMA).
  • Viruses packaged with these polymers were shown to be less vulnerable to antibody attacks and circulated longer in human blood, thereby extending the exposure time of target cells.
  • Packaging of these viruses requires the addition of new cell binding ligands as the packaged particles have become non-infectious.
  • Addition of ligands such as antibodies, etc relies on the interaction of amide-groups present on the polymer and reactive groups on the ligand. This means that ligands can be bound in several ways to the polymer-coated viruses. Some of these bindings may even abolish the antigen binding capacity of the ligand reducing the overall retargeting capacity of the virus-ligand.
  • the amide-groups on the polymer are also required for coating the viral particles, resulting in a competition for binding places of both virus and ligand.
  • ligands to viral particles using the BTX-peptide interaction provides a method that allows all ligands to bind in a similar way, leaving the antigen-binding site intact. Furthermore, the system allows a more effective coating of the viral particles, as there is no competition for amide- groups on the polymer.
  • Example 4.1 Synthesis of the multivalent reactive polymer bearing BTX binding peptide The synthesis of the reactive precursor - statistical copolymer
  • PoIy(HPMA-Co-Ma-GG-TT) was described earlier (Subr, V., Ulbrich, K. (2006) Synthesis and properties of new N-(2-hydroxypropyl)methacrylamide copolymers containing thiazolidine-2-thione reactive groups. React. Fund. Polym. 66, 1525-1538.).
  • the BTX peptide (GSGGSGGTGYRSWRYYESSLEPYPD) was prepared by standard solid phase Fmoc/t-Bu peptide synthesis.
  • the pH of the reaction mixture was adjusted and maintained at 7.4 by continuous addition of a saturated aqueous Na2B4 ⁇ 7 solution using pH stat (Radiometer).
  • the course of the coupling reaction was monitored by HPLC (C18 Chromolith column, eluent water-acetonitrile, 0.1 % TFA, gradient 0-100 % acetonitrile).
  • acetic acid 0.1 ml was added to the reaction mixture and the polymer conjugate was purified by SEC chromatography on Sephadex G 25 in water. The polymer fraction was lyophilized.
  • the content of remaining TT groups was 4.1 mol % as determined spectrophotometricaly at 305 nm (absorption coefficient 10 500 1 mol 1 cm 1 in methanol).
  • Example 4.2 coating adenoviral particles with the PHMA-BTX peptide polymer and prostate specific scFv J591/BTX fusion protein for PSMA specific infection.
  • Adenovirus encoding luciferase was mixed with PHPMA-BTX binding peptide in 5OmM pH7.4 HEPES at room temperature for 20min, to give Ad+PHPMA- BTXbp. Excess unreacted polymer was then purified away using filtration through an s400 minispin column (GE healthcare). The anti-PSMA BTX-scFv fusion protein was then added to the polymer coated Ad, to give Ad+PHPMA- BTXbp+ BTX-scFv. Recovery was calculated using a picogreen assay (Molecular Probes).
  • Ad+PHPMA-BTXbp or Ad+PHPMA-BTXbp+BTXscFv were added to a suspension of 200,000 LNCap cells (PSMA P0S ) or PC-3 cells (PSMA NEG ). After 20min incubation at 37 0 C cells and media were separated, cells were washed in ImI of PBS before resuspension in lOOul of media. 50ul of cell suspension was taken and incubated overnight at 37 0 C before being assayed for luciferase gene expression and 50ul was immediately assayed for genome content by QPCR.
  • Ad+PHPMA-BTXbp+BTXscFv (J591) mediated specific infection of PSMA P0S LNCap cells only.
  • No enhancement of luciferase gene expression or viral copies could be detected when PSMA NEG PC-3 cells were incubated with Ad+PHPMA-BTXbp+BTXscFv.
  • the enhanced luciferase expression and particle count was due to incubation with the scFv/BTX fusion protein.
  • Example 5 Targeting cytotoxic drugs
  • BTX-peptide binding pair Another application of the BTX-peptide binding pair is targeting cytotoxic drugs to specific cells. To this end both the 13 AA peptide bound by BTX and the cytotoxic drug doxorubicin were coupled to the PHMA polymer.
  • the BTX peptide (GSGGSGGTGYRSWRYYESSLEPYPD) was prepared by standard solid phase Fmoc/t-Bu peptide synthesis.
  • the pH of the reaction mixture was adjusted and maintained at 7.4 by continuous addition of a saturated aqueous Na2B4U7 solution using pH stat (Radiometer).
  • the course of the coupling reaction was monitored by HPLC (C 18 Chromolith column, eluent water-acetonitrile, 0.1 % TFA, gradient 0-100 % acetonitrile).
  • the polymer fraction was evaporated to dryness and lyophilized from water yielding 55 mg of the title polymer conjugate.
  • Content of Dox was 5.7 wt % as determined by HPLC of the reaction mixture at the beginning and the end of the coupling reaction comparing the area of the peaks corresponding to the free Dox.
  • the content of the peptide in the polymer conjugate (12.5 wt %) was determined analogically.
  • M w 110 000 by SEC on Superose 6 using 0.3 M sodium acetate, pH 6.5 as an eluent and light-scattering detector.
  • Example 6 Using the binding pair for affinity chromatography.
  • the binding pair may also be used for affinity chromatography, as an alternative to presently used affinity tags such as: c-myc, V5, HA, 6 x His and Streptag.
  • the proteins to be purified may be expressed as fusions with the 13 amino-acid peptide, or as fusions with the BTX protein. Resins coupled to the BTX protein or 13 amino-acid peptide can then capture the fusion proteins.
  • peptide-phage display distinct sequences were identified that bind the BTX protein with affinities ranging from 10 4 M to 10 9 M. This allows for tight optimisation of binding and elution strategies of the fusion proteins.
  • the PSMA specific scFv/BTX protein expressed in the periplasm of SEl was purified using this strategy.
  • streptavidin coated beads were incubated with a biotinilated peptide with the Torpedo AcChoR alpha- subunit (WVYYTCCPDTPYL) and 10 6 M affinity for BTX.
  • Serial washings with PBS then removed excess peptide.
  • Fusion proteins were released from the periplasm of SEl bacteria that were induced for 4 hour with IPTG to express the fusion protein, by addition of PBS/EDTA.
  • the periplasmic fraction was the incubated with the peptide-loaded beads and incubated for 15 min at room temperature. After several washes with PBS the protein was eluted by adding the high affinity peptide.
  • Example 7 Using the binding pair in combination with viriotherapy for radiotherapy
  • an artificial receptor including the BTX protein that has no homologues in humans, might be ideal for nuclear imaging and radiotherapy mediated by viral delivery.
  • a receptor was made for the extracellular expression of BTX, allowing it to bind its ligand, the 13 amino-acid peptide. Labeling the peptide with radio nucleotides may allow preferential uptake of radio nucleotides that are either useful for imaging or therapy.

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Abstract

La présente invention concerne un procédé pour préparer un médicament apte au ciblage comprenant un composé actif et un fragment de ciblage, comprenant la production d’une paire de liaison constituée de la protéine bungarotoxine (BTX) en tant que premier élément et un peptide comprenant le site de liaison de la bungarotoxine en tant que second élément de ladite paire de liaison ; (b) la production dudit composé actif avec ledit premier ou second élément de ladite paire de liaison pour produire un composé actif modifié ; (c) la production dudit fragment de ciblage avec l’élément complémentaire de ladite paire de liaison pour produire un fragment de ciblage modifié, et (d) la liaison dudit composé actif modifié avec ledit fragment de ciblage modifié par mise en contact desdits premier et second éléments de ladite paire de liaison, de manière à produire le médicament apte au ciblage.
PCT/NL2008/050236 2008-04-23 2008-04-23 Lieur contenant de la bungarotoxine et un peptide de liaison WO2009131435A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
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US20130183307A1 (en) * 2012-01-13 2013-07-18 Johan Renes Aberrant cell-restricted immunoglobulins provided with a toxic moiety
US10543232B2 (en) 2014-05-14 2020-01-28 Targimmune Therapeutics Ag Polyplex of double-stranded RNA and polymeric conjugate
EP2658872B1 (fr) * 2010-12-27 2020-09-30 Apo-T B.V. Polypeptide se liant à des cellules aberrantes et induisant l'apoptose
US11098115B2 (en) 2011-09-29 2021-08-24 Apo-T B.V. Multi-specific binding molecules targeting aberrant cells
WO2022074152A1 (fr) 2020-10-08 2022-04-14 Targimmune Therapeutics Ag Immunothérapie pour le traitement du cancer
WO2023079142A2 (fr) 2021-11-05 2023-05-11 Targimmune Therapeutics Ag Conjugués linéaires ciblés comprenant du polyéthylèneimine et du polyéthylène glycol et polyplexes comprenant ceux-ci
WO2024100046A1 (fr) 2022-11-07 2024-05-16 Targimmune Therapeutics Ag Conjugués linéaires ciblés comprenant du polyéthylèneimine et du polyéthylène glycol et polyplexes comprenant ceux-ci
WO2024100040A1 (fr) 2022-11-07 2024-05-16 Targimmune Therapeutics Ag Conjugués linéaires ciblant psma comprenant du polyéthylèneimine et du polyéthylène glycol et polyplexes comprenant ceux-ci
WO2024100044A1 (fr) 2022-11-07 2024-05-16 Targimmune Therapeutics Ag Polyplexes d'acides nucléiques et conjugués ciblés comprenant de la polyéthylèneimine et du polyéthylène glycol

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2658872B1 (fr) * 2010-12-27 2020-09-30 Apo-T B.V. Polypeptide se liant à des cellules aberrantes et induisant l'apoptose
US11098115B2 (en) 2011-09-29 2021-08-24 Apo-T B.V. Multi-specific binding molecules targeting aberrant cells
US20130183307A1 (en) * 2012-01-13 2013-07-18 Johan Renes Aberrant cell-restricted immunoglobulins provided with a toxic moiety
EP3470434A1 (fr) 2012-01-13 2019-04-17 Apo-T B.V. Immunoglobulines restreintes de cellules aberrantes dotées d'une fraction toxique
US10946104B2 (en) 2012-01-13 2021-03-16 Apo-Tb.V. Aberrant cell-restricted immunoglobulins provided with a toxic moiety
US10543232B2 (en) 2014-05-14 2020-01-28 Targimmune Therapeutics Ag Polyplex of double-stranded RNA and polymeric conjugate
US11298376B2 (en) 2014-05-14 2022-04-12 Targimmune Therapeutics Ag Method of treating cancer
WO2022074152A1 (fr) 2020-10-08 2022-04-14 Targimmune Therapeutics Ag Immunothérapie pour le traitement du cancer
WO2023079142A2 (fr) 2021-11-05 2023-05-11 Targimmune Therapeutics Ag Conjugués linéaires ciblés comprenant du polyéthylèneimine et du polyéthylène glycol et polyplexes comprenant ceux-ci
WO2024100046A1 (fr) 2022-11-07 2024-05-16 Targimmune Therapeutics Ag Conjugués linéaires ciblés comprenant du polyéthylèneimine et du polyéthylène glycol et polyplexes comprenant ceux-ci
WO2024100040A1 (fr) 2022-11-07 2024-05-16 Targimmune Therapeutics Ag Conjugués linéaires ciblant psma comprenant du polyéthylèneimine et du polyéthylène glycol et polyplexes comprenant ceux-ci
WO2024100044A1 (fr) 2022-11-07 2024-05-16 Targimmune Therapeutics Ag Polyplexes d'acides nucléiques et conjugués ciblés comprenant de la polyéthylèneimine et du polyéthylène glycol

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