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WO2009053700A1 - Modification d'entités biologiques contenant des acides nucléiques - Google Patents

Modification d'entités biologiques contenant des acides nucléiques Download PDF

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WO2009053700A1
WO2009053700A1 PCT/GB2008/003594 GB2008003594W WO2009053700A1 WO 2009053700 A1 WO2009053700 A1 WO 2009053700A1 GB 2008003594 W GB2008003594 W GB 2008003594W WO 2009053700 A1 WO2009053700 A1 WO 2009053700A1
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biological entity
polymer
virus
polymerisation
modified
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Leonard William Seymour
Kerry Fisher
Simon Stephen Briggs
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Cancer Research Technology Ltd
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Cancer Research Technology Ltd
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A61K47/6455—Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
    • 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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10321—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10345—Special targeting system for viral vectors
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
    • C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
    • C12N2810/851—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from growth factors; from growth regulators

Definitions

  • the present invention relates to methods of modifying nucleic acid-containing biological entities.
  • the invention also relates to such nucleic acid-containing biological entities that have been modified in accordance with the invention and their use in various fields, including agriculture, the petrochemical industry, environmental science and medicine including vaccination.
  • the nucleic acid-containing biological entity is a bacterium or virus.
  • Micro-organisms including viruses, find many applications throughout the broad fields of biotechnology. They are involved in medicine, agriculture, industrial production processes (including notably the oil and brewing industries) and bioremediation. Many useful applications and functions have been identified and developed for such biological agents. However, often the development or enhancement of their activities is limited by their precise physical properties, restricting their ability to fulfil tasks that are theoretically possible but practically beyond their scope. In this situation, which is quite commonly encountered, it would often be desirable to re-engineer the physical properties of the virus or microorganism, notably the surface properties, to endow it with properties more appropriate for its required purpose.
  • biological insecticides for example such as baculoviruses may be restricted in their usefulness through inappropriate target specificity and adverse survival characteristics in the environment; sulphur metabolising bacteria may be limited in their useful application in the petrochemical industry through inadequate patterns of dispersion and distribution; in the context of human or veterinary gene therapy, viruses intended to mediate delivery of therapeutic genes maybe limited in their usefulness through inefficiency of delivery or transgene expression in target tissues; in the context of microbial cancer therapies, tumour-killing viruses or bacteria may be limited in their usefulness by inefficient delivery to tumours or (in the case of viruses) inefficient infection of target tissues; finally in the context of viral vaccination (where a harmless virus is used to express a pathogen-related transgene, in order to stimulate an anti-pathogen response), activity may be limited by inefficient infection of target cells leading to poor induction of anti-pathogen immune response.
  • viruses intended to mediate delivery of therapeutic genes maybe limited in their usefulness through inefficiency of delivery or transgene expression in target tissues
  • somatic cell gene therapy has attracted major interest in recent years because it promises to improve treatment for many different types of disease, including both genetic diseases (eg. cystic fibrosis, muscular dystrophy, enzyme deficiencies) and diseases resulting from age or damage related physiological deterioration (cancer, heart disease, mature onset diabetics).
  • genetic diseases eg. cystic fibrosis, muscular dystrophy, enzyme deficiencies
  • cancer heart disease, mature onset diabetics
  • cancer heart disease, mature onset diabetics
  • Viruses are the obvious choice as vectors for gene delivery since this is essentially their sole function in nature. Consequently viruses have seen considerable use in gene therapy to date, forming the majority of vectors employed in clinical studies.
  • the main feature of adenoviruses that limits their successful application is their neutralisation and/or removal by the immune system. Although they are professional pathogens, evolved over millions of years as highly efficient gene delivery vectors, their hosts have similarly developed very effective protection mechanisms. Serum and ascites fluid from cancer patients contain neutralising antibodies that can completely prevent viral infection in vitro even at high dilution (see below). Typical human protocols involving adenovirus lead to significant inflammatory responses (typically flu-like symptoms), as well as inefficient infection of target cells.
  • non- viral vector systems have a good safety record, and are easier to produce in large quantities, they have low specific transfection activity and efficiency of gene expression in target tissues has been a major problem.
  • a central limitation to successful application of presently available vectors for treatment of disease is a requirement' for their administration directly to the site of disease, either by direct application or by intra-arterial administration.
  • Presently available vectors are not capable of targeted infection following intravenous injection, except perhaps to lung, liver or spleen.
  • La addition type 5 adenovirus binds to human (but not murine) blood cells in vitro and in vivo, preventing receptor-mediated infection of target cells. Particle kinetics are also damaged by unwanted receptor- mediated infection of non-target cells; for example type 5 adenovirus infects hepatocytes following intravenous injection to mice, further depleting virus in the circulation and potentially also providing unwanted transgene expression and toxicity when hepatocytes are not the intended target cell.
  • non- viral vectors lose their transfection activity in the presence of physiological concentrations of plasma proteins, thought to reflect formation of non- transfection active aggregates.
  • neutralising antibodies that can efficiently prevent infection. This is a particular challenge for viruses found in the normal environment, such as type 5 adenovirus, to which most humans have a lifelong exposure and consequent strong antibody protection even prior to treatment.
  • most treatment regimes require repeated administration, and even viral vectors to which the patient is initially na ⁇ ve will stimulate strong neutralising responses after the first injection. Accordingly neutralising antibodies in the bloodstream present a real challenge to gaining transduction of peripheral targets using viral vectors.
  • 'oncolytic' viruses can be engineered by placing key genes of lytic viruses under control of tumour-associated promoters, or alternatively by deleting virus genes that are essential in normal tissues but largely redundant in tumours, where tumour-associated changes provide functional complementation of the deleted gene and enable virus replication and cell lysis.
  • tumour-associated promoters or alternatively by deleting virus genes that are essential in normal tissues but largely redundant in tumours, where tumour-associated changes provide functional complementation of the deleted gene and enable virus replication and cell lysis.
  • tumour-associated promoters include the series developed and evaluated by
  • Onyx virus 'Onyx 015' also known as dll520
  • Onyx virus 'Onyx 015' also known as dll520
  • efficacy is mainly seen only following direct intratumoural injection, and clinical usefulness is limited by the inability to deliver such oncolytic viruses intravenously to access metastatic tumour deposits.
  • oncolytic viruses for ' virotherapy' are presently limited by poor systemic kinetics, largely because of their interaction with the innate and adaptive immune system.
  • Wild type (non genetically-modified) viruses also often show selective replication in cancer cells, thought to reflect deficiencies in the cellular innate defence mechanisms. More powerful than most genetically-modified viruses, wild type versions can be very useful for virotherapy and have already been subject to several clinical trials. However their usefulness is limited by their interaction with the immune system, including poor systemic circulation kinetics following intravenous delivery.
  • Bacteria are also being used as tumour-killing agents.
  • Bifidobacterium, Clostridium and salmonella all replicate selectively within tumours, perhaps reflecting hypoxia or aberrant immune surveillance, and are showing some promise as anticancer agents.
  • they are limited by poor delivery to tumours following intravenous administration, mainly reflecting rapid scavenging and clearance by phagocytes.
  • tumour-killing viruses or bacteria may be appropriate for some disease settings, for example in treatment of individual non-resectable tumour nodules, clinical usefulness would be dramatically enhanced by the possibility of intravenous delivery to access disseminated malignant disease.
  • Viral vaccines designed to express one or more transgene antigens in specific target cells (usually antigen-presenting cells or muscle cells), are being developed as promising new vaccines for HTV, tuberculosis, malaria, flu, cancer and other diseases.
  • target cells usually antigen-presenting cells or muscle cells
  • prime boost regimes i.e by multiple administrations, sometimes using different vectors to deliver the same antigen transgene
  • adjuvants they can generate powerful and robust anti-disease cytotoxic T-cell responses in mice.
  • these remarkable preclinical demonstrations of efficacy have largely failed to translate into the clinics, predominantly because of neutralisation of the viral vectors by antibodies, preventing infection of target cells and expression of disease antigens.
  • Anti-vector antibodies may be pre-existing following natural exposure to virus in the environment, or may be generated rapidly after initial vaccination, preventing further 'boost' administrations.
  • vaccinia a widely used vector for viral vaccination, prior vaccination against smallpox endows many of the population with powerful vaccinia-neutralising antibodies.
  • Eukaryotic cells notably stem cells
  • stem cells are showing promise in tissue engineering, . «.'”7— however their usefulness is restricted by poor delivery properties in vivo, and in genetically-non-identical recipients their use is hampered by immune recognition and early rejection.
  • Eukaryotic cells are also being developed as agents to confer immune protection, either as transgene-expressing vaccines (eg. antigen presenting cells displaying MHC-restricted disease-related epitopes) or as active immune components (eg epitope-specific T cells).
  • transgene-expressing vaccines eg. antigen presenting cells displaying MHC-restricted disease-related epitopes
  • active immune components eg epitope-specific T cells
  • WO 00/74722 describes a method of modifying the biological and/or physicochemical properties of a biological element such as a virus by providing it with a coating of a multivalent polymer having multiple reactive groups.
  • This approach can enable some biological elements to be targeted or re-targeted to particular sites in a host biological system and can be useful in connection with viral vectors for gene therapy or antitumour therapy.
  • this method and that described in WO 98/44143 may provide some protection from neutralising antibodies, they do not provide sufficient steric protection to withstand the neutralising potency of blood components in many patients.
  • the steric protection which can be imparted by the methods of WO 98/44143 and WO 00/74722, is both hard to control and restricted by the methods themselves. Problems arise because reactive polymers that become linked to the biological surface provide a steric layer that inhibits access of subsequent reactive polymers to the surface during the coating process. This makes the coating reaction self-limiting and the coating does not, therefore, completely cover the surface of the entity.
  • the biological elements, whilst partially protected, are not fully encased in polymer. Oriented addition of branched polymers by this method would be particularly problematic.
  • Steric shielding using multivalent reactive polymers while providing a different steric 'footprint' from monovalent polymers by flattening the polymer against the surface, also risks crosslmking surface proteins so that unpackaging or other biological activity is impaired.
  • a further problem with the methods referenced above is encountered when attempting to purify the coated element from any remaining unreacted polymer.
  • a problem arises because the polymer and polymer-coated element are both large, and therefore difficult to separate using conventional separation methodologies. This can therefore lead to contamination of coated elements with non-degradable high molecular weight contaminants that may decrease definition and could be toxic.
  • the inventors have devised a new method for modifying the surface of nucleic acid- containing biological entities, comprising growing polymer chains from one or more initiation points on said surface, in the presence of one or more catalysts, wherein the nucleic acid-containing biological entity is typically a virus, a bacteriophage, a prokaryotic cell, a bacterium, an archaeum, a eukaryotic cell, a fungus, a spore, a nucleic acid-containing viral core with its outer membrane(s) and/or capsids removed, a membrane-stripped virus, a viral core, a eukaryotic cell nucleus, a mitochondrion, a mammalian cell nucleus, a complex comprising a nucleic acid and a condensing agent or a complex comprising siRNA and a condensing agent; preferably a virus or bacterium; more preferably an adenovirus, adenovirus core,
  • the invention also provides a polymer modified biological entity obtainable or obtained by the method of the invention.
  • the invention also provides a polymer modified biological entity comprising a nucleic acid containing biological entity having attached to the surface thereof one or more branched polymer chains, wherein the one or more branched polymer chains are typically hyperbranched, dendritic or brush polymer chains; and/or wherein the number of polymer chains linked to the surface of the biological entity is typically 20,000 per square ⁇ m of surface area of the biological entity or less; and/or wherein the steric size of each polymer chain typically increases further away from the point of attachment of the polymer chain to the biological entity.
  • the method of the invention involves growing polymer chains directly from the biological entity.
  • individual monomers are added to the ends of growing polymer chains that are anchored distally to the biological entity.
  • This reduces the issues of steric inhibition of coating that are associated with existing polymer coating methods and provides a higher degree of steric protection than has previously been available.
  • This in turn allows the formation of a denser and more structured polymer layer, providing more effective modification of surface properties and protection from the environment.
  • the shielding of the biological entity in this manner also provides improved stability of the entity, notably increased shelf life and better resistance to low pH.
  • branching of the polymers can be induced at the growing ends of the polymer chains, affording the possibility of brush, hyper-branched and dendrimer structures and thus maximizing the level of end-group display and steric protection achieved for each point of surface modification of the biological entity. In this way, complete coverage of the surface can be achieved with only a relatively small number of points of linkage to the surface of the biological entity, and viability of the modified entity is more likely to be maintained.
  • these approaches are conducive to efficient un-packing following activation of the virus protease in the endosome, since the capsid proteins are not cross-linked by the polymer and can be easily shed from the virus core, facilitating maintenance of infectious potential.
  • the method of the invention also provides a means of detargeting normal receptor- mediated tropisms of the biological entities, by steric occlusion of receptor-binding regions, hi addition, the invention provides the possibility of retargeting the biological entity by addition of functionalised end groups to the growing polymer chains. Maximising surface coverage of the entity also has the effect of ensuring that functional groups are retained at the surface of the modified polymer and do not collapse in towards the biological entity. This in turn increases the efficiency of retargeting.
  • a further advantage of the present invention is that the polymer modified biological entity can be easily purified.
  • the materials used in the polymerisation are typically small individual monomer subunits having relatively low molecular weights. These can be separated from the biological entity using standard techniques, thus removing potentially toxic impurities.
  • the method of the invention will facilitate large scale production of coated viruses meeting current Good Manufacturing Practice (cGMP) Regulation of the USFDA.
  • cGMP Good Manufacturing Practice
  • the present controlled polymerisation process will decrease polydispersity, which assists in meeting these standards.
  • the method of the invention has implications for all aspects of gene therapy and microbiological treatments.
  • the coating of a biological entity using the method of the invention prevents recognition of the biological entity by components of the innate and adaptive immune system of the patient and enables delivery of genetic medicines that has not previously been possible. Further, prevention of rapid inactivation by the immune system and removal of normal, unwanted, tropisms provides a platform for efficient retargeting of the biological entity to specific cellular targets following intravenous injection. This is particularly valuable in the treatment of cancer and other such widespread diseases where localised application is impossible.
  • the invention is not limited to such uses and can be used to provide effective delivery of nucleic acid-containing biological entities in a range of applications.
  • the invention therefore also provides a method of microbial treatment, vaccination or gene therapy, which method comprises administering to a patient in need of such therapy an effective amount of a polymer modified biological entity of the invention.
  • Microbial therapy includes virotherapy and cancer therapy using bacteria.
  • the polymer modified biological entities of the invention are particularly useful in vaccination and in the treatment of cancer.
  • a method of delivering nucleic acid to cells of an individual comprising administering a polymer-modified biological entity of the invention which comprises the nucleic acid.
  • polymer modified biological entity of the invention in the manufacture of a medicament for use in microbial treatment, vaccination or gene therapy; and a polymer modified biological entity of the invention for use in microbial treatment, vaccination or gene therapy.
  • the invention also provides a composition comprising a polymer modified biological entity of the invention in association with a pharmaceutically acceptable carrier or diluent.
  • This composition is appropriate for use in gene therapy, vaccination or microbial treatments, e.g. virotherapy or bacterial treatment of cancer.
  • Certain naturally occurring microorganisms e.g. bacteria, fungi and yeasts, can biodegrade oil pollutants. This may involve breaking down hydrocarbons to water and carbon dioxide.
  • the existing native oil- degrading population can be augmented with additional microorganisms.
  • Polymer modified biological entities of the present invention can be particularly useful for this, for example by virtue of hydrophobic groups incorporated into the polymer coating.
  • the invention also provides the use of a polymer modified biological entity of the invention in the treatment of oil pollutants or for delivery of biological pesticides to pathogens in the agricultural industry.
  • Figure 1 shows a typical synthesis of a polymerisation initiation unit for use in the process of the present invention.
  • a typical reactive initiator (described in Example 1) is shown as Product X.
  • Figure 2 shows TEM images of polymer modified Adenovirus particles and unmodified Adenovirus.
  • the left hand image shows a single plane of view showing one single and one double coated virus prepared according to the method of the invention.
  • the right hand image shows unmodified viruses.
  • AU TEM images of virus particles were stained with methylamine tungstate (2% solution) using the drop method.
  • Figure 3 shows the results of PCS analysis, to determine hydrodynamic diameter, performed on polymer modified Adenoviruses prepared according to the method of the invention, unmodified Adenoviruses and Adenoviruses bound only to polymerisation initiator units.
  • the y-axis represents hydrodynamic diameter in run.
  • the first column of the x-axis represents Adenovirus Ad5; the second, Adenovirus treated with HEG-sulfoNHS initiator (10 mg/ml, Example 5); the third, Adenovirus treated with HEG-sulfoNHS initiator (20 mg/ml, Example 6); the fourth, HEG- sulfoNHS initiator (10 mg/ml)-modified Adenovirus further modified using AGET ATRP (described in Example 9); the fifth, HEG-sulfoNHS initiator (20 mg/ml)- modified Adenovirus further modified using AGET ATRP (described in Example 9).
  • Figure 4 shows the results of laser Doppler velocometry, to determine zeta potential, performed on polymer modified Adenoviruses prepared according to the method of the invention, unmodified Adenoviruses and Adenoviruses bound only to polymerisation initiator units.
  • the x-axis represents zeta potential.
  • the bottom bar represents Adenovirus 5; the second bar (from the bottom) Adenovirus treated with HEG-sulfoNHS initiator (10 mg/ml, Example 5); the third, Adenovirus treated with HEG-sulfoNHS initiator (20 mg/ml, Example 6); the fourth, HEG-sulfoNHS initiator (10 mg/ml)-modified Adenovirus further modified using AGET ATRP (described in Example 9); the fifth, HEG-sulfoNHS initiator (20 mg/ml)-modified Adenovirus further modified using AGET ATRP (described in Example 9).
  • Figure 5 shows the results of PCS analysis, to determine hydrodynamic diameter, performed on polymer modified Adenoviruses prepared according to the method of the invention, unmodified Adenoviruses and Adenoviruses bound only to polymerisation initiator units.
  • the y-axis represents hydrodynamic diameter in nm.
  • the first column of the x-axis represents Adenovirus Adluc; the second, Adenovirus and DMSO; the third, Adenovirus modified according to example 3; the fourth, Adenovirus modified according to example 4; the fifth, Adenovirus modified according to example 7 and the sixth, Adenovirus modified according to example 8.
  • Figure 6 shows the results of laser Doppler velocometry, to determine zeta potential, performed on polymer modified Adenoviruses prepared according to the method of the invention, unmodified Adenoviruses and Adenoviruses bound only to polymerisation initiator units.
  • the y-axis represents zeta potential.
  • the first column of the x-axis represents Adenovirus Adluc; the second, Adenovirus and DMSO; the third, Adenovirus modified according to example 3; the fourth, Adenovirus modified according to example 4; the fifth, Adenovirus modified according to example 7 and the sixth, Adenovirus modified according to example 8.
  • Figures 7 and 8 demonstrate the biological activity of modified Adenovirus according to the invention in terms of transfection activity measured by luciferase assay and reported per 2500 cells.
  • Figure 5 shows transfection efficiency of Ad Luc at MOI of 1000.
  • Figure 6 shows transfection efficiency of Ad Luc of varying MOIs following ATRP polymerisation not linked to the virus capsid.
  • Figure 9 shows the result of an assessment of antibody-binding by polymer coated viruses, comparing binding to unmodified type 5 adenovirus (AdWT) and binding to two virus preparations that were coated in accordance with the invention (Examples 9 and 10) using a sandwich ELISA technique.
  • AdWT unmodified type 5 adenovirus
  • nucleic acid-containing biological entity biological entity
  • biological entity biological entity
  • entity entity
  • a nucleic acid-containing biological entity is a micro-organism or cell that contains genetic information or, in some cases, is a fragment or component thereof that contains genetic information.
  • the nucleic acid-containing biological entity is typically chosen from a virus, a bacteriophage, a prokaryotic cell, a bacterium (including a bacterial spore), an archaeum, a eukaryotic cell, a fungus, a spore, a nucleic acid-containing viral core with its outer membrane(s) and/or capsids removed, a membrane-stripped virus, a viral core, a eukaryotic cell nucleus, a mitochondrion and a mammalian cell nucleus.
  • the nucleic acid-containing biological entity may be a complex comprising a nucleic acid and a condensing agent.
  • nucleic acid includes synthetic molecules such as siRNA molecules, as well as DNA or RNA.
  • the synthetic molecules including the siRNA molecules may or may not be charged.
  • the condensing agent may be a cationic condensing agent, a polycation, a cationic lipid or a copolymer.
  • Preferred polycations include copolymers formed by oxidative polycondensation of thiol-terrninated oligopeptide blocks, such as:
  • the condensing agent maybe poly(ethylenimine) (linear (22 KDa) or branched (25
  • biodegradable poly(ethylenimine) eg based on internal disulfides
  • Suitable cationic lipids include: DOTAP, lipofectin, lipofectamine, oligofectamine and transfectam.
  • Preferred complexes include cationic polymer/siRNA complexes formed using lower molecular weight polymers based on CH 6 K 3 H 6 C (weight average molecular weight 30-80 kDa) at a w/w ratio around 10:1 (Nitrogen:Phosphate ratio 3.8). Under these conditions 80 k His6 RPC can form 86-92 nm particles (at w/w 8:1 (N:P 3:1).
  • the nucleic acid-containing biological entity is a virus or bacterium.
  • Bacterial biological entities used in carrying out the invention may include, for example, bacteria used in experimental cancer therapy (e.g. salmonella, Clostridium, bifidobacterium), bacteria or baculovirus used as a biological pesticide (e.g. nuclear polyhedrosis virus NPD, nonocclude virus NV, granulosis virus or bacillus thuringiensis), a bacteria strain useful for degrading oil sludges/spills or a genetically modified version thereof (e.g.
  • bacteria used in experimental cancer therapy e.g. salmonella, Clostridium, bifidobacterium
  • bacteria or baculovirus used as a biological pesticide e.g. nuclear polyhedrosis virus NPD, nonocclude virus NV, granulosis virus or bacillus thuringiensis
  • a bacteria strain useful for degrading oil sludges/spills or a genetically modified version thereof e.g.
  • enterobacteriaceae anitratum, pseudomonas, micrococcus, comamonas, zanthomonas, achromobacter or vidrio-aeromonas
  • a bacterial strain responsible for reducing sulphur to H 2 S in oil e.g. petrotoga mobilis, petrotoga miotherma, desulfotomaculum nigrificans, desulphovibrio
  • a bacterial strain capable of oxidising sulphur from oil e.g. rhodococcus sp. Strain ECRD-I).
  • a further example of a bacterium that is suitable for use as the biological entity is one selected from Rickettsiella popiliae, Bacillus popiliae, B. thuringiensis (including its subspecies israelensis, kurstaki and B. sphaericus), B. lentimorbus, B. sphaericus, Clostridium malacosome, Pseudomonas aeruginosa and Xenorhabdus nematophilus.
  • a further example of a bacterium that is suitable for use as the biological entity is one selected from Rickettsiella popiliae, Bacillus popiliae, B. thuringiensis (including its subspecies israelensis, kurstaki and B. sphaericus), B. lentimorbus, B. sphaericus, Clostridium malacosome, Pseudomonas aeruginosa and Xenorhabdus nematophilus.
  • any virus may be used in the present invention as the biological entity.
  • the virus is preferably a recombinant genetically engineered virus.
  • the recombinant virus optionally contains a transgene.
  • transgene is used herein to denote a nucleic acid that is not native to a virus.
  • a transgene could encode a biologically functional protein or peptide, an antisense molecule, or a marker molecule.
  • the virus may be a wild type lytic virus or a genetically modified version, including conditionally replicating forms that have been 'armed' with transgenes designed to modify virus activity (e.g.
  • transgenes encoding proteins intended to enhance virus spread through solid tumours) or to improve anticancer activity (e.g. transgenes encoding an immune stimulating protein or GDEPT protein), and also including viruses engineered to encode other viruses and produce them upon infection.
  • the virus is often genetically modified adenovirus (including non human adenovirus), herpes virus or vaccinia encoding transgenes as antigens.
  • the virus is either an RJSfA or DNA virus and is optionally from one of the following families and groups: Adenoviridae; Alfamoviruses; Bromoviridae; Alphacryptovirases; Partitiviridae: Baculoviridae; Badnaviruses; Betacryptoviruses; Partitiviridae; Bigeminiviruses; Geminiviridae; Birnaviridae; Bromoviruses; Bromoviridae; Bymoviruses; Potyviridae; Bunyaviridae; Caliciviridae; Capillovirus group; Carlavirus group; Carmovirus group; Caulimovirus group; Closterovirus group; Commelina yellow mottle virus group; Comovirus virus group; Coronaviridae; PM2 phage group; Corcicoviridae; Cryptic virus group; Cryptovirus group; Cucumovirus virus ⁇ 6 phage group; Cystoviridae; Cytorhabdo
  • a particularly preferred virus for use in the invention is an adenovirus, adenoassociated virus, baculovirus, herpesvirus, papovavirus, retrovirus (including lentivirus) or poxvirus.
  • Adenovirus and poxvirus are especially preferred, including unusual adenovirus serotypes and non-human adenovirus (e.g. Simian adenovirus), and pox viruses based on Wyeth and Western Reserve backgrounds.
  • a component of a micro-organism that is suitable for use as the biological entity may be provided by, for example, a viral core or a provirus (from e.g. pox viruses).
  • a viral core is an adenovirus core that is preparable by the method disclosed in Russell, W. C, M., K., Skehel, J. J. (1972)."The preparation and properties of adenovirus cores” Journal of General Virology 11, 35-46 and modifications thereto.
  • the biological entity is an adenovirus, adenovirus core, pox virus or pox virus core.
  • a phage which is suitable for use as the biological entity is for example one from one of the following families: Cyanophages, Lambdoid phages, Inovirus, Leviviridae, Styloviridae, Microviridae, Plectrovirus, Plasmaviridae, Corticoviridae, Satellite bacteriophage. Myoviridae, Podoviridae, T-even phages.
  • An example of a particular phage is MV-L3, PI, P2, P22, ⁇ 29, SPOI, T4, T7, MV-L2, PM2, Fl, MV-L51, ⁇ 174, ⁇ 6, MS2, M13, Q ⁇ , tectiviridae (e.g. PRDl).
  • a fungus that is suitable for use as the biological entity is for example one from familv Basidiomycetes (which make basidiospores, which include classes such as Gasteromycetes, hymenomycetes. urediniomycetes, ustilaginomycetes), Beauveria, Metarrhizium, Entomophthora or Coelomomyces .
  • a spore that is suitable for use as the biological entity is a basidiospore, actinomyceres, arthrobacter, microbacterium, Clostridium, Rhodococcus, Thermomonospora or Aspergillus fumigatus.
  • a eukaryotic cell suitable for use as the nucleic acid-containing biological entity is a blood cell (erythrocyte, leukocyte including B lymphocyte and T lymphocyte), progenitor cells isolated from bone marrow, haemopoietic stem cells, mesenchymal stem cells, embryonic stem cells, cord blood-derived stem cells or cells derived from progenitor cells or stem cells.
  • erythrocyte leukocyte including B lymphocyte and T lymphocyte
  • progenitor cells isolated from bone marrow
  • haemopoietic stem cells mesenchymal stem cells
  • embryonic stem cells embryonic stem cells
  • the process of growing polymer chains from one or more initiation points on the surface of a biological entity comprises a process of polymerisation from one or more initiation points on the surface of the biological entity in the presence of the monomers from which the polymer will be formed and also in the presence of one or more catalysts that facilitate the polymerisation.
  • growing a polymer from the surface of the entity means building up a polymer at one or more initiation points from individual monomer building blocks.
  • an initiation point is a group, typically a particular functional group, on the surface of the biological entity from which a polymer may grow in the presence of a catalyst, hi one embodiment, such initiation points are naturally present on the surface of the biological entity. Growth of the polymer can therefore be directly carried out from such naturally occurring initiation points. Examples of such naturally occurring initiation points include free hydroxyl groups, saccharides or thiol groups.
  • the method of the invention involves a first step (a) wherein initiation points are introduced to the surface of the biological entity either by genetic engineering or by chemical modification. Polymer chains are then grown from the initiation points in a second step (b). A combination of one or more types of initiation point selected from naturally occurring, genetically engineered and chemically modified may be employed. Further, each initiation point may be introduced by use of a combination of genetic engineering and chemical modification.
  • initiation points by genetic engineering of the biological entity may involve, for example, engineering an adenovirus to contain cysteine residues bearing free thiols in its fibre molecules. These free thiols can be used to initiate the growth of a polymer chain.
  • Suitable processes for introducing initiation points by genetic engineering will be apparent to those skilled in the art and include, for example, the processes described iiiKreppel, F. et al, Molecular Therapy, 12, 1, 2005. Kreppel, et al describes the genetic introduction of cysteine residues in adenovirus capsids.
  • a biological entity e.g. adenovirus
  • a biological entity maybe genetically engineered to contain biotin or sugar groups on its surface, which may act as initiation points in the process of the present invention, either with or without further chemical modification.
  • the method of the invention comprises a first step (a) of linking one or more polymerisation initiator units to the surface of the entity followed by a step (b) of growing polymer chains from the polymerization initiator units.
  • Step (a) in this case typically involves reacting the nucleic acid containing biological entity with one or more polymerisation initiator units under aqueous conditions.
  • the conditions employed should be compatible with the viability and integrity of the biological entity.
  • step (a) is typically carried out at room temperature, although any temperature can be used so long as biological integrity of the biological entity is maintained.
  • a polymerisation initiator unit is a molecule having at least one group capable of bonding to the surface of a biological entity and further having one or more groups capable of acting as an initiation point in a polymerisation reaction.
  • Polymerisation initiator units typically consist of molecules of general formula A n -L- B m , wherein A is a group capable of bonding to the surface of the biological entity, B is a group capable of acting as the initiation point in a polymerisation reaction, and L is a linker.
  • the linker moiety is not particularly limited and may be any moiety capable of linking A and B.
  • L does not contain functional groups reactive under the conditions employed by the present invention.
  • the physical properties of L eg. degree of hydrophilicity may serve to maximise the reaction of group A with the surface of the biological entity.
  • L is preferably hydrophilic, thus improving the solubility of the polymerisation initiator unit in physiological solvents. L may also be absent depending on the properties of A and B. n is from 1 to 5, preferably 1. m is from 1 to 5, preferably 1.
  • L is typically a polymer comprising a plurality of monomer units U.
  • U is preferably a hydrophilic monomer unit, more preferably -CH 2 O- or -CH 2 CH 2 O-, most preferably -CH 2 CH 2 O-.
  • L is an amino acid or peptide.
  • Preferably L comprises one or more monomer units U, more preferably 5 or more monomer units U.
  • initiators having large molecular weights e.g. compounds having at least 100, at least 1000 or at least 3000 monomer units U.
  • PEG poly(ethylene glycol)
  • PEG 5000 may be preferred.
  • Use of initiator molecules having a large size prevents the initiator from penetrating the outer layer of the biological entity and entering the vulnerable regions within.
  • the polymerisation initiator unit is water-soluble.
  • the at least one group capable of bonding to the surface of a biological entity (A) is at least one group capable of forming a covalent bond with a group present on the surface of the biological entity, for example with an amine group, thiol, hydroxy group, aldehyde, ketone, tyrosine residue, carboxylic acid or sugar group.
  • group A is capable of forming a covalent bond with an amine group on the surface of the biological entity.
  • suitable types of group A in this embodiment include acid chlorides, acyl-thiazolidine-2-thiones, maleimides, N-hydroxy-succinimide esters (NHS esters) sulfo-N-hydroxy-succinimide esters (Sulfo-NHS esters), 4-nitrophenol esters, epoxides, 2-immo-2-methoxyethyl-l- thioglycosides, cyanuric chlorides, imidazolyl formates, succinimidyl succinates, succinimidyl glutarates, acyl azides, acyl nitriles, dichlorotriazines, 2,4,5- trichlorophenols, azlactones and chloroformates.
  • group A is capable of forming a covalent bond with a thiol group on the surface of the biological entity.
  • suitable types of group A in this embodiment include alkyl halides, haloacetamides, and maleimides.
  • a reducible group may be introduced using pyridyldithiopropionyl mixed disulfides, thus enabling cleavage of the initiator from the biological entity following cellular uptake. Modification of the capsid using initiators which can be cleaved from the capsid such as disulphides may also be used to permit intracellular release of the polymer.
  • group A is capable of forming a covalent bond with a hydroxyl group on the surface of the biological entity.
  • suitable types of group A in this embodiment include chloroformates and acid halides.
  • hydroxyl groups on the surface of the biological entity can be oxidised with an oxidizing agent, e.g. periodate, followed by reaction with polymerisation intiator units having A groups that include hydrazines, hydroxylamines or amines.
  • group A is capable of forming a covalent bond with a tyrosine residue on the surface of the biological entity.
  • suitable types of group A in this embodiment include sulfonyl chlorides and iodoacetamides.
  • group A is capable of forming a covalent bond with an aldehyde or ketone group on the surface of the biological entity.
  • suitable types of group A in this embodiment include hydrazides, semicarbazides primary aliphatic amines, aromatic amines and carbohydrazides.
  • group A is capable of forming a covalent bond with a carboxylic acid on the surface of the biological entity. This can be effected by, for example, activating a carboxylic acid using the water soluble carbodiimide, 1-ethyl- 3-(3-dimethylaminopropyl)carbodiimide hydrochloride followed by reaction with a polymerisation initiator unit having an amine as group A.
  • group A is capable of reacting with a sugar on the surface of the biological entity resulting in the formation of a covalent bond.
  • This can be effected by, for example, enzyme-mediated oxidation of the sugar with galactose oxidase to form an aldehyde followed by reaction with a polymerisation intiator unit having an aldehyde reactive compound such as a hydrazide at group A.
  • the at least one group capable of bonding to the surface of a biological entity (A) is at least one group capable of bonding to the surface of the biological entity by hydrophobic interaction.
  • the group capable of bonding to the surface of the biological entity (A) is typically a hydrophobic moiety such as cholesterol or a long chain lipid such as a stearyl group.
  • the group capable of acting as the initiation point in a polymerisation reaction may be any group from which a polymer will grow when reacted with a plurality of suitable monomer units in the presence of a suitable catalyst.
  • the functionality possessed by the initiation point depends on the polymerisation methodology employed. It may initiate chemical polymerizations, or it may act as a primer for enzymatic polymerizations.
  • the initiation point typically comprises a short primer suitable for recognition by the enzyme.
  • primers could be, for example, sialylated oligosaccharides for recognition by polysialyltransferase, or short ⁇ 1-4 linked oligomers of D-glucose (e.g. maltotetraose) for recognition by glycogen synthase.
  • D-glucose e.g. maltotetraose
  • Many enzymes are suitable for catalysing, and each requires a suitable primer to initiate polymerization.
  • the appropriate primer typically requires conjugation to the surface of the biological entity by linkage of a polymerization initiation unit to the biological entity. However, the primer may be naturally present on the surface of the entity, or may be there by virtue of genetic engineering.
  • the initiation point(s) are typically selected from alkyl halides and aryl halides.
  • an alkyl halide is an alkyl fluoride, alkyl chloride, alkyl bromide or an alkyl iodide.
  • Alkyl chlorides are preferred.
  • Tertiary alkyl halides are more preferred.
  • the number of initiation points (e.g. the number of polymerisation initiator units linked to the surface of each biological entity) is usually 20,000 per square micrometre of surface area of the biological entity or less, for example 2000 or less, 500 or less, 100 or less, or 50 or less.
  • the branching nature of the polymers gives a high steric protection to the biological entity, while the low number of initiation points (or polymerisation initiator units attached) minimises damage to the biological entity, which could otherwise reduce its activity at a target site.
  • the initiation points (or polymerisation initiator units) on any one biological entity may be the same or different, but are typically the same.
  • the number of initiation points (e.g. the number of polymerisation initiator units linked to the surface of each biological entity) is usually 20,000 per square micrometre of surface area of the biological entity or more, for example 50,000 or more, or 100,000 or more.
  • the high number of initiation points (or polymerisation initiator units attached) allows production of a dense coating layer that provides a high level of steric protection.
  • the initiation points (or polymerisation initiator units) on any one biological entity may be the same or different, but are typically the same.
  • the process of the invention involves an additional purification step after step (a) and before step (b) in order to remove, e.g. any unreacted polymerisation initiator unit.
  • Any appropriate means of purification may be used, for example size exclusion chromatography. This may involve the use of size exclusion centrifugation spin columns or, alternatively, dialysis.
  • a method employing size exclusion centrifugation spin columns offers rapid and efficient purification, particularly of viruses and modified viruses. Dialysis is a useful alternative method in the event of non-specific adherence of the modified biological element to the spin columns. High purity of the biological entity is important to ensure that polymerisation is only initiated on the entity itself and not from any free initiator in solution.
  • Step (a) is not limited to a single reaction step.
  • step (a) could comprise two or more steps.
  • a first step might involve reacting the biological entity with a pre- polymerisation initiator unit or a genetic engineering step.
  • a subsequent step could involve functionalising the pre-polymerisation initiator unit bound to the surface of the biological entity, or the genetically engineered entity, so that it was capable of acting as an initiator in a polymerisation reaction.
  • This series of steps could also involve one or more purification steps.
  • a pre-polymerisation initiator unit is a molecule having at least one group capable of bonding to the surface of the biological entity and being capable of further reaction to provide a group capable of acting as an initiation point in a polymerisation reaction.
  • the polymerisation step of the present invention typically involves reacting the biological entity having one or more initiation points on its surface with a plurality of monomers, which maybe the same or different, using a suitable method of polymerisation, in the presence of one or more catalysts. Any appropriate method of polymerisation may be used, although the conditions should be compatible with the viability of the biological entity.
  • the method of the invention is normally conducted in a solvent that is non-destructive to the biological entity, allowing maintenance of sufficient biological integrity for the modified biological entity to fulfil its purpose.
  • the catalyst(s) may include any catalyst appropriate for the type of polymerisation employed. Typically a chemical or biological catalyst is used.
  • a biological catalyst is typically an enzyme, e.g. polysialyltransferase or glycogen synthase.
  • An enzymatic approach to the polymerisation typically involves using one or more synthetic enzymes to catalyse the growth of polymer chains from the initiation points on the surface of the initiator-bound biological entity, hi the case of polysialylytransferase (PST), where the growing polymer chain will be polysialic acid, PSTs transfer oligomers of sialic acid onto a terminal sialic acid residue on the polymerisation initiator unit.
  • PST polysialylytransferase
  • glycogen synthase where the growing polymer will be glycogen
  • glycogen synthase catalyzes the reaction between UDP glucose and the non-reducing end of the growing glycogen chain, hence the requirement for a short surface-bound primer of glucose units.
  • Branching can be introduced, if required, using a small quantity of the glycogen synthase branching enzyme, amylo-(l,4 - 1,6)- transglycosylase, thus increasing the steric barrier surrounding the biological entity, e.g. virus.
  • This enzyme transfers a terminal fragment of 6-7 glucose residues (from a polymer at least 11 glucose residues long) to an internal glucose residue at the C-6 hydroxyl position. Branching could occur continually during linear synthesis, or the branching enzyme could be introduced at a specific time.
  • the polymerisation step comprises polymerising the plurality of monomers using controlled radical polymerisation (CRP).
  • Controlled radical polymerisation is a term well known to the person skilled in the art and typically refers to free radical addition polymerization of monomers wherein there is a degree of control of molecular weight of the growing polymer with respect to time.
  • CRPs include atom transfer radical polymerisation (ATRP), atom generated by electron transfer (AGET) ATRP, and reversible addition-fragmentation chain transfer (RAPT).
  • Polymerisation methodologies are not exclusively defined as CRPs but may include free radical polymerisation (FRP) and ring opening metathesis polymerisation (ROMP)
  • ATRP is used as the polymerisation technique.
  • ATRP maybe carried out in an aqueous environment or optionally in a mixture of water and another solvent, which may be, for example, methanol, DMSO, THF, DMF or NMP.
  • ATRP polymerisation is preferably carried out under substantially oxygen free conditions. Typically, ATRP is carried out at room temperature.
  • Catalysts for use in ATRP polymerisation are typically transition metals or transition metal salts complexed with one or more ligands.
  • a ligand is a compound that co-ordinates to a metal ion (usually a transition metal ion).
  • Suitable transition metals include copper, cobalt, molybdenum, rhodium, osmium, ruthenium, palladium, nickel and rhenium. Copper is preferred.
  • Suitable ligands for copper coordinating catalysts include 2,2'-bipyridine (bpy), 4,4'-di(5-nonyl)-2,2- bipyridine (dNbpy), N,N,N',N'-tetramethylethylenediamine (TMEDA), N-propyl(2 ⁇ ⁇ yridyl)methanimine (NPrPMI), 2,2':6',2"-ter ⁇ yridme (tpy), 4,4',4"-tris(5-nonyl)- 2,2' : 6',2"-terpyridine (tNtpy), N,N,N',N",N"-pentamethyldiethylenetriamine (PMDETA), N,N-bis(2-pyridylmethyl)octylamine (BPMOA), 1,1,4,7,10,10- hexamethyltriethylenetetramine (HMTETA), tris[2-(dimethylamino)ethyl]amine (
  • AGET is used as the polymerisation technique.
  • AGET is tolerant of low concentrations of O 2 .
  • Suitable catalysts and ligands for AGET include those suitable catalysts and ligands detailed above for ATRP. However, a lower concentration of the catalyst can be used. Mild oxidants such as Cu(O), Sn(EH 2 ) and ascorbic acid are typically added to the reaction mixture in order to oxidatively regenerate the catalyst.
  • the polymerisation may be controlled by the addition of a sacrificial initiator, which may consist of a group capable of initiating polymerisation, e.g. a group B as defined above, which is bound to a solid phase e.g. a solid support such as a polystyrene bead, which can be easily removed a the end of the reaction.
  • a sacrificial initiator which may consist of a group capable of initiating polymerisation, e.g. a group B as defined above, which is bound to a solid phase e.g. a solid support such as a polystyrene bead, which can be easily removed a the end of the reaction.
  • Sacrificial initiators are used to maintain a suitable reaction equilibrium between the activated catalyst and deactivated catalyst complex by ensuring that there are sufficient polymer chains "growing.” This maintains the low polydispersity of the resulting polymer by keeping the rate of polymerization constant. A low concentration of growing polymer chains will result in erratic polymer extension and give rise to high polydispersity polymers. This effect is described in, for example, Bontempo, D, et al, J. Am. Chem. Soc. 127, 6508-6509, 2005, which demonstrates the polydispersity of growing polymer chains with and without the sacrificial initiator present.
  • the polymerisation may employ one or more different types of monomer, wherein a monomer is any individual unit used to form the polymer.
  • the monomers each include one or more polymerisable groups and are typically selected from monofunctional monomers, difunctional monomers, macromonomers, and derivatives of biologically active agents.
  • a macromonomer is typically a monomer having a molecular weight of 500 or more, 750 or more, 1000 or more, 1500 or more, 2000 or more or 5000 or more.
  • the polymerisable group used for radical based polymerisations is preferably an acrylate/acrylamide or methacrylate/methacrylamide.
  • Suitable monofunctional monomers are 2-hydroxypropyl methacrylamide (HPMA) and hydroxyethylmethacrylate (HEMA). Methacrylate/methacrylamide monomers are preferred for RAFT catalysed polymerisations. These monofunctional monomers can be used to provide a basic polymer structure.
  • Difunctional monomers for example dimethacrylates, or macromonomers can be used to introduce branching and/or cross-linking into the polymer structure. This is described further in terms of some particular embodiments below.
  • Further functional groups may be incorporated into the monomers as desired.
  • reactive functional groups are incorporated which will not take part in the polymerisation.
  • Such reactive functional groups can be utilised in post- polymerisation functionalisation of the polymer chains, for example by linking biologically active agents to the polymer to provide targeting of the modified biological entity.
  • Glycidylmethacrylates are examples of monomers that enable post- polymerisation functionalisation to be carried out, for example by reaction of a biologically active agent containing a reactive amine with the glycidyl group.
  • Such monomers containing reactive functional groups may, for example, be added to the polymerisation mixture towards the end of polymerisation so that the functional groups are located on the outside of the coated entity and are accessible for further reaction.
  • a monomer in another embodiment, includes a hydrophobic group so that such groups can be incorporated into the polymer. This has the effect of modifying the solubility or partition co-efficient characteristics of the biological entity in nonaqueous media.
  • a radioisotope is incorporated in the growing polymer in order to allow the detection of the biological entity e.g. in a biological environment. This incorporation may involve, for example, synthesizing a monomer unit which is bound to or encapsulates the radioisotope, using covalent or non-covalent interactions.
  • the polymerisation step involves the growth of branched and/or cross-linked polymers.
  • Branched polymers include hyperbranched polymers and brush polymers.
  • Hyperbranched polymers (including dendrimers) have tree-like structures in which one or more branches from the main polymer backbone are themselves branched.
  • Brush (or bottle brush) polymers have a plurality of side chains (typically linear side chains) attached to the main polymer backbone.
  • cross-linking may occur during polymerisation or may be induced afterwards by change of chemical environment of addition of cross-linking group.
  • Cross-linked structures encapsulating individual biological entities may be referred to as 'nano gels'.
  • One embodiment which exemplifies the generation of a branched structure involves the introduction of preformed side-chains into the growing polymer to increase the steric protection of the biological entity.
  • Macromonomers such as monomethoxypolyethylene glycol methacrylate may simply be added to the reaction mixture, typically at later stages of the polymerisation reaction, where they will compete with the residual free monomer for incorporation into the growing chain.
  • the polymerisation may be halted, in the case of ATRP by oxygenating the reaction mixture, and the partially modified biological entity purified.
  • Polymerisation can then be restarted with the macromonomers to generate a brush or "bottle brush” structure wherein the polymer is substantially linear in the portion attached to the biological entity, and contains one or more long branches formed from the macromonomers in the terminal portion of the polymer chain.
  • the polymerisation comprises the sequential steps of (i) reacting the biological entity with a plurality of monomers to create a partially modified biological entity, (ii) terminating the initial polymerisation reaction, (iii) optionally purifying the partially modified biological entity, and (iv) reacting the partially modified biological entity with a plurality of macromonomers.
  • the macromonomers used in this embodiment typically have a molecular weight of 1000 or more, preferably 2000 or more, more preferably 5000 or more.
  • Step (i) may, for example comprise reacting the biological entity with a plurality of monomers having a molecular weight of less than 1000, preferably less than 750, more preferably less than 500 to create a partially modified biological entity.
  • step (b) comprises the sequential steps of (i) reacting the biological entity with a plurality of monomers, and (ii) adding to the polymerisation mixture a plurality of macromonomers.
  • the macromonomers used in this embodiment typically have a molecular weight of 1000 or more, preferably 2000 or more, more preferably 5000 or more.
  • Step (i) may, for example, comprise reacting the initiator-bound biological entity with a plurality of monomers having a molecular weight of less than 1000, preferably less than 750, more preferably less than 500 .
  • Step (ii) is typically carried out at the later stages of the polymerisation reaction.
  • Suitable macromonomers are readily synthesized from the corresponding semi- telechelic polymers (i.e. where one end of a linear polymer is functionalised), for example methacrylated macromonomers may be produced by modification with methacryloyl chloride, hi addition, a number of methacrylated PEG macromonomers of various molecular weights are available commercially. The brash or "bottle brush" effect can alternatively be introduced by post- polymerization derivitisation. Functional monomers such as glycidyl methacrylate may be included in the polymer main chain and, after polymerisation is complete, macromolecules linked to the glycidyl functional groups.
  • Appropriate macromolecules include PEG-NH 2 and semitelechelic HPMA and poly(HEMA/HPMA).
  • the macromolecules must have a group which will react with the functionalised polymer.
  • Amine groups for example, can be used to react with a glycidyl-functionalised polymer. The amine-glycidyl reactions can be carried out in aqueous solution and with stereochemical retention if this is important for subsequent biological ligand functionality.
  • hyperbranched polymers may be produced by carrying out polymerisation in the presence of a plurality of difunctional monomers, which may be the same or different, and a chain transfer agent.
  • the chain transfer agent is typically in stoichiometric excess over the dif ⁇ nctional monomer.
  • a non-cross-linked hyper-branched polymer results. This can be accomplished using either a conventional chain transfer agent (e.g. 2- aminoethanethiol) or a catalytic chain transfer species (e.g. ATRP metal centre).
  • branching can be introduced by adding a small quantity of a branching enzyme as detailed above.
  • the degree of branching of the polymer(s) attached to the biological entity of the invention increases further away from the point of attachment of the polymer(s) to the biological entity.
  • the above- described hyper-branching embodiments provide polymer(s) attached to the biological entity that are dendritic in nature.
  • polymerisation is performed in the presence of a difunctional monomer or macromonomer in the absence of a chain-transfer agent.
  • This allows cross-linking of the extending polymer chain, improving the steric resistance imparted by the polymer coating.
  • This forms a nanogel structure, a cross-linked polymer wherein the holes between the cross-linked chains are of nanoscale dimensions.
  • Each nanogel encapsulates an individual biological entity, resulting in improved resistance to neutralization by antibodies while retaining potential for biological activity.
  • Suitable difunctional monomers include bismethacrylates or bismethacrylamides, e.g. PEG bismethacrylates or other difunctional PEG groups. Variation of the length of the PEG chain enables the mesh size of the cross-linked polymer to be controlled.
  • any of the above-described branching and/or cross-linking embodiments preferably results in the steric size of each polymer increasing further away from the point of attachment of the polymer to the biological entity.
  • the polymer, and/or inter-polymer cross-links and/or the linkages between the polymer and the biological entity are optionally 'bioreversible' for example they may be reducible, or hydrolytically or enzymatically degradable. Instability provided by hydro lytic degradability can be desirable since it permits regulation of the time for which the biological entity is protected.
  • the polymer is provided with a tissue-specific targeting group, the polymer (or the linkage between the polymer and the biological entity) can be designed so that the polymer protects the biological entity for as long as it takes for the modified biological entity to reach the appropriate location within the target tissue before disintegrating, freeing the biological entity to interact with the tissue.
  • the polymer could be designed to disintegrate at a rate yielding optimal kinetics of release of the biological entity.
  • Instability provided by enzymatic degradability can be desirable since it permits the polymer (or the linkage between the polymer and the biological entity) to be designed for cleavage selectively by chosen enzymes.
  • Such enzymes could be present at the target site, endowing the modified biological entity with the possibility of triggered disintegration at the target site, thereby releasing the biological entity for interaction with the target tissue.
  • the enzymes may also be intracellular enzymes, which can bring about disintegration of the modified biological entity in selected cellular compartments of a target cell to enhance the activity of the biological entity.
  • enzyme-cleavage sites may be designed to promote disintegration of the modified biological entity in response to appropriate biological activity (e.g. arrival of an invading or metastatic tumour cell expressing metalloproteinase).
  • enzymes capable of activating the modified biological entity may be administered at the appropriate time or site to mediate required disintegration of the modified biological entity and subsequent interaction of the biological entity with the tissue.
  • the nanogel mentioned above may be hydrolytically unstable or is degradable by an enzyme, for example matrix metalloproteinases 2 or 9. This is in order that the biological entities are immobilised within the nanogel and so that the release of the biological entities can be regulated.
  • Infectivity mediated through specific cell surface receptors may be restored or replaced in certain embodiments by incorporating one or more biologically active agents in the growing polymer or by reacting a biologically active agent with a functionalised polymer.
  • one or more derivatives of biologically active agents can be incorporated in the growing polymer coating.
  • macromonomers of the desired biologically active agents are synthesized by incorporation of an appropriate polymerisable group, for example by methacryloylation.
  • the macromonomers are preferably added at the end of the polymerisation to ensure that the biologically active agent does not interfere with production of the steric coating, and to provide the desired biological functionality in an accessible site on the outside of the polymer modified biological entity.
  • This embodiment is not restricted to the use of individual biologically active agents and may include e.g. mixtures of biologically active agents being incorporated onto a single coated biological entity.
  • a multi-purpose agent may be used as the biologically active agent to permit subsequent attachment of desired molecules.
  • monomers containing reactive functional groups are incorporated into the polymer chain and (after optional purification of the polymer) post-polymerisation functionalisation is then carried by adding a biologically active agent capable of reacting with the reactive functional group, for example by way of an amine group.
  • biologically active agents are preferably incorporated in accordance with the invention to improve targeting, tissue penetration, pharmacokinetics or immune stimulation or suppression.
  • the biologically active agent may be, for example, a growth factor or cytokine, a sugar, a hormone, a lipid, a phospholipid, a fat, an apolipoprotein, a cell adhesion promoter, an enzyme, a toxin, a peptide, a glycoprotein, a serum protein, a vitamin, a mineral, a ligand for a Toll-lie receptor, an adjuvant molecule, a nucleic acid, an immunomodulatory element or an antibody recognising a receptor, for example a growth factor receptor or recognising a tissue- specific antigen or tumour-associated antigen.
  • An antibody is preferably used as the biologically active agent to re-target modified biological entities to a different target site which may comprise, for example, various receptors, different cells, extracellular environments and other proteins.
  • a different target site which may comprise, for example, various receptors, different cells, extracellular environments and other proteins.
  • a wide range of different forms of antibody may be used including monoclonal antibodies, polyclonal antibodies, diabodies, chimeric antibodies, humanised antibodies, bi- specific antibodies, camalid antibodies, Fab fragments, Fc fragments and Fv molecules.
  • a suitable biologically active agent is for example an antibody recognizing a cancer associated antigen such as a carcinoembryonic antigen or ⁇ - fetoprotein, tenascin, HER-2 proto-oncogene, prostate specific antigen or MUC- 1 or an antibody recognising an antigen associated with tumour-associated endothelial cells, such as receptors for vascular endothelial growth factor (VEGF), Tiel, Tie2, P-selectin, E-selectin or prostate-specific membrane antigen (PSMA).
  • VEGF vascular endothelial growth factor
  • Tiel Tiel
  • Tie2 P-selectin
  • E-selectin E-selectin
  • PSMA prostate-specific membrane antigen
  • a suitable multi-purpose protein for use as the biologically active agent to act as a generic linker permitting flexibility of application is protein G (this will bind an antibody, allowing surface modification with any IgG class antibody from most species), protein A (which has properties similar to protein G), avidin (which binds biotin with very high affinity allowing the incorporation of any biotin labelled element onto the surface), streptavidin (which has properties similar to avidin), extravidin (which has properties similar to avidin), bungaratoxin-binding peptide (which binds to bungaratoxin fusion proteins), wheat germ agglutinin (which binds sugars), hexahistidine (which allows for gentle purification on nickel chelate columns), GST (which allows gentle purification by affinity chromatography).
  • protein G this will bind an antibody, allowing surface modification with any IgG class antibody from most species
  • protein A which has properties similar to protein G
  • avidin which binds biotin with very high affinity allowing the incorporation of any bio
  • a suitable growth factor or cytokine for use as the biologically active agent is for example Brain derived neurotrophic factor, Cilary neurotrophic factor, b-Endothelial growth factor, Epidermal growth factor (EGF), Fibroblast growth factor Acidic (aFGF), Fibroblast growth factor Basic (bFGF), Granulocyte colony-stimulating factor, Granulocyte macrophage colony- stimulating factor, Growth hormone releasing hormone, Hepatocyte growth factor, Insulin like growth factor-I, Insulin like growth factor-II, Interleukin-II, Interleukin-la, Interleukin-lb, Merleukin 2, Merleukin 3.
  • Merleukin 4 Merleukin 5, Interleukin 6, Interleukin 7, Merleukin 8, Merleukin 9, Merleukin 10, Merleukin 11.
  • Merleukin 12 Merleukin 13. Keratinocyte growth factor, Leptin, Liver cell growth Factor, Macrophage Colony stimulating factor, Macrophage inflammatory protein Ia, Macrophage inflammatory protein Ib,
  • a suitable sugar for use as the biologically active agent for incorporation is a monosaccharide, disaccharide or polysaccharide including a branched polysaccharide is, for example, D-Galactose, D-Mannose, D-Glucose, L- Glucose, L-Fucose, and Lactose. Sugars are typically incorporated by amino derivitisation.
  • a hormone which is suitable for use as the biologically active agent is, for example, Adrenomedullin, Adrenocorticotropic hormone, Chorionic gonadotropic hormone, Corticosterone, Estradiol, Estriol, Follicle stimulating hormone, Gastrin 1, Glucagon, Gonadotrophin, Growth hormone, Hydrocortisone, Insulin, Leptin, Melanocyte stimulating hormone, Melatonin, Oxytocin, Parathyroid hormone, Prolactin, Progesterone, Secretin, Thrombopoetin, Thyrotropin, Thyroid stimulating hormone, and Vasopressin.
  • a suitable lipid, fat or phospholipid for use as the biologically active agent for targeting the polymer modified biological entity or for providing steric protection is, for example, Cholesterol, Glycerol, a Glycolipid, a long chain fatty acid, particularly an unsaturated fatty acid e.g. Oleic acid, Platelet activating factor, Sphingomylin, Phosphatidyl choline, or Phosphatidyl serine.
  • a suitable cell adhesion promoter for use as the biologically active agent can be provided by, for example, Fibronectin, Larninin, Thrombospondin, Vitronectin, polycations, integrins or by oligopeptide sequences binding integrins or tetraspan proteins.
  • a suitable apoliproprotein for use as the biologically active agent that may also provide steric protection is, for example, a high-density lipoprotein or a low-density lipoprotein, or a component thereof.
  • a suitable enzyme for use as the biologically active agent, for example, to promote mobility of the modified biological entity through a particular environment is an enzyme capable of degrading the extracellular matrix (for example a gelatinase, e.g. matrix metallopro teases type 1 to 11, or a hyaluronidase), an enzyme capable of degrading nucleic acids (for example Deoxyribonuclease I, Deoxyribonuclease ⁇ , Nuclease, Ribonuclease A), an enzyme capable of degrading protein (for example Carboxypeptidase, plasmin, Cathepsins, Endoproteinase, Pepsin, Proteinase K, Thrombin, Trypsin, Tissue type plasminogen activator or Urokinase type plasminogen activator), an enzyme facilitating detection (for example Luciferase, Peroxidase, b-galactosidase), or other useful enzymes such as Amylase,
  • a suitable peptide for use as the biologically active agent may be provided by, for example, transferrin, Green/blue/yellow fluorescent protein, Adrenomedullin, Amyloid peptide, Angiotensin I, Angiotensin II, Arg-Gly-Asp, Atriopeptin, Endothelin, Fibrinopeptide A, Fibrinopeptide B, Galanin, Gastrin, Glutathione, Laminin, Neuropeptide, Asn-Gly-Arg, Peptides containing integrin binding motifs, targeting peptides identified using phage libraries, peptides containing nuclear localisation sequences and peptides containing mitochondrial homing sequences.
  • a suitable immunomodulatory element for use as the biologically active agent may be provided by any ligand for a 'Toll-like receptor' (TLR), notably: TLR2 ligands such as: Lipoglycans, Lipopolysacchari.de, Lipotechoic acids, peptidoglycans, or synthetic lipoproteins such as Pam2cys, Pam3cys, or Zymosan.
  • TLR2 ligands such as: Lipoglycans, Lipopolysacchari.de, Lipotechoic acids, peptidoglycans, or synthetic lipoproteins such as Pam2cys, Pam3cys, or Zymosan.
  • TLR3 ligands such as: double stranded RNA including poly I: C
  • TLR4 ligands including: negative lipopolysaccharide (LPS), Lipid A and analogues
  • TLR5 ligands including: Flagellin TLR7/8 ligands including GardiquimodT and Lniquimod (imidazoquinoline compounds), Loxoribine (guanosine) single stranded RNA, CLO75 thiazoloquinoline compound), CLO97 (imidazolquinoline compound)
  • TLR9 DNA sequences containing CpG islands
  • a suitable serum protein for use as the biologically active agent is, for example, Albumin, Complement proteins, Transferrin, Fibrinogen, or Plasminogen.
  • a suitable vitamin or mineral for use as a biologically active agent is, for example, Vitamin B 12, Vitamin B 16 or folic acid.
  • a polymer modified biological entity in accordance with the present invention can be synthesised so as to be targeted to a highly specific set of cells, e.g. tumour cells.
  • polymer modified biological entities in accordance with the present invention are not generally rendered inactive by neutralising antibodies. This is believed to be because the modified biological entity is shielded by the polymer.
  • the polymer modified biological entities of the invention can be purified using more aggressive technology than that which is feasible with existing unmodified biological entities.
  • the process of the invention comprises an additional purification step after polymerisation in order to remove any unreacted monomer and catalyst.
  • This purification step ensures that the product of the invention is not contaminated by molecules that might prove harmful in vivo.
  • This purification step can be carried out using similar techniques to those described above for the purification step between steps (a) and (b). As the size difference between unreacted monomers and modified biological entity is large, this purification step is readily achieved.
  • the present invention provides a nucleic acid-containing biological entity modified with a polymeric layer synthesized from the surface that is able to resist recognition by and binding of antibodies.
  • the determinative assay to show the lack of binding of biological macromolecules is that of antibody binding to the surface of the biological entity, typically determined by ELISA using immobilized nucleic acid-containing biological entities.
  • the polymer modified biological entity of the invention typically has a polymer coating that masks the normal infectivity of the biological entity by inhibiting the ability of the biological entity to bind to sites or receptors to which it would otherwise normally bind.
  • the polymer modified biological entity of the invention retains its viability.
  • the polymer-modified nucleic acid-containing biological entity is preferably modified with one or more branched polymer chains to improve steric protection of the entity.
  • the one or more branched chains attached to the surface of the polymer modified biological entity of the invention are typically hyperbranched, dendritic or brush polymer chains.
  • the number of hyperbranched, dendritic or brush polymer chains linked to the surface of the polymer modified biological entity of the invention is 20,000 per square micrometre of surface area of the biological entity or less, e.g. 2000 or less, 500 or less, 100 or less or 50 or less.
  • the modified biological entity can be characterised using EUipsometry and AFM.
  • the degree of protection of the surface of the biological entity can be evaluated using Surface Plasmon Resonance (SPR) and Quartz Crystal Microbalance (QCM) wherein the polymer modified surfaces are challenged with serum proteins and biological fluids.
  • SPR Surface Plasmon Resonance
  • QCM Quartz Crystal Microbalance
  • the physical properties and integrity of the polymer modified biological entities are determined by gel permeation chromatography (GPC), photon correlation spectroscopy (PCS) and by transmission electron microscopy (TEM).
  • GPC gel permeation chromatography
  • PCS photon correlation spectroscopy
  • TEM transmission electron microscopy
  • capsid integrity can be determined by its ability to exclude Picogreen.
  • the effects of polymer coating on surface charge are measured by zeta potentiometry, for example by laser Doppler velocometry.
  • the individual polymers formed on the surface of the biological entity are characterized following digestion of the entity, for example in the case of a virus with proteinase K, which degrades protein to tetrapeptides. Digestion is followed by purification and GPC (LALS) to determine the absolute molecular weight of the polymer chains. Protease digestion may leave some amino acids attached to the polymer chains, and these can be determined using amino acid analysis; this information can then be used to correct the molecular weight of the polymer chains. Levels of branching are assessed by the determination of the number of bromine- containing terminating groups which terminate the end of each growing polymer chain using ICP-MS. In this way, the number of chains can be determined and, by comparing this to the molecular weight of the polymer, therefore the degree of branching of the polymer can be calculated.
  • LALS GPC
  • the present invention provides a composition comprising a polymer modified biological entity according to the invention and a pharmaceutically acceptable carrier or diluent, which can be used in the microbial treatment, vaccination and gene therapy methods of the invention.
  • a composition comprising a polymer modified biological entity according to the invention and a pharmaceutically acceptable carrier or diluent, which can be used in the microbial treatment, vaccination and gene therapy methods of the invention.
  • Preferred compositions are free of contamination micro-organisms and pyrogens.
  • the polymer modified biological entities of the invention may be administered in a variety of dosage forms. Thus, they can be administered orally, for example as aqueous or oily suspensions.
  • the polymer modified biological entities of the invention may also be administered parenterally, either subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques.
  • the polymer modified biological entities may be administered by inhalation in the form of an aerosol via an inhaler or nebuliser.
  • the formulations for oral administration may contain, together with the polymer modified biological entity, solubilising agents, e.g. cyclodextrins or modified cyclodextrins; diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g.
  • solubilising agents e.g. cyclodextrins or modified cyclodextrins
  • diluents e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch
  • lubricants e.g. si
  • starch alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations.
  • Liquid dispersions for oral administration may be solutions, syrups, emulsions and suspensions.
  • the solutions may contain solubilising agents e.g. cyclodextrins or modified cyclodextrins.
  • the syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
  • Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol.
  • the suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol; solubilising agents, e.g. cyclodextrins or modified cyclodextrins, and if desired, a suitable amount of lidocaine hydrochloride.
  • a pharmaceutically acceptable carrier e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol
  • solubilising agents e.g. cyclodextrins or modified cyclodextrins, and if desired, a suitable amount of
  • Solutions for intravenous or infusions may contain as carrier, for example, sterile water and solubilising agents, e.g. cyclodextrins or modified cyclodextrins or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
  • solubilising agents e.g. cyclodextrins or modified cyclodextrins or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
  • a therapeutically effective amount of a polymer modified biological entity of the invention is administered to a patient.
  • typical doses would contain 10 -10 virus particles, depending on the individual virus.
  • the polymer modified biological entity of the invention is typically administered to the patient in a non-toxic amount.
  • the polymer modified biological entities of the present invention are useful for in vivo delivery of therapeutic genetic material to a patient, in carrying out gene therapy or genetic-vaccination treatment for example, wherein the polymer modified biological entity is a polymer modified biological entity in accordance with the invention which includes the therapeutic genetic material.
  • therapeutic genetic material is used herein to denote any genetic material or nucleic acid administered for obtaining a therapeutic effect, e.g. by expression of therapeutically useful proteins or RNA' s.
  • Gene therapy has applications across the whole field of human disease including, but not limited to, the treatment of cancer (including locally accessible tumour nodules suitable for direct injection, as well as metastatic cancer requiring systemic treatment), Parkinson's disease, X-SCID, Sickle Cell Disease, Lesch-Nyhan syndrome, phenylketonuria (PKU), Huntington's chorea, Duchenne muscular dystrophy, hemophilia, cystic fibrosis, lysosomal storage diseases, cardiovascular diseases and diabetes.
  • cancer including locally accessible tumour nodules suitable for direct injection, as well as metastatic cancer requiring systemic treatment
  • Parkinson's disease X-SCID
  • Sickle Cell Disease X-SCID
  • Sickle Cell Disease X-SCID
  • Lesch-Nyhan syndrome phenylketonuria
  • PKU phenylketonuria
  • Huntington's chorea Duchenne muscular dystrophy
  • hemophilia cystic fibrosis
  • lysosomal storage diseases cardiovascular diseases and diabetes
  • the polymer-modified biological entities of the invention may also be used for the delivery of viral vaccines.
  • Vaccination against HIV, tuberculosis, malaria, flu, cancer and other diseases are envisaged.
  • Vaccines may be given in prime boost regimes (i.e. by multiple administrations) or in combination with adjuvants.
  • the polymer modified biological entities of the present invention are useful for in vzvo delivery of therapeutic agents to a patient, in carrying out microbial therapy including virotherapy for example, wherein the polymer modified biological entity is a polymer modified biological entity in accordance with the invention.
  • the polymer modified biological entity of the present invention may be used in combination with other medicaments, e.g. other medicaments effective in the treatment of cancer.
  • the resultant acid (200 mg) was activated in THF (anhydrous, 2 mL) at 0°C by sequential addition of DMAP (catalytic amount), dicyclohexylcarbodiimide (95 mg) and thiazolidine-2-thione (54 mg).
  • the reaction was stirred for 6 hours at room temperature followed by addition of 50 ⁇ L of glacial acetic acid.
  • the reaction was stirred for a further 1 hour followed by filtration and concentration in vacuo. 5 mL anhydrous EtOAc was added and the mixture was left at 4 °C overnight. A precipitate formed and was removed by filtration followed by concentration in vacuo.
  • the product formed (shown as product X in figure 1) was used without additional purification.
  • Example 2 synthesis of HEG Sulfo-NHS based initiator unit Hexaethylene glycol (500 mg), and diisopropylethylamine (450 mg) in 5 mL anhydrous dichloromethane (DCM), was cooled to 0°C in a salt ice bath. 2-bromo-2- methyl propionyl bromide in DCM (2 mL) was added dropwise with stirring. The reaction was stirred at 0°C for 1 hour and allowed to return to room temperature. The reaction was continued overnight at room temperature. The mixture was concentrated in vacuo and purified by column chromatography (silica with 10% MeOH:90% EtOAc). Yield 325 mg, 42%.
  • DCM anhydrous dichloromethane
  • the product was activated using a 10 x molar concentration of phosgene (20% in toluene) overnight in anhydrous DCM followed by removal of excess phosgene in vacuo.
  • the chloroformate was immediately reacted with 1 equivalent of sulfo-N- hydroxysuccinimide(NHS) in anhydrous THF and reacted for 2 hours. A solid precipitate formed and was removed by filtration.
  • the product (structure below) was isolated following concentration in vacuo.
  • a catalyst solution was prepared by dissolving CuBr (6.4mg) and 2,2-bipyridine (14.0 mg) in 500 ⁇ L of degassed water. 2-Hydroxypropyl methacrylamide (HPMA) (45mg) was added to 150 ⁇ L of degassed water in an argon atmosphere. To this was 594
  • ATRP was carried out as described in Example 7, except that the initiator-bound Adenovirus used was prepared according to Example 4.
  • Example 9 AGET ATRP initiated from the surface of the virus capsid [1]
  • a catalyst solution was prepared containing CuBr 2 (0.45 mg), TPMA (0.58 mg), PEGMa 300 (60 mg) in degassed water (225 ⁇ L). To this solution was added the purified initiator modified virus (100 ⁇ L) (Example 5) and degassed using argon. The reaction was started using an ascorbic acid solution (35.2 ⁇ L, lmg/mL). The reaction vessel was purged with argon and allowed to react for 2 hours at room temperature (22° C), before purification using sephacryl S400 HR spin columns (GE Healthcare). The resulting particles were further purified using CsCl density centrifugation followed by dialysis against virus storage buffer (PBS, pH 7.8, CaCl (0.1 mg/mL), MgCl (0.1 mg/mL) glycerol (10%)).
  • PBS virus storage buffer
  • Example 10 AGET ATRP initiated from the surface of the virus capsid [21
  • AGET ATRP was carried out as described in Example 9, except that the initiator- bound Adenovirus used was prepared according to Example 6. 94
  • Example 11 TEM images of modified and unmodified Adenovirus particles
  • TEM images of the polymer modified Adenovirus particles were stained using methylamine tungstate (2% solution) using the drop method and compared with TEM images of unmodified Adenovirus particles stained with methylamine tungstate (2% solution) also using the drop method.
  • the TEM images are displayed in Figure 2.
  • the images in Figure 2 show that there are significant differences between unmodified adenovirus and the polymer modified adenovirus.
  • the polymer modified adenovirus samples were much more difficult to image as the polymer coating decreased binding to the carbon coated grids.
  • the modified Adenovirus produced in Examples 3 to 10 was characterized by both photon correlation spectroscopy (PCS) and laser Doppler velocometry (for zeta potential) to determine what changes had occurred during each step of the modification.
  • PCS and zetapotentiometry were carried out using a Malvern Zetasizer 3000Hs in filtered PBS (0.2 ⁇ m) and 10 mM HEPES pH 7.4 respectively.
  • the PCS analysis determined the hydrodynamic diameter of the virus particles and the results of this analysis are shown in Figures 3 and 5.
  • the increase in hydrodynamic diameter of viruses modified according to the present invention shows that ATRP polymerisation from the surface of the virus has taken place successfully.
  • the results of the zeta potential experiment are shown in Figures 4 and 6.
  • the zeta potential of the virus particles tends towards more negative values following modification with an amino reactive initiator. This observation reflects the fact that the initiator reacts with primary amino groups on the surface thus reducing the effective positive charge and increasing the negative zeta potential of the particle.
  • the shear plane (the region at which zeta potential is determined) is shifted away from the core of the particle and thus the decrease/absence of any charge measured is consistent with the presence of a densely packed polymer coating on the surface of the viral particle
  • Polymerisation was also carried out in the same solution as the virus with a water solution, non-reactive initiator. As anticipated, without direct attachment of the initiator to the surface of the capsid, there is no change in hydrodynamic size or charge of the virus.
  • Example 13 Virus infectivity and viability
  • the modified adenoviruses of Examples 3, 4, 7 and 8 were incubated with A549 cells (90 min, MOI 1000 particles/cell); the cells were washed and reincubated in medium containing fetal calf serum. Luciferase expression was measured in cell extracts after 48h.
  • FIG. 7 The results for the initiator modified viruses of Examples 3 and 4, together with some controls, are shown in Figure 7 ((a) blank; (b) DMSO control; (c) initiator modified virus of Example 3 using lmg/mL initiator; (d) initiator modified virus of Example 4 using lOmg/mL initiator; and (d) virus alone).
  • Coating of the virus with an initiator knocks down transgene expression significantly at both lmg/mL and lOmg/mL initiator. This knockdown is due to modification of the fibre proteins which bind the cell receptors (CAR) and consequently there is decreased binding and uptake of the modified virus.
  • CAR cell receptors
  • transgene expression of the polymer-modified viruses of Examples 7 and 8 showed no activity above background. This reflects steric blocking of all receptor- binding sites on the virus, preventing uptake and entry into cells.
  • Adenovirus encoding luciferase was modified with the initiator as described in Example 3. The resulting virus was analysed by picogreen assay against a DNA standard curve and diluted to 1 x 10 12 particles per mL.
  • the initiator modified virus solution was mixed with water (150 ⁇ L), 2-(Dimethylamino)ethyl methacrylate (1 mg) and polyethylene glycol methacrylate (PEGMA) (Mw 300) (44 mg).
  • the mixture was degassed by purging with argon followed by addition of 50 ⁇ L of a catalyst solution (6.4 mg CuBr and 14.0 mg 2,2 bipyridine in 500 ⁇ L water).
  • the reaction proceeded for 30 minutes before the polymerization was quenched by purging the solution with air.
  • the polymer modified virus was purified by extensive dialysis against PBS to removed unreacted monomer and the catalyst.
  • the hydrodynamic diameter of the resulting particles was determined using photon correlation spectroscopy. 10 ⁇ L of the virus solution was added to 600 ⁇ L of filtered (0.2 ⁇ m) PBS for measurement. The hydrodynamic diameter was measured 10 times per run and the run repeated 3 times. The average diameter of these modified particles was 286 ⁇ 35 nm.
  • the zetapotential of the modified virus was determined in 10 mM HEPES pH 7.4. 10 ⁇ L of the virus solution was added to 2.5mL of the HEPES solution and injected into the zeta potentiometer. The zetapotential was measured 5 times and averaged. The average zetapotential was measured to be +28 ⁇ 7 mV indicating that the dimethylaminoethylmethacrylate had copolymerized with the PEGMA on the surface of the virus.
  • the modified virus was incubated with A549 cells for 90 min, at an MOI of 1000, the cells were then washed and reincubated in fresh medium containing fetal calf serum. Luciferase expression was measured after 48 h, compared with non- transduced cells, cells incubated with the same number of unmodified virus particles.
  • the dimethylaminoethylmethacrylate-modif ⁇ ed virus showed 1.1 x 10 8 RLU per 2500 cells, compared with 8.8 x 10 8 PvLU for the unmodified virus. This demonstrates that the inclusion of positive charges on the surface of the modified virus can restore cell uptake and infectivity, using virus-cell binding interactions different from the parental virus.
  • Example 15 in vivo trials
  • the biodistribution of polymer modified viruses in vivo is evaluated following intravenous injection in Balb/c mice.
  • the mice are actively immunised using heat- inactivated adenovirus, to maximise relevance to the clinical situation.
  • Normal viruses are known to be cleared rapidly into hepatic Kupffer cells following opsonization by antibodies in the plasma. This is avoided with the polymer modified viruses of the invention.
  • the polymer modified viruses of the invention achieve extended plasma circulation. Blood samples are taken at 10, 30, 300 minutes after IV injection and virus genomes in plasma measured by QPCR. Using an El -deleted virus expressing luciferase, the animals are imaged after 24, 48, 96 hours (using an IvislOO luminescence imaging system) to quantify patterns of infection in vivo.
  • FGF- and EGF- retargeting of polymer modified viruses of the invention to infect receptor-positive human tumours is assessed in MFl nude mice following IV injection in vivo, using subcutaneous A431 cancer xenografts with non-invasive imaging of transgene expression in the tumour using the IvislOO. These mice are passively immunized by pre-injection of human serum (IP). Reporter gene expression in the tumour is then measured and compared with the number of virus genomes present (measured by QPCR post mortem) to assess the efficiency of the retargeted polymer modified viruses in reaching the tumour.
  • IP human serum
  • Example 16 Resistance to binding of anti-adenovirus antibodies
  • the polymer coated virus produced in Examples 5, 6, 9 and 10 was characterised by picogreen assay (to measure the concentration of intact virus particles) and used in a capture ELISA assay to measure the ability of the coating polymer to protect against binding of anti-adenovirus antibodies.
  • Polymer-coated viruses that had been formed using 10 mg/ml and 20 mg/ml HEG Sulfo-NHS based initiator units were compared with unmodified virus (AdWT).
  • Figure 9 shows that the viruses coated in accordance with this invention (Star 10Ad and Star20Ad) completely prevented binding of anti- adenovirus antibodies, showing total protection from neutralising antibodies.
  • Adenovirus was modified and coated as described in Examples 5, 6, 9 and 10, and its concentration was determined using a modified picogreen assay. Virus particles were then treated with proteinase K and cell lysis buffer before DNA extraction and quantification by real time PCR, using primers recognising the knob-turn domain of 2008/003594
  • Example 18 Polymer modification of polyelectrolyte complexes containing siRNA.
  • Polyelectrolyte complexes were formed by mixing siRNA molecules with high molecular weight 'reducible polycations', formed by oxidative polycondensation of oligocations containing histidine, lysine and cysteine as described by Stevenson M. et al. (Delivery of siRNA mediated by histidine-containing reducible polycations. J Control Release. 2008 Aug 25;130(l):46-56.).
  • Such polymers are based on CH 6 K 3 H 6 C, made into a high molecular weight linear polymer by oxidative polycondensation.
  • the terminal cysteine groups function to enable polymerization, the lysine groups are strongly charged and provide an electrostatic attachment to the negatively charged siRNA, and the histidine groups provide endosomal-buffering activity which improves transfer of the complex from the endosome into the cytoplasm. Following entry into the cytoplasm the disulphide bonds are reduced, due to the intracellular reducing environment, and free siRNA is released into the cytoplasm to mediate its intended function.
  • Free siRNA was removed using spin columns, particles were crosslinked using disulphide-containing amino-crosslinking agent (Dimethyl dithiobispropionimidate (DTBP)) and polymerisation initiators were then reacted onto the surface as described in Examples 3 to 6. Polymerisation was then carried out as described in Examples 7 to 10. The resulting particles showed discrete size distribution and extended plasma circulation in mice following intravenous injection, enabling their use of targeted delivery to disseminated sites.
  • DTBP disulphide-containing amino-crosslinking agent

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Abstract

L'invention concerne un procédé de modification de la surface d'une entité biologique contenant des acides nucléiques. Le procédé selon l'invention comprend la croissance de chaînes polymères à partir d'un ou de plusieurs points d'initiation sur ladite surface, en présence d'un ou de plusieurs catalyseurs. Selon l'invention, l'entité biologique contenant des acides nucléiques est éventuellement un virus, un bactériophage, une cellule procaryote, une bactérie, un archaeum, une cellule eucaryote, un champignon, un spore, un core viral contenant des acides nucléiques, sa ou ses membranes externes et/ou ses capsides étant éliminés, une mitochondrie, un noyau de cellule mammalienne, un complexe comprenant un acide nucléique et un agent de condensation ou un complexe comprenant de petits ARNi et un agent de condensation ; de préférence un virus ou une bactérie ; de manière davantage préférée un adénovirus, un core d'adénovirus, un poxvirus, un core de poxvirus ou un virus de l'herpès.
PCT/GB2008/003594 2007-10-23 2008-10-23 Modification d'entités biologiques contenant des acides nucléiques Ceased WO2009053700A1 (fr)

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CN105289759A (zh) * 2015-11-13 2016-02-03 西北大学 一种基于废弃固定化酶的强阳离子交换剂及其制备方法与应用
RU2575603C2 (ru) * 2010-07-30 2016-02-20 Куревак Гмбх Получение комплексов нуклеиновых кислот и поперечно сшитых дисульфидными связями катионных компонентов, предназначенных для трансфекции и иммуностимуляции
US9314535B2 (en) 2009-09-03 2016-04-19 Curevac Ag Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids
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CN115960323A (zh) * 2021-10-12 2023-04-14 南京大学 一种细胞表面聚糖的可激活型标记方法
CN115960324A (zh) * 2021-10-12 2023-04-14 南京大学 一种用于细胞环境中选择性标记游离糖蛋白的聚糖重构方法
US11690910B2 (en) 2012-01-31 2023-07-04 CureVac SE Pharmaceutical composition comprising a polymeric carrier cargo complex and at least one protein or peptide antigen
US11739125B2 (en) 2013-08-21 2023-08-29 Cure Vac SE Respiratory syncytial virus (RSV) vaccine

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