WO1998044143A1 - Polymer-modified viruses - Google Patents
Polymer-modified viruses Download PDFInfo
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- WO1998044143A1 WO1998044143A1 PCT/US1998/006609 US9806609W WO9844143A1 WO 1998044143 A1 WO1998044143 A1 WO 1998044143A1 US 9806609 W US9806609 W US 9806609W WO 9844143 A1 WO9844143 A1 WO 9844143A1
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—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
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- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P35/00—Antineoplastic agents
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- 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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- C12N2710/00011—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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- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
- C12N2710/16641—Use of virus, viral particle or viral elements as a vector
- C12N2710/16645—Special targeting system for viral vectors
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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
- C12N2710/24011—Poxviridae
- C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
- C12N2710/24141—Use of virus, viral particle or viral elements as a vector
- C12N2710/24145—Special targeting system for viral vectors
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- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
- C12N2740/13041—Use of virus, viral particle or viral elements as a vector
- C12N2740/13045—Special targeting system for viral vectors
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- C12N2810/00—Vectors comprising a targeting moiety
- C12N2810/10—Vectors comprising a non-peptidic targeting moiety
Definitions
- Viruses have many potential therapeutic uses, for example in gene therapy, whereby the viral genome is used as a vector for foreign genes, as well as in vaccination and cancer therapy, for example by exploiting the phenomenon of viral oncolysis, which exploits cell destruction following selective virus replication in certain tumors.
- clinical use of viruses presents certain problems. For example, many human subjects are pre-immune to common viruses such as adeno viruses, and thus have circulating antibodies. In cases in which the circulating antibodies are neutralizing in nature, the administered viral particles may have reduced or no infectivity. Repeated administration may exacerbate this problem, since most viruses are highly immunogenic. Immune responses may also contribute to the toxicity of viral administration, and in cases in which cellular immunity is involved, some profound tissue damage may result.
- Polymer modification has been shown, in the context of polymer-protein and polymer-liposome constructs, to have the potential to solve many problems.
- polymer cover has been demonstrated to reduce antigenicity and immunogenicity.
- light polymer cover can turn an antigen into a tolerogen.
- Polymer cover can also ameliorate reticuloendothelial system (RES) uptake of particulates.
- RES reticuloendothelial system
- polymer can serve as a linker to couple targeting devices to the surface of other molecules or macromolecular structures to target them to specific sites.
- living viruses are very different in their characteristics to proteins and liposomes. The surface structures involved in infectivity might well be compromised by polymer modification. Virtually all clinical applications of viruses require infectivity to be maintained.
- viral particles can be polymer modified and yet retain infectivity. It has also been discovered that polymer modification of viruses results in the acquisition of beneficial properties such as improved capacity to infect in the presence of neutralizing antibodies.
- the present invention provides viruses modified by polymers.
- the polymer is polyethylene glycol (PEG).
- the polymer is directly covalently attached to the virus.
- the polymer is indirectly covalently attached to the virus via an intermediate coupling moiety.
- the polymer is indirectly noncovalently attached to the virus via a ligand.
- the ligand has specificity for a viral surface component.
- the ligand may be an antibody.
- the present invention further provides a method of making viruses modified by polymers, whereby the modified viruses retain infectivity.
- Another embodiment of the present invention provides a method for introducing a transgene into a target cell comprising contacting the target cell with a polymer-modified virus, wherein the virus comprises the transgene.
- Fig. 2 is a graph of the time course of mobility change on capillary electropherographs of adenovirus treated with 3 % (w/v) TMPEG.
- Fig. 3A-D shows photon correlation spectroscopy results demonstrating the change in viral particle size during PEGylation.
- Fig. 10A and B shows graphs of an antibody neutralization assay for the impact of stepwise additions of 5% PEG 5000 on neutralization of infectivity (chemiluminescence,
- Fig. 15 shows an SDS-PAGE gel showing immunoprecipitation of adenoviral hexon by PEGylated anti-hexon antibody.
- Fig. 18A-C shows the elution profile of control and TMPEG-treated virus from DEAE ion exchange resin following chromatography.
- Fig. 19 A-C shows the elution profile of untreated (panel 19a), MPEG treated (panel 19b) and TMPEG treated (panel 19C) Adenovirus ONYX-015 from 1 ml Resource Q column (Pharmacia).
- Fig. 20 depicts infectivity assay results (ELISA for hexon protein) following stepwise additions of 5% TMPEG 5000 or MPEG 5000 to Adenovirus ONYX-015.
- Fig. 21 A-F shows a laser copy of photographs demonstrating cytophatic effect (CPE) for untreated Adenovirus ONYX-015 (panels A-B) and ONYX-015 incubated with 5% MPEG 5000 (panels C-D) or TMPEG 5000 (panels E-F).
- CPE cytophatic effect
- Fig. 23 shows the infectivity measured by plaque assay of vaccinia virus following stepwise addition of MPEG500 or TMPEG 5000 .
- Fig. 26 demonstrates the expression of late genes (IL- 1 ⁇ receptor) following infection with vaccinia virus which had been incubated with MPEG 5000 or TMPEG 5000
- Fig. 30 A-B shows the infectivity measured by plaque assay of Herpesvirus following step-wise addition of MPEG 5000 or TMPEG 5000 .
- Fig. 31 A-B shows the elution profile of ONYX-015 incubated with PVP (panel 32a) and activated PVP (panel 32b) from 1 ml Resource Q column (Pharmacia).
- Fig. 32 shows immunofluorescent staining of liver (A) and tumor sections (B and C) taken from nude mice bearing LS174T human colon carcinoma injected with PEGylated virus (A and B) or control virus (C).
- Fig. 33 shows transgene expression in mice infected with PEGylated or sham treated adeno viral vectors.
- polymers are generally large non- immunogenic, biologically inert molecules comprising a chain of smaller molecules linked by covalent bonds.
- Polymers useful in accordance with the present invention are those polymers which, when covalently or noncovalently bound to a virus, provide a polymer-modified virus that retains detectable levels of infectivity and is substantially non-immunogenic.
- the polymers preferably have an average molecular weight of from about 200 to about 20,000 daltons.
- the polymers are biocompatible, and may be linear or branched.
- the polymers may be homopolymers or heteropolymers.
- Suitable polymers for use in the present invention include polyalkalene compounds such as polyalkalene oxides and glycols.
- Polyalkalene compounds include polyoxymethylene, polyethylene glycols (PEG) and oxides, and methoxypolyethyleneglycols, and derivatives thereof including for example polymethyl-ethyleneglycol, polyhydroxypropyleneglycol, polypropylene glycol, polymethylpropylene glycol, polyhydroxypropylene oxide and poly vinyl pyrrolidone (PVP).
- PEG polyethylene glycols
- PVP poly vinyl pyrrolidone
- a preferred polymer in accordance with the present invention is PEG.
- PEG is a water-soluble polymer having the formula H(OCH 2 CH 2 ) n OH, wherein n is the number of repeating units and determines the average molecular weight.
- PEGs having average molecular weights of from 200 to 20,000 daltons are commercially available.
- PEG having an average molecular weight of from 200 (PEG 200 ) to 20,000 (PEG 20000 ) may be used to prepare viruses modified by PEG.
- the PEG has an average molecular weight of from about 2000 to about 12,000. In a more preferred embodiment, the PEG has an average molecular weight of about 5000.
- the polymer-modified viruses have utility in medical therapy and diagnosis in medical and veterinary practice and in agriculture. They are of particular use in gene therapy (for example the delivery of genes for the localized expression of a desired gene product) and for non-gene therapy applications such as, but without limitation, viral oncolysis.
- the viruses are useful, for example, to deliver genes, toxins and/or diagnostic markers.
- An additional application is in the creation of tolerogens for viral antigens. More specifically, the present invention is directed to a virus selected -from RNA and
- the virus used is selected from the following families and groups: Adenoviridae; Birnaviridae; Bunyaviridae; Caliciviridae; Capillovirus group; Carlavirus group; Carmovirus virus group; Group Caulimovirus; Closterovirus Group; Commelina yellow mottle virus group; Comovirus virus group; Coronaviridae; PM2 phage group: Corcicoviridae; Group Cryptic virus; group Cryptovirus; Cucumovirus virus group Family ⁇ 6 phage group; Cystoviridae; Group Carnation ringspot; Dianthovirus virus group; Group Broad bean wilt; Fabavirus virus group; Filoviridae; Flaviviridae; Furovirus group; Group Gemini virus; Group Giardiavirus; Hepadnaviridae; Herpesviridae; Hordeivirus virus group; Ilarvirus virus group; Inoviridae; Iridoviridae; Leviviridae; Lipoth, Adenovirid
- Nodaviridae Orthomyxoviridae; Papovaviridae including adeno-associated viruses;
- Paramyxoviridae Parsnip yellow fleck virus group; Partitiviridae; Parvoviridae; Pea enation mosaic virus group; Phycodnaviridae; Picornaviridae; Plasmaviridae; Podoviridae; Polydnaviridae; Potexvirus group; Potyvirus; Poxviridae; Reoviridae;
- Retroviridae Rhabdoviridae; Group Rhizidio virus; Siphoviridae; Sobemovirus group;
- Tobravirus Togaviridae; Group Tombusvirus; Group Toro virus; Totiviridae; Group
- Tymovirus Plant virus satellites.
- viruses for the purpose of delivery of transgenes include, for example, retrovirus, adenovirus, adenoassociated virus, herpesvirus and poxvirus.
- Adenovirus is particularly preferred.
- the term virus includes recombinant genetically engineered viruses.
- the virus may be a virus that has been engineered such that it is incapable of replicating and exhibits minimal gene expression.
- the recombinant viruses may contain transgenes.
- Transgenes are defined herein as nucleic acids that are not native to the virus.
- a transgene may encode a biologically functional protein or peptide, an antisense molecule, or a marker molecule.
- the polymer-modified viruses of the present invention may be provided by direct covalent, indirect covalent, or indirect noncovalent attachment of the polymer to the virus.
- polymer may be attached via direct covalent coupling to the viral surface; 2) polymer may be attached via indirect covalent coupling (e.g. via an intermediate coupling moiety which links the polymer to the viral surface); or 3) attached via an indirect non-covalent linkage using, for example, a suitable PEGylated ligand.
- Suitable ligands are not restricted to antibodies to surface proteins or lipid and could include hydrophobic ligands for viral particles with hydrophobic surface components such as envelope viruses.
- the polymer may be attached via direct or indirect covalent coupling to the viral surface by methods that are generally known in the art for covalent attachment of polymers to other molecules, such as proteins.
- Targets for polymer modification include reactive groups on the viral surface with which the polymer or coupling agent can interact, including for example primary and secondary amino groups, thiol groups and aromatic hydroxy groups.
- the preferred method for polymer modification of a virus depends upon the available target sites on the surface of the particular virus. The specificity of particular methods of polymer modification for particular target groups is well-known, and thus the ordinarily skilled artisan can select a method suitable for the desired target.
- Non-enveloped virus The surface of a non-enveloped virus is a protein shell, or capsid, often containing multiple types of polypeptides.
- Representative non-enveloped viruses include adenovirus, parvovirus and picornavirus.
- enveloped viruses the protein capsid is enclosed by a lipid bilayer that contains viral-encoded polypeptides.
- Representative enveloped viruses include herpesvirus, poxvirus and baculovirus. Both the capsid and the envelope polypeptides provide targets for polymer modification.
- a nonenveloped virus such as adenovirus
- the hexon, penton cell base, and fiber proteins are targets for polymer modification.
- the polymer is activated by converting a terminal moiety of the polymer to an activated moiety, or by attaching an activated coupling moiety to the polymer.
- the activated polymer is then coupled to the target via the activated moiety.
- the activated moiety or activated coupling moiety can be selected based upon its affinity for the desired target site on the viral surface.
- the hydroxyl end groups of PEG may be converted into reactive functional group or attached to an activated coupling moiety to provide a molecule known as "activated" PEG.
- activated PEG Various forms of activated PEG are known in the art and are commercially available.
- the covalent attachment of PEG to the viral surface is accomplished by incubating the virus with the activated PEG, for example TMPEG.
- the activated PEG for example TMPEG.
- Several incubation regimes may be used.
- a single addition of the activated polymer with or without gentle mixing can be used.
- the optimal ratios of TMPEG to viral particles to achieve modified virus having reduced antigenicity with maintenance of infectivity may be determined by performing the assays described below.
- virus and activated TMPEG are combined at molar ratios of activated PEG to e-amino termini of lysine residues of from about 1 : 1 to about 400: 1.
- stepwise addition is that viral particles tend to aggregate and this is exacerbated by certain activated polymers, e.g. TMPEG, especially at high concentrations.
- TMPEG certain activated polymers
- initial PEGylation at low polymer concentration can serve to reduce the tendency to aggregate at subsequent higher polymer concentrations and hence help to achieve a higher degree of PEGylation.
- the reaction may be quenched by dialysis or by addition of excess lysine, for example from 10 to 100-fold excess lysine.
- the reaction might be run to completion (i.e. the point at which the activated PEG, such as TMPEG, is either completely consumed in the PEGylation reaction or rendered inactive by hydrolysis).
- the activated PEG such as TMPEG
- hexon affinity resin may be useful to separate the PEGylated antibody from unreacted PEG.
- separation of modified from unmodified virus may be performed by partitioning in an aqueous biphasic polyalkylene glycol solution.
- phase partitioning in an aqueous biphasic system of PEG and dextran may allow the separation of PEG-modified virus from unmodified virus. Partitioning may be performed by counter-current distribution.
- the phase system is prepared by mixing solutions of dextran and PEG.
- PEG and PEG-modified virus are incorporated into the phase system, mixed by inversion or rotation, and allowed to separate.
- PEG modified virus partitions into the PEG phase
- unmodified virus partitions into the dextran phase may be desirable to separate.
- PEGylation The modification of virus by PEG (“PEGylation") may be evaluated by methods known in the art, including ion exchange chromatography , capillary electrophoresis
- CE photon correlation spectroscopy
- PCS photon correlation spectroscopy
- Ion exchange chromatography for example, DEAE-chromatography
- DEAE-chromatography can be performed by standard methods to evaluate the modified viruses based upon altered charge.
- Whole virus CE provides a means to monitor the modification of virus by polymer as a function of altered surface charge. For example, covalent attachment of PEG to the virus surface seems to result in shrouding of the negative surface charges on the viral particle and thus this polymer-modified virus displays a more neutral mobility to the virus.
- CE may be performed by methods known to those of ordinary skill in the art.
- a ramped low-high voltage pre-treatment is used to electrophorese the highly mobile salt ions in which the virus may be formulated for stability, before true, high voltage separation begins.
- virus particles with PEG covalently attached run at a position closer to the neutral point than virus without covalently attached PEG.
- CE may be conveniently used to assess the influence of various conditions, including molar ratios, concentrations and incubation times, on the covalent attachment of PEG to the virus particles.
- Increasing neutrality reflects increasing PEG- chain density on the virus surface.
- PCS uses the relationship between particle size and movement in suspension (via Brownian motion) to gain accurate measurements on the size of the particles. This method is widely applied to monitor polymer attachment to particles including liposomes, microspheres and nanoparticles by measuring their increase in size. These data suggest that covalently attached PEG at relatively low density forms globular "mushroom” shapes and thus the increase in size is relatively small. Altering the conditions under which one would expect to increase the density of covalently attached PEG chains results in a more extended conformation of the polymer or "brush" shapes which is reflected by a relatively larger increase in particle size. Thus PCS may be used using methods known to those of ordinary skill in the art to monitor the size changes of the virus particle under different reaction conditions.
- the ELISA analysis of a biotinylated PEG can provide the most quantitative assessment of the number of molecules of PEG covalently bound to a virus particle. The ELISA can be performed by standard methods known in the art.
- the recombinant adenoviral vector contains a transgene, including for example the cystic fibrosis transmembrane conductance regulator (CFTR) gene.
- CFTR cystic fibrosis transmembrane conductance regulator
- the polymer modified virus is a recombinant adenovirus that can induce tumor-specific cyto lysis also known as viral oncolysis.
- adenovirus that are useful for viral oncolysis are disclosed by Bischoff et al. (1996) Science 274:373; Heise et al. (1997) Nature Medicine 3:630; and EP689447A, the disclosures of which are incorporated herein by reference.
- the polymer is indirectly noncovalently attached to the virus via a suitable polymer-modified ligand.
- Suitable ligands are not restricted to those having specificity for a viral surface component such as a viral surface protein or lipid, and may include hydrophobic ligands for viral particles with hydrophobic surface components such as envelope viruses and also ionic ligands.
- the ligand is an antibody or antibody fragment, including for example a non-neutralizing anti- virus antibody or fragment therefrom.
- the term antibody includes monoclonal and polyclonal antibodies.
- the ligand is a non-neutralizing anti-hexon antibody.
- Such antibodies are commercially available and include, for example, MAb 8052 and MAb 805 available from Chemicon International, Temecula, CA, USA.
- Indirect non-covalent attachment of polymer to the virus is accomplished by incubation of the virus with a suitable ligand that has been modified by the covalent attachment of polymer.
- the polymer may be covalently attached to the ligand by standard methods as described herein above.
- a non-neutralizing anti-virus antibody such as anti-hexon antibody may be PEGylated using an activated PEG molecule as described above.
- anti-hexon antibody is modified using TMPEG.
- the polymer-modified viruses of the present invention maintain infectivity and exhibit reduced antigenicity. It has been discovered in accordance with the present invention that viral infectivity eventually decreases upon additional polymer modification.
- standard assays including the following assays, to assess infectivity and antigenicity, those of ordinary skill in the art can determine the method and conditions of polymer modification that allow retention of infectivity and reduction in antigenicity. Under conditions designed to provide direct TMPEG polymer modified adenovirus, the methods correlating with PEGylation due to exposure to TMPEG of about 5-20%o w/v are preferred, with a concentration of about 10%> w/v being most preferred.
- the product of the transgene can be assessed by colorimetric, chemiluminescence or fluorescence assays, or immunoassays.
- Retention of infectivity is defined herein as an infectivity level sufficient to have therapeutic value, for example at least about 20%> infective relative to unmodified virus.
- the polymer-modified virus maintains at least 60%) infectivity.
- the polymer-modified virus is preferred to maintain at least 80%> infectivity. Lower percent infectivity of at least 5%> may be therapeutically useful for applications such as viral oncolysis.
- Another embodiment of the present invention provides a method for introducing a transgene into a target cell.
- the method comprises introducing into the target cell a polymer-modified virus of the present invention, wherein the virus is a recombinant viral vector comprising the transgene.
- Use of the present polymer-modified viruses to deliver a transgene to a target cell is useful for the treatment of various disorders, for example in which the transgene product is absent, insufficient, or nonfunctional.
- the expression of the transgene may serve to block the expression or function of an undesired gene or gene product in the target cell.
- the polymer-modified virus is introduced into the host cell by methods known in the art, including for example infection. Infection of a target cell in vivo is accomplished by contacting the target cell with the polymer-modified virus.
- the polymer-modified virus is delivered as a composition in combination with a physiologically acceptable carrier.
- physiologically acceptable carrier includes any and all solvents, diluents, isotonic agents, and the like. The use of such media and agents for compositions is well known in the art.
- the polymer-modified viruses of the invention may be delivered to the target cell by methods appropriate for the target cell, including for example by ingestion, injection, aerosol, inhalation, and the like.
- compositions may be delivered intravenously, by injection into tissue, such a brain or tumor, or by injection into a body cavity such as pleura or peritoneum.
- the transgene is a DNA molecule encoding CFTR or an analog or variant thereof which provides functional regulated chloride channel activity in target cells, and the complex is delivered to the airway epithelium by inhalation.
- DNA molecules encoding CFTR are well known in the art and disclosed for example in W094/12649 and W095/25796, the disclosures of which are incorporated herein by reference.
- the present invention further provides a method for delivering a virus to a tumor, comprising administering a polymer-modified virus of the invention to a subject in need of such treatment under conditions whereby the polymer-modified virus localizes to a tumor.
- Tumors have leaky vasculature and thus long circulating particles have the opportunity to leave the circulation and enter the tumor parenchyma via the holes in tumor blood vessels.
- Tumors lack lymphatics which is the main system for removal of macromolecules and particles from the tissues (the basis for the Retention element in EPR).
- PEG has been used to enhance the passive targeting of liposomes to tumors via increased circulation time.
- this approach leads to unfavorable properties such as unacceptable low tumor to blood ratios (i.e. less than 1) for much of the lifetime of the product.
- the present invention provides a means of improving the tumor localization of virus particles. This is relevant to both gene therapy applications where viral vectors are used to deliver genes and for non-gene therapy applications.
- the polymer-modified virus is administered to a subject as a composition of polymer-modified virus in combination with a physiologically acceptable carrier as described hereinabove.
- the composition may be administered by methods appropriate in view of the location of the tumor, including for example ingestion, injection, aerosol, inhalation, and the like.
- the compositions are delivered intravenously.
- the present invention further provides compositions comprising the polymer- modified viruses and further comprising a physiologically acceptable carrier.
- the polymer-modified virus is a recombinant viral vector modified by covalent attachment of PEG.
- the formulation of compositions is generally known in the art and reference can conveniently be made to Remington's Pharmaceutical Sciences, 17 th ed., Mack Publishing Co., Easton, PA.
- the forms of the present complexes suitable for administration include sterile aqueous solutions and dispersions.
- the subject polymer-modified viruses are compounded for convenient and effective administration in effective amounts with a suitable physiologically acceptable carrier and/or diluent.
- Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated, each unit containing a predetermined quantity of active material calculated to produce the desired effect in association with the required carrier.
- the specification for the novel dosage unit forms of the invention are dictated by and directly depend on the unique characteristics of the polymer-modified viruses and the limitations inherent in the art of compounding. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the ingredients.
- the stock solution used contained 6.4x10 10 infectious units per ml (4.8x10" particles/ml).
- the virus stock was made 3%>w/v by the addition of dry TMPEG, typically 3.0mg to 100/-il of stock. The samples were incubated at 25 °C with rotary mixing for 24h.
- a preliminary 1.5min wash in 1M NaOH and second wash in running buffer (20mM phosphate buffer pH 7.0, 5.0mM NaCl) were performed.
- the samples were transferred to the CE machine where the auto sampler removed a few nanolitres by a pressure injection setting of 10s and separation was achieved using 2 minute voltage ramping to a final of 17Kv.
- Whole virus CE monitors the changes in surface charge of the virus upon treatment with PEG. Incubation with PEG correlates with a progressively increased more neutral mobility to the virus. Increasing neutrality is consistent with an increased PEG- chain density on the virus surface.
- Figure 2 shows the time course of the change in electrophoretic mobility of virus with duration of exposure to TMPEG 3%> (w/v), prepared essentially as described, above using 300 ⁇ l of virus stock and 3%(w/v) TMPEG.
- the %> mobility was calculated as follows: (mobility of modified virus peak- mobility of neutral position)/(mobility of unmodified virus peak-mobility of neutral position) X 100. Since the reaction co-product can influence the running buffer, this was renewed at the point arrowed: lOO l of reaction mixture was analyzed up to this point (using the repeat sampling function of the CE machine, i.e. without mixing) and a fresh lOO ⁇ l aliquot of the reaction mixture was used thereafter.
- Viral particle size was monitored using photon correlation spectroscopy (PCS) in a Malvern Instrument's ZetaMaster 5.
- Figures 3a and 3b show the diameter versus time for TMPEG treated and untreated virus respectively. Results are expressed as % time 0 values.
- Figures 3c and 3d show measurements taken during a PEGylation reaction over a longer time period. Reaction with TMPEG is shown in Figure 3d and sham treatment with MPEG is shown in Figure 3c. Treatment with TMPEG results in an increase in particle size (Figs. 3b and 3d) which is not seen in the control untreated virus (Fig. 3a) or in the MPEG treated virus
- stepwise addition was also used (the objective being to achieve higher ultimate PEGylation).
- the rationale behind step wise addition is that viral particles tend to aggregate and this is exacerbated by PEG, especially at high concentrations.
- PEGylation has been shown, in the context of other particles (e.g. liposomes), to prevent aggregation.
- initial PEGylation at low polymer concentration can serve to reduce the tendency to aggregate at subsequent higher polymer concentrations and hence achieve a higher degree of PEGylation.
- TMPEG or MPEG were added every thirty min to viral stock solution (prepared as in Example 1 ) to increase the polymer concentration by 3%>, 5% or 8%> in the reaction mixture.
- Viral stocks used for these experiments ranged from 1.35-7.6x10 10 infectious units per ml and 9.3-20x10" particles per ml.
- a maximum of four additions of dry polymer were made, equating to final polymer concentrations of 12 %>, 20 %> and 32% ( ⁇ w/v, i.e. not correcting for the volume of the polymer).
- the 4th addition was sampled after 30 mins and a further incubation time (giving 5 reaction conditions).
- lysis buffer 15 % triton X-100, 25OmM Tris-HCl, pH 7.0
- lysis buffer 15 % triton X-100, 25OmM Tris-HCl, pH 7.0
- ⁇ -gal standards 5.5 units in lysis buffer and doubling dilutions in lysis buffer
- TMPEG and MPEG Single and stepwise additions of TMPEG and MPEG were prepared as in Example 3 and analyzed with respect to infectivity using a chemiluminesent reporter assay system for the detection of the virally encoded ⁇ -galactosidase (Galacto-LightTM).
- This assay system uses a chemiluminescent substrate and was performed in accordance with the manufacturer's instructions.
- Figure 6a-c compares the effects of 3%>, 5% and 8%> incremental additions of TMPEG 5000 (filled circles) or MPEG 5000 (open circles) on viral infectivity. Note that in Figure 6a and b the MPEG and TMPEG treated viral samples show similar infectivity. A modest decline in infectivity with treatment with either MPEG or TMPEG was observed. In subsequent experiments with no-PEG controls these showed a similar decline in infectivity, suggesting that this was a handling effect and not due to PEG. In Figure 6c the MPEG and the TMPEG treated virus performed similarly. Thus, this experiment shows that treatment with TMPEG or MPEG does not result in loss of infectivity.
- Transgene expression was monitored in the presence and absence of a polyclonal neutralizing antibody purified from rabbit anti-hexon serum using a hexon affinity resin.
- the polyclonal antibody was titered with untreated virus and the ratio was established where 30 to 50%> infectivity was retained in the presence of the neutralizing antibody.
- Two antibody titers were used 10,000:1 (-30%) or 5,000:1 (-40-50%) (antibody molecules to virus particles) where indicated.
- Figures 10-12 show the impact of incremental additions of 5% TMPEG 5000 ( Figures 10 and 11) and TMPEG 12000 ( Figure 12) on antibody neutralisation.
- Antibody treatment is shown by the filled symbols and MPEG treatment by circles and TMPEG treatment by squares. In the lower panels, hatched bars indicate TMPEG treatment.
- protection is defined as there being a statistically significant difference in transgene expression in the presence of the immune agent under test (e.g. antibody or cell suspension) as compared with the expression observed in untreated control.
- the single addition of 3% TMPEG 5000 showed some protection after 4h and 6h incubation in two independent assays.
- the present invention relates to polymer-modified viruses, processes for obtaining them and their use.
- the invention also provides means of attaching polymer molecules to viral particles whilst retaining infectivity of the modified virus.
- Initial experiments on the PEGylation of an anti-hexon antibody were performed using commercially available anti-hexon antibodies from Chemicon (Mab 8052). Two types of activated PEGs were tested for their ability to PEGylate the antibody namely cyanuric chloride activated PEG and PEG-tresylate (TMPEG).
- TMPEG 5000 was obtained from Shearwater Polymers, Huntsville, AL. PEGylation of an anti-hexon antibody using TMPEG was accomplished as follows.
- TMPEG modified Mab 8052 (modified at a ratio of 100:1 PEG:lysine as prepared in Example 6) and unmodified antibody were incubated with a detergent solubilized fraction of adenovirus for 2 hrs at 4 °C. Antibody antigen complexes were captured with
- Table 4 shows that antibody PEGylated with TMPEG at the ratios of PEG:lysine of 10:1 and 100:1 could still effectively compete with the biotinylated parental antibody for virus. This resulted in less biotinylated antibody bound to the virus and hence a lower titre value.
- Antibody PEGylated with TMPEG at a ratio of 200:1 PEG:lysine was ineffective at competing with the biotinylated parental antibody suggesting that at this high ratio of PEG the antigen binding site of the antibody is compromised.
- the antibody preparation did not contain any significant proportion of residual unmodified antibody (note the lack of a subsidiary peak in the unmodified position). Incubation of the antibody with increasing concentrations of TMPEG-5K lead to a progressive displacement of the protein elution peak from circa 11.1ml to circa 9.5ml, 9.1ml and 8.95ml, indicative of increasing degree of modification
- the displacement of the protein elution peak by PEGylation was more marked for the conjugates obtained with TMPEG- 12K than that observed for conjugates prepared with TMPEG-5K.
- the conjugates obtained with TMPEG- 12K have an overall hydrodynamic radius grater than that of the conjugates obtained with TMPEG-5K.
- a greater hydrodynamic radius could indicate: either a) greater impact per PEG chain for the TMPEG- 12K than for the TMPEG-5K, or b) greater number of PEG chains attached with TMPEG- 12K than with TMPEG-5K.
- the chromatograms do not allow to discriminate between these two possibilities.
- the plates were incubated for 1 h at 37 °C and then the wells were washed 3 times with 400 ⁇ l of wash buffer.
- the biotinylated antibody bound to the virus was then quantified using a standard streptavidin-HRP assay.
- the stock inactivated adenovirus type 2 was obtained in lyophilized form, 200 ⁇ g/vial, from Lee Biomolecular Research, San Diego CA, Cat No.405001.
- the coating buffer was 100 mM carbonate pH 9.2 (Pierce).
- Blocking buffer was PBS containing 0.05% Tween 20, 0.5 % BSA (Pierce 10X).
- Wash buffer was PBS containing 0.05%) Tween 20.
- the biotinylated antibody was at a concentration of 10.8 ⁇ M.
- Ad2/ ⁇ -gal 2 vector (U.S. Patent No. 5,670,488 and described by Zabner et al. (1996) J. Virol. 70 : 6994) was covalently modified by PEG with 0.01%, 0.1%, 1.0% or 5.0%) biotinylated NHS-PEG 5000 (Shearwater Polymers). PEGylated vector proteins were analyzed by SDS-PAGE. SDS-PAGE demonstrated that the hexon, penton base and fiber were the primary targets for covalent modification by PEG, and increasing concentration of PEG led to modification of additional proteins.
- Type 2 adenovirus (genetically modified to carry the ⁇ -gal reporter gene) was prepared by banding with isopycnic CsCl density centrifugation then extensively dialysed against phosphate buffered saline (PBS pH 7.2).
- Three different types of mPEGs were tested for their ability to PEGylate adenovirus namely a) cyanuric chloride activated mPEG 5000 b) TMPEG 5000 and c) amino-PEG 5000 .
- the mPEGs were obtained from Shearwater Polymers. Activation of mPEG with cyanuric chloride couples one triazine ring per mPEG molecule. This activated mPEG can react with amino groups on proteins.
- Table 8 expresses the size of the two peaks (expressed as area under peak) in relation to the PEG:lysine ratios used during PEGylation.
- ion exchange chromatography may be used to resolve heterogeneous populations of PEGylated virus particles and may be used to separate highly PEGylated virus particles from lightly PEGylated particles on the basis of charge differences.
- TMPEG 5000 and MPEG 5000 were prepared as in Example 15 and the preparations were monitored by IEC for PEGylation.
- Figure 20a-d shows the effect of 5%> additions of TMPEG 5000 and MPEG 5000 on adenovirus ONYX-015 infectivity.
- the infectivity of virus treated with 5 or 10%> PEG is similar for each treated virus sample (open circles MPEG; closed circles TMPEG) and the untreated sample (triangles), whereas at 15 and 20%> PEG the infectivity of the TMPEG treated virus is reduced with respect to the other two samples, but is still maintained at a significant level.
- Purified virus stocks were prepared by sedimentation through a sucrose cushion, dialysed against PBS overnight at 4°C, and titrated by plaque assay in TK " 143B cells (provided by Dr. Alcami). Titres of 6x10 9 pfu/ml were obtained.
- HSV-I Infectivity was assessed following treatment with TMPEG as follows. Vero cells and BHK cells were trypsinised using standard procedures, and maintained on ice. Serial 10 fold dilutions of the untreated, MPEG treated and TMPEG treated vims samples were prepared in GMEM, containing 2%> FCS. 2 x 10 6 Vero cells and 3 x 10 7 BHK cells were added to each vims dilution (10 "3 to 10 "8 ), and the cells were infected by shaking gently at 37°C.
- Sections taken from tumor tissues showed distribution of PEGylated and control vims within the tissue ( Figures 32B and C). Sections taken from the liver tissue showed no localization of viras in either PEGylated virus ( Figure 32 A) or control vims (data not shown). The localization of the PEGylated vims in the tumor is shown in Figure 32B. Some tumor localization was also seen for the sham PEGylated viras ( Figure 32C). ( Figures 32B and C are at the same magnification).
- Ad2/ ⁇ -gal 4 vims (U.S. Patent No. 5,670,488) was PEGylated with 10% tresyl mPEG (TMPEG - Sigma Chemicals, St. Louis, MO) as already described. PEGylated virus was purified from unreacted TMPEG by banding on cesium chloride gradients (Rich et al., Human Gene Therapy 4:461-476, 1993). The purified PEGylated virus was dialysed into phosphate buffered saline (PBS), 5%> sucrose and the titre was determined by end point dilution on HEK293 cells using fluorescent isothiocyanate (FITC)- conjugated anti-hexon antibody (Rich et al., 1993).
- PBS phosphate buffered saline
- FITC fluorescent isothiocyanate
Abstract
Description
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Priority Applications (6)
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EP98914467A EP0994959A1 (en) | 1997-04-03 | 1998-04-03 | Polymer-modified viruses |
CA002285416A CA2285416A1 (en) | 1997-04-03 | 1998-04-03 | Polymer-modified viruses |
JP54202198A JP2001521381A (en) | 1997-04-03 | 1998-04-03 | Polymer-modified virus |
AU68817/98A AU745056B2 (en) | 1997-04-03 | 1998-04-03 | Polymer-modified viruses |
US09/409,803 US6569426B2 (en) | 1997-04-03 | 1999-09-30 | Tresyl-monomethoxypolyethylene glycol-modified viruses having viral infectivity |
US10/349,630 US20030180261A1 (en) | 1997-04-03 | 2003-01-22 | Polymer-modified viruses |
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GB9706735.9 | 1997-04-03 | ||
GBGB9706735.9A GB9706735D0 (en) | 1997-04-03 | 1997-04-03 | Particles |
GB9719625.7 | 1997-09-15 | ||
GBGB9719625.7A GB9719625D0 (en) | 1997-09-15 | 1997-09-15 | Particles |
GBGB9722316.8A GB9722316D0 (en) | 1997-10-22 | 1997-10-22 | Particles |
GB9722316.8 | 1997-10-22 |
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US09/409,803 Continuation US6569426B2 (en) | 1997-04-03 | 1999-09-30 | Tresyl-monomethoxypolyethylene glycol-modified viruses having viral infectivity |
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JP (1) | JP2001521381A (en) |
AU (1) | AU745056B2 (en) |
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Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000011202A1 (en) * | 1998-08-24 | 2000-03-02 | Genzyme Corporation | Cationic complexes of polymer-modified adenovirus |
WO2000043044A1 (en) * | 1999-01-19 | 2000-07-27 | The Children's Hospital Of Philadelphia | Compositions and methods for controlled delivery of virus vectors |
WO2000074722A2 (en) * | 1999-06-09 | 2000-12-14 | Hybrid Systems Limited | Modification of biological elements |
WO2001023001A2 (en) * | 1999-09-29 | 2001-04-05 | The Trustees Of The University Of Pennsylvania | Rapid peg-modification |
JP2003507348A (en) * | 1999-08-19 | 2003-02-25 | ユニバーシティ オブ サザン カリフォルニア | Targeted artificial gene delivery |
WO2004072289A1 (en) * | 2003-02-17 | 2004-08-26 | Fuso Pharmaceutical Industries, Ltd. | Novel virus vector |
WO2005106046A1 (en) * | 2004-05-03 | 2005-11-10 | Stefan Kochanek | Modified viral vector particles |
US7223388B2 (en) * | 2001-08-03 | 2007-05-29 | Board Of Regents, The Univeristy Of Texas System | Modified reoviral therapy |
WO2010067081A2 (en) | 2008-12-11 | 2010-06-17 | Hybrid Biosystems Limited | Modification of nucleic acid vectors |
US7776322B2 (en) | 2004-08-16 | 2010-08-17 | Stefan Kochanek | Modified viral vector particles |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1996014874A1 (en) * | 1994-11-10 | 1996-05-23 | University Of Manitoba | Method for gene therapy involving suppression of an immune response |
WO1996021036A2 (en) * | 1994-12-30 | 1996-07-11 | Chiron Viagene, Inc. | Nucleic acid condensing agents with reduced immunogenicity |
-
1998
- 1998-04-03 CA CA002285416A patent/CA2285416A1/en not_active Abandoned
- 1998-04-03 AU AU68817/98A patent/AU745056B2/en not_active Ceased
- 1998-04-03 JP JP54202198A patent/JP2001521381A/en not_active Ceased
- 1998-04-03 EP EP98914467A patent/EP0994959A1/en not_active Withdrawn
- 1998-04-03 WO PCT/US1998/006609 patent/WO1998044143A1/en not_active Application Discontinuation
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996014874A1 (en) * | 1994-11-10 | 1996-05-23 | University Of Manitoba | Method for gene therapy involving suppression of an immune response |
WO1996021036A2 (en) * | 1994-12-30 | 1996-07-11 | Chiron Viagene, Inc. | Nucleic acid condensing agents with reduced immunogenicity |
Cited By (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000011202A1 (en) * | 1998-08-24 | 2000-03-02 | Genzyme Corporation | Cationic complexes of polymer-modified adenovirus |
WO2000043044A1 (en) * | 1999-01-19 | 2000-07-27 | The Children's Hospital Of Philadelphia | Compositions and methods for controlled delivery of virus vectors |
WO2000074722A2 (en) * | 1999-06-09 | 2000-12-14 | Hybrid Systems Limited | Modification of biological elements |
WO2000074722A3 (en) * | 1999-06-09 | 2001-07-12 | Hybrid Systems Ltd | Modification of biological elements |
JP2003501060A (en) * | 1999-06-09 | 2003-01-14 | ハイブリッド・システムズ・リミテッド | Modification of biological elements |
JP2003507348A (en) * | 1999-08-19 | 2003-02-25 | ユニバーシティ オブ サザン カリフォルニア | Targeted artificial gene delivery |
US6399385B1 (en) | 1999-09-29 | 2002-06-04 | The Trustees Of The University Of Pennsylvania | Methods for rapid PEG-modification of viral vectors, compositions for enhanced gene transduction, compositions with enhanced physical stability, and uses therefor |
WO2001023001A3 (en) * | 1999-09-29 | 2002-03-14 | Univ Pennsylvania | Rapid peg-modification |
WO2001023001A2 (en) * | 1999-09-29 | 2001-04-05 | The Trustees Of The University Of Pennsylvania | Rapid peg-modification |
US7223388B2 (en) * | 2001-08-03 | 2007-05-29 | Board Of Regents, The Univeristy Of Texas System | Modified reoviral therapy |
WO2004072289A1 (en) * | 2003-02-17 | 2004-08-26 | Fuso Pharmaceutical Industries, Ltd. | Novel virus vector |
WO2005106046A1 (en) * | 2004-05-03 | 2005-11-10 | Stefan Kochanek | Modified viral vector particles |
US7776322B2 (en) | 2004-08-16 | 2010-08-17 | Stefan Kochanek | Modified viral vector particles |
US8715642B2 (en) | 2004-08-16 | 2014-05-06 | Stefan Kochanek | Modified viral vector particles |
WO2010067081A2 (en) | 2008-12-11 | 2010-06-17 | Hybrid Biosystems Limited | Modification of nucleic acid vectors |
Also Published As
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JP2001521381A (en) | 2001-11-06 |
WO1998044143A9 (en) | 1999-04-22 |
AU6881798A (en) | 1998-10-22 |
AU745056B2 (en) | 2002-03-07 |
CA2285416A1 (en) | 1998-10-08 |
EP0994959A1 (en) | 2000-04-26 |
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