Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • 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
  • C12N2710/10351—Methods of production or purification of viral material

Definitions

• the present invention relates to a formulation for the preservation of viral particles and viral vectors, which is directly injectable into an organism. It relates more particularly to a formulation for a recombinant adenovirus vector that optimally enhances the vector titer, or stabilizes the vector at refrigerator or room temperature, or both.
• the invention relates to compositions comprising a recombinant adenovirus vector and a concentration of human serum albumin (HSA) effective to stabilize the adenovirus vector at a temperature above the freezing point of water or to enhance a titer of the adenovirus vector compared to a titer in the absence of HSA, or both, in an aqueous buffer.
• HSA human serum albumin
• Human serum albumin is a non-glycosylated monomeric protein of 585 amino acids, with a molecular weight of 66 kD. Its globular structure is maintained by 17 disulphide bridges, which create a sequential series of 9 double loops (Brown, J.R., 1977). "Albumin structure, function and uses",
• HSA histone deacetylase
• the genes encoding HSA are known to be highly polymorphic, and more than 30 apparently different genetic variants have been identified by electrophoretic analysis under varied conditions (Weitkamp, L.R. et al., 1973. Ann. Hum. Genet., 37:219-226).
• the HSA gene comprises 15 exons and 14 introns comprising 16,961 nucieotides, from the supposed "capping" site up to the first site of addition of poly(A).
• Human albumin is synthesized in the hepatocytes of the liver, and then secreted into the peripheral blood. This synthesis leads, in a first instance, to a precursor, prepro-HSA, which contains a signal sequence of 18 amino acids directing the nascent polypeptide in the secretory pathway.
• HSA is the most abundant blood protein, with a concentration of about 40 grams per liter of serum. Therefore, there are about 160 grams of circulating albumin in the human body at any one time.
• the most important role of HSA is to maintain a normal osmolarity of the blood. It also has an exceptional binding capacity for various substances and plays a role both in the endogenous transport of hydrophobic molecules, such as steroids and bile salts, and in that of different therapeutic substances, which may thus be transported to their respective sites of action.
• HSA has been recently implicated in the breakdown of the prostaglandins.
• HSA has been previously shown to stabilize solutions of proteins, including protein antigens, and small organic molecules, such as hemin (Paige, A.G. et al., 1995. Pharmaceutical Res., 12:1883-1888; Chang, A.-C. and R.K. Gupta, J., 1996. Pharm. Sci., 85:129-132; Niemeijer, N. R.. et al, 1996. Ann. Allergy Asthma Immunol., 76:535-540; and Cannon, J.B. et al., 1995. PDA:J. Pharm. Sci.
• HSA is purified from material source of human serum, or can be obtained from genetic engineering, whether by fermentation of recombinant cells (bacteria, yeast, or mammalian cells), or by expression in transgenic animals, particularly from mammary tissues. HSA has also been used to preserve biological materials for freezing (W097/33975). However, this use has not been described for room temperature preservation and storage of adenoviruses.
• adenovirus formulations such as formulation 1 (Example 1, infra) require storage at -70°C to remain stable.
• the requirement to keep viral vector preparations at these temperatures necessitates acquisition by clinicians of freezers that maintain -70°C temperatures.
• Another complication arises during shipment of the vectors from the manufacturing site to the clinic.
• the present invention addresses and overcomes these deficiencies in the art, and unexpectedly provides, for the first time, formulations that provide for long term stability of adenovirus vectors at such higher temperatures than have been achieved to date.
• the present invention advantageously provides a formulation for the preservation and/or storage of a recombinant virus vector, particularly and preferably an adenovirus vector, that optimally enhances the vector titer, or stabilizes the vector at refrigerator or room temperature, or both.
• the invention provides a composition comprising a recombinant adenovirus vector and a concentration of serum albumin effective to stabilize the adenovirus vector at a temperature above the freezing point of water or to enhance a titer of the adenovirus vector compared to a titer in the absence of serum albumin, or both, in an aqueous buffer at a pH effective to stabilize the adenovirus vector.
• the serum albumin is human serum albumin (HSA).
• HSA human serum albumin
• the concentration of HSA is from about 0.01% to about 25% (w/v).
• the concentration of HSA is from about 0.1% to about 15%. More preferably, the concentration of HSA is from about 1% to about 10%. Most preferably, the concentration of HSA is about 5%.
• HSA can be purified from natural sources, or, more preferably, obtained by genetic engineering.
• the advantage of such a formulation stems from the fact that the solution is available for administration immediately after removal from the storage temperature, without any further manipulation being necessary. It then becomes possible to carry out the removal from storage conditions directly in the clinic, thereby reducing the time between storage and use, which also makes it possible to remain constantly in a sterile formulation and therefore to reduce to a minimum the risks of external contamination.
• the pH of the composition is greater than or equal to
• the pH is greater than 7.5.
• the pH can be 7.6, 7.7, 7.8, or 7.9. More preferably, the pH is greater than 8.0, e.g., 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, or 8.9.
• the pH is 8.2.
• the pH is 8.4.
• the concentration of HSA is 1% to 10%; more preferably 5%.
• the concentration of HSA is 5%.
• the pH is 8.4, the concentration of HSA is 5%.
• the aqueous buffer of the invention comprises a physiological buffer, such as, but by no means, limited to phosphate buffer, Tris-HCl buffer, Hepes buffer, and the like.
• a suitable buffer for use according to the invention is pharmaceutically acceptable, i.e., compatible with in vivo delivery.
• the buffer is a Tris-HCl buffer.
• the buffer is set at a pH that stabilized adenovirus vectors.
• An aqueous buffer for use in the invention comprises salts, e.g., calcium chloride (CaCl 2 ), magnesium chloride (MgCl 2 ), and sodium chloride (NaCl).
• the buffer may contain about 2.0 mM MgCl 2 and 150 mM NaCl.
• the aqueous buffer contains a physiological concentration of salt.
• a composition or formulation of the invention may contain additional components in addition to HSA to further stabilize the recombinant adenovirus.
• any constituent of a composition of the invention is pharmaceutically acceptable.
• compositions of the invention are particularly suitable for formulation of a recombinant adenovirus for gene therapy.
• the recombinant adenovirus expresses a 5 heterologous protein.
• heterologous proteins include, but are by no means limited to, tumor suppressor proteins such as p53; suicide genes such as herpes simplex virus thymidine kinase (HSV-tk); growth factors such as acidic fibroblast growth factor (FGF); angiogenic factors such as FGF or vascular endothelial growth factor (VEGF); trophic factors such at nerve growth factor (NGF), neurotrophic factor-3 (NT-3), NT-4, glial-derived neurotrophic factor (GDNF), and ciliary neurotrophic factor
• tumor suppressor proteins such as p53
• suicide genes such as herpes simplex virus thymidine kinase (HSV-tk)
• growth factors such as acidic fibroblast growth factor (FGF); angiogenic factors such as F
• heterologous proteins for expression in a vector formulated in accordance with the present invention can be found infra.
• the heterologous protein is p53.
• the heterologous protein is HSV-TK.
• the present invention further provides a method for preparing a recombinant adenovirus vector formulation comprising preparing an admixture of a recombinant adenovirus and a
• the temperature is greater than or equal to 4°C and less than 37°C. In a further embodiment, the temperature is greater than or equal to 20°C. Preferably, when the temperature is greater than 4°C, and particularly when the temperature is greater than 20°C, the
• the present invention provides a method for stabilizing an adenovirus vector at about 20°C by preparing an admixture of the adenovirus vector in an aqueous composition of Dulbecco's phosphate buffered saline, from about 5% to 15% glycerol, from about 0.25 to 2.0 mM CaCl 2 , and from about 0.1 to 1.0 mM MgCl 2 .
• the concentration of glycerol is
• the concentration of CaCl 2 is about 1.0 mM, and the concentration of MgCl, is about 0.5 mM.
• FIG. 3 Bioactivity Assay - +4°C Bioactivity for 4 Formulations.
• Figure 4. Bioactivity Assay - +20°C Bioactivity for 4 Formulations.
• Figure 5. Effect of HSA and Sucrose on Viral Titers at -20°C.
• Figure 6. Effect of Buffer, Salt and/or Cations on Viral Titers at +2-10°C.
• Figure 7. Effect of HSA on Viral Titer at +2-10°C.
• Figure 8 Activity of AdCMVp53 Stored in Formulation 4 at Various pH/1 week.
• Figure 9. Comparison of HSA and BSA in viral formulations.
• Figure 10. Stability of virus at 37°C for 1 week in different formulations.
• Figure 11. Stability of AVI .OHSVTK Stored in Formulation 19 at -70°C, -20°C, +4°C, and +20°C for 1.5, 3.5, and 8.5 months.
• the present invention advantageously provides a new type of medium which makes it possible to preserve virus vectors, particularly adenovirus vectors, using a formulation that optimally enhances the recombinant virus vector titer, or stabilizes the vector at refrigerator or room temperature, or both.
• a second formulation comprising lOmM Tris-HCL, pH 8.4, 5% HSA, 5% sucrose, 2.0mM MgCl 2 , 150mM NaCl was tested for its ability to stabilize adenovirus vector preparations for at least 8.5 months, relative to a control stored at -70°C. It was found that this lOmM Tris-HCl, pH 8.4, 5% HSA, 5% sucrose, 2.0mM MgCl 2 , 150mM NaCl formulation was also very stable, preserving viral particle integrity and infectivity (titer) for at least 8.5 months at +4°C and preserving viral particle integrity for at least 8.5 months at +20°C.
• the critical variables for a formulation to preserve adenovirus vectors were HSA and pH; sucrose was also found to enhance stability.
• the invention provides a formulation for the preservation and/or storage of viruses comprising a concentration of HSA effective to stabilize an adenovirus vector at a temperature above the freezing point of water or to enhance a titer of the adenovirus vector compared to a titer in the absence of HSA, or both, in an aqueous buffer.
• the invention provides a composition comprising a recombinant adenovirus vector and a concentration of HSA effective to stabilize the adenovirus vector at a temperature above the freezing point of water or to enhance a titer of the adenovirus vector compared to a titer in the absence of HSA, or both, in an aqueous buffer.
• the pH of the composition is greater than or equal to 5.0 and less than or equal to 9.0, and preferably, the pH is greater than 7.5.
• the pH is 8.2 and the concentration of HSA is 5%.
• the pH is 8.4 and the concentration of HSA is 5%.
• the term “about” or “approximately” means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.
• corresponding to is used herein to refer similar or homologous sequences, whether the exact position is identical or different from the molecule to which the similarity or homology is measured.
• a nucleic acid or amino acid sequence alignment may include spaces.
• corresponding to refers to the sequence similarity, and not the numbering of the amino acid residues or nucleotide bases.
• a “formulation” refers to an aqueous or solution medium for the preservation of viral particles and viral vectors, which is directly injectable into an organism. It relates more particularly to a formulation for a recombinant adenovirus vector that optimally enhances the vector titer, or stabilizes the vector at refrigerator or room temperature, or both. It also relates to compositions comprising a recombinant adenovirus vector and a concentration of HSA effective to stabilize the adenovirus vector at a temperature above the freezing point of water or to enhance a titer of the adenovirus vector compared to a titer in the absence of HSA, or both, in an aqueous buffer.
• the aqueous buffer will include salts or sugars, or both, at about an isotonic concentration.
• a “gene” refers to an assembly of nucieotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acids.
• HSA Human serum albumin
• the genes encoding HSA are known to be highly polymorphic, and more than 30 apparently different genetic variants have been identified by electrophoretic analysis under varied conditions (Weitkamp, L.R. et al., 1973. Ann. Hum. Genet., 37:219-226).
• the HSA gene comprises 15 exons and 14 introns comprising 16,961 nucieotides, from the supposed "capping" site up to the first site of addition of poly(A).
• phrases “pharmaceutically acceptable” refers to molecular entities, at particular concentrations, and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, fever, dizziness and the like, when administered to a human or non-human animal.
• pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in humans or non-human animals.
• a "recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation, i.e., genetic engineering.
• a "subject” is a human or a non-human animal who may receive a vector formulated in a composition of the invention.
• a “vector” is any means for the transfer of a nucleic acid into a host cell.
• a vector may be a replicon to which another DNA segment may be attached so as to bring about the replication of the attached segment.
• a “replicon” is any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., capable of replication under its own control.
• the term "vector” as used herein specifically refers to viral means for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo.
• Viral vectors include retrovirus, adeno-associated virus, pox, baculovirus, vaccinia, herpes simplex, Epstein-Barr and adenovirus vectors, as set forth in greater detail below.
• the nucleic acid contains a coding region for a gene of interest.
• the coding region is operably associated with an expression control sequences, e.g., a promoter.
• a vector may also contain one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (transfer to which tissues, duration of expression, etc.).
• HSA Human Serum Albumin
• the HSA used within the framework of the present invention may be either of natural origin (purified HSA) or of the recombinant origin (rHSA).
• rHSA recombinant origin
• the present invention is described primarily in the context of human serum albumin, it has been found infra that serum albumin from other species is equally effective.
• the term HSA should be considered to encompass any serum albumin, such as bovine serum albumin (BSA) and murine serum albumin (MSA).
• BSA bovine serum albumin
• MSA murine serum albumin
• an autologous serum albumin for delivery of a formulation in vivo for gene therapy, it is preferable to use an autologous serum albumin.
• human serum albumin is desirable and preferred.
• a recombinant or natural HSA is advantageously used which meets certain quality criteria (e.g., homogenetic, purity, stability).
• the pharmacopoeia sets a number of parameters for the plasma albumin solutions, namely a pH value, a protein content, a polymer and aggregate content, an alkaline phosphatase content and a certain protein composition. It imposes, furthermore, a certain absorbance, the compliance with a test of sterility, with a test for pyrogens and for toxicity (see “Albumini humai solutio", European Pharmacocpoeia (1984), 255).
• compositions according to the invention comprise a purified human plasma albumin or a recombinant human albumin, preferably produced in a eukaryotic host.
• HSA comprises, for the purpose of the invention, any natural variant of human albumin, resulting from the polymorphism of this protein. It is also possible to use an HSA equivalent, that is to say, any HSA derivative conserving the properties of HSA. These derivatives may be especially amino- (N-) terminal fragments of HSA.
• Natural HSA is generally produced by purification from biological material of human origin. In particular, it is obtained by conventional techniques for fractionation of plasma obtained from blood donations (Cohn et al., 1946. J. Am. Chem. Soc, 68:459 pp.), or by extraction from the human placenta, according to the technique described by J. Liautraud et al. (1973, 13 th International IABS Conference, Budapest; A: "Purification of proteins. Development of biological standard", Karger (ed.), Bale, 27:107 pp).
• the purified albumin used within the framework of the present invention is a plasma albumin. Most particularly, a commercial plasma albumin may be used.
• HSA Fermentation More particularly, as regards the bacterial hosts, genetic engineering can be accomplished in a bacterium, for example, Escherichia coli, as a host organism. European patents EP 236 210, EP 200 590, or EP 198 745 describe processes for the production of HSA in E. coli using different expression vectors, different transcriptional promoters, and different secretory signals. Subsequently, secretion of HSA in Bacillus subtilis was also carried out (Saunders et al., 1987. J. Bacteriol., 169:2917). As regards the eukaryotic hosts, processes for the production of HSA have been developed using yeasts as a host organism.
• HSA human serum albumin sequence
• the DNA constructs comprise a 5' regulatory sequence which directs the expression and secretion of HSA protein in the milk of a transgenic animal.
• the recombinant HSA of the invention is RecombuminTM (Centeon).
• RecombuminTM is a yeast-derived recombinant human albumin, which is structurally identical to plasma-derived human albumin.
• RecombuminTM is produced using a hybrid secretion leader sequence conjugated to a cDNA which encodes human serum albumin.
• the cDNA sequence is disclosed in EP 0 073 646 and the leader sequence is disclosed in US Patent No. 5,302,697.
• the leader sequence is cloned into an expression cassette in a disintegration vector which is described in US Patent No. 5,637,504.
• the host yeast cell used to make RecombuminTM includes various mutations, such as gene disruptions of the yeast aspartyl protease gene (WO 95/23857) and gene disruptions of the heat shock protein 150 gene (US 5,783,423).
• the formulations according to the invention may be prepared in various ways.
• the different components may be mixed together, and then the viral particle or vector is added to the mixture. It is also possible to mix one or several of the components with the viral particle or vector and then to add the remaining component(s).
• a formulation comprising all of the components is prepared, to which the viral particle or vector is then added.
• the preparation of the formulation and the addition of the viral particles or viral vectors are performed under sterile conditions.
• the viral particles and viral vectors more particularly relevant to the present invention are those which may have be used in gene therapy.
• a large number of viruses may have their genome modified, on the one hand, so that they lose their ability to multiply while retaining their infectivity, and on the other hand, so as to insert into their genome a nucleic acid sequence of therapeutic interest which will be expressed in the infected cells.
• viruses there may be mentioned more particularly, the adenoviruses, the adeno-associated viruses (AAVs), the retroviruses, the herpes viruses and the like.
• the present formulation for relatively high temperature storage of a viral particle or vector was specifically developed for storage of an adenovirus vector.
• the invention contemplates that the HSA formulation, particularly with the preferred pH range, may also stabilize or enhance infectivity, or both, of other vectors.
• Adenovirus vectors In a preferred embodiment, the vector is an adenovirus vector.
• Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a nucleic acid of the invention to a variety of cell types.
• Various serotypes of adenovirus exist. Of these serotypes, preference is given, within the scope of the present invention, to using type 2 or type 5 human adenoviruses (Ad 2 or Ad 5) or adenoviruses of animal origin (see W094/26914).
• adenoviruses of animal origin which can be used within the scope of the present invention include adenoviruses of canine, bovine, murine (example: Mavl, Beard et al., Virology 75 (1990) 81), ovine, porcine, avian, and simian (example: SAV) origin.
• the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus (e.g., Manhattan or A26/61 strain (ATCC VR-800), for example).
• the replication defective adenoviral vectors of the invention comprise the ITRs, an encapsidation sequence and the nucleic acid of interest. Still more preferably, at least the El region of the adenoviral vector is non-functional. The deletion in the El region preferably extends from nucieotides 455 to 3329 in the sequence of the Ad5 adenovirus (PvuII-Bglll fragment) or 382 to 3446 (HinfII-Sau3A fragment).
• E3 region WO95/02697
• E2 region W094/28938
• E4 region W094/28152, W094/12649 and WO95/02697
• the adenoviral vector has a deletion in the El region (Ad 1.0). Examples of El-deleted adenoviruses are disclosed in EP 185 573, the contents of which are incorporated herein by reference. In another preferred embodiment, the adenoviral vector has a deletion in the El and E4 regions (Ad 3.0). Examples of El/E4-deleted adenoviruses are disclosed in WO95/02697 and W096/22378, the contents of which are incorporated herein by reference. In still another preferred embodiment, the adenoviral vector has a deletion in the El region into which the E4 region and the nucleic acid sequence are inserted (see FR94 13355, the contents of which are incorporated herein by reference).
• the replication defective recombinant adenoviruses according to the invention can be prepared by any technique known to the person skilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham, EMBO J. 3 (1984) 2917). In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid which carries, inter alia, the DNA sequence of interest. The homologous recombination is effected following cotransfection of the said adenovirus and plasmid into an appropriate cell line.
• the cell line which is employed should preferably (i) be transformable by the elements, and (ii) contain the sequences which are able to complement the part of the genome of the replication defective adenovirus, preferably in integrated form in order to avoid the risks of recombination.
• Examples of cell lines which may be used are the human embryonic kidney cell line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which contains the left-hand portion of the genome of an Ad5 adenovirus (12%) integrated into its genome, and cell lines which are able to complement the El and E4 functions, as described in applications W094/26914 and WO95/02697.
• adeno-associated viruses are DNA viruses of relatively small size which can integrate, in a stable and site-specific manner, into the genome of the cells which they infect. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, mo ⁇ hology or differentiation, and they do not appear to be involved in human pathologies.
• the AAV genome has been cloned, sequenced and characterized.
• ITR inverted terminal repeat
• the replication defective recombinant AAVs according to the invention can be prepared by cotransfecting a plasmid containing the nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions, and a plasmid carrying the AAV encapsidation genes (rep and cap genes), into a cell line which is infected with a human helper virus (for example an adenovirus).
• ITR inverted terminal repeat
• the gene can be introduced in a retroviral vector, e.g., as described in Anderson et al., U.S. Patent No. 5,399,346; Mann et al., 1983, Cell 33:153; Temin et al., U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No. 4,980,289; Markowitz et al., 1988, J. Virol. 62: 1120; Temin et al., U.S. Patent No. 5,124,263; EP 453242, EP178220; Bernstein et al. Genet. Eng. 7 (1985) 235;
• the retroviruses are integrating viruses which infect dividing cells.
• the retrovirus genome includes two LTRs, an encapsidation sequence and three coding regions (gag, pol and env).
• the gag, pol and env genes are generally deleted, in whole or in part, and replaced with a heterologous nucleic acid sequence of interest.
• vectors can be constructed from different types of retrovirus, such as, HIV, MoMuLV ("murine Moloney leukaemia virus” MSV ("murine Moloney sarcoma virus”), HaSV ("Harvey sarcoma virus”); SNV ("spleen necrosis virus”); RSV ("Rous sarcoma virus”) and Friend virus.
• Defective retroviral vectors are disclosed in WO95/02697.
• Regulatory Regions Expression of a polypeptide from a vector of the invention may be controlled by any regulatory region, i.e., promoter/enhancer element known in the art, but these regulatory elements must be functional in the host tissue, such as a target tumor, selected for expression.
• the regulatory regions may comprise a promoter region for functional transcription in the host cell, as well as a region situated 3' of the gene of interest, and which specifies a signal for termination of transcription and a polyadenylation site. All these elements constitute an expression cassette.
• Promoters that may be used in the present invention include both constitutive promoters and regulated (inducible) promoters.
• the promoter may be naturally responsible for the expression of the nucleic acid. It may also be from a heterologous source. In particular, it may be promoter sequences of eukaryotic or viral genes. For example, it may be promoter sequences derived from the genome of the cell which it is desired to infect. Likewise, it may be promoter sequences derived from the genome of a virus, including the adenovirus used. In this regard, there may be mentioned, for example, the promoters of the El A, MLP, CMV and RSV genes and the like.
• the promoter may be modified by addition of activating or regulatory sequences or sequences allowing a tissue-specific or predominant expression (enolase and GFAP promoters and the like).
• the nucleic acid may be inserted, such as into the virus genome downstream of such a sequence.
• promoters useful for practice of this invention are ubiquitous promoters (e.g., HPRT, vimentin, actin, tubulin), intermediate filament promoters (e.g., desmin, neurofi laments, keratin, GFAP), therapeutic gene promoters (e.g., MDR type, CFTR, factor VIII), tissue-specific promoters (e.g., actin promoter in smooth muscle cells), promoters which are preferentially activated in dividing cells, promoters which respond to a stimulus (e.g., steroid hormone receptor, retinoic acid receptor), tetracycline-regulated transcriptional modulators, cytomegalovirus immediate-early, retroviral LTR, metallothionein, SV-40, El a, and MLP promoters. Tetracycline-regulated transcriptional modulators and CMV promoters are described in WO 96/01313, US 5,168,062 and 5,385,839, the contents of which are inco ⁇ orated herein by
• the promoters which may be used to control gene expression include, but are not limited to, the cytomegalovirus (CMV) promoter, the SV40 early promoter region (Benoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the he ⁇ es thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.
• prokaryotic expression vectors such as the b-lactamase promoter (Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A.
• eful proteins from recombinant bacteria in Scientific American, 1980, 242:74-94; promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter; and the animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region 5 which is active in pancreatic acinar cells (Swift et al, 1984, Cell 38:639-646; Ornitz et al, 1986, Cold Spring Harbor Symp. Quant. Biol.
• mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al, 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert et al, 1987, Genes and Devel. 1 :268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al, 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al, 1987, Science 235:53-58), alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al, 1987, Genes and Devel.
• beta-globin gene control region which is active in myeloid cells
• heterologous proteins expressed by vectors include, but are by no means limited to, tumor suppressor proteins such as p53; suicide genes such as he ⁇ es simplex virus thymidine kinase (HSV-tk); growth factors such as acidic fibroblast growth factor (FGF); angiogenic factors such as FGF or vascular endothelial growth factor (VEGF); trophic factors such at nerve growth
• tumor suppressor proteins such as p53
• suicide genes such as he ⁇ es simplex virus thymidine kinase (HSV-tk)
• growth factors such as acidic fibroblast growth factor (FGF); angiogenic factors such as FGF or vascular endothelial growth factor (VEGF); trophic factors such at nerve growth
• FGF acidic fibroblast growth factor
• VEGF vascular endothelial growth factor
• the heterologous protein is p53. In a second specific embodiment, the heterologous protein is HSV-TK.
• the present invention further provides a method for preparing a recombinant adenovirus formulation comprising preparing an admixture of a recombinant adenovirus and a
• the temperature is greater than or equal to 4°C and less than 37°C. In a further embodiment, the temperature is greater than or equal to 20°C. Preferably, when the temperature is greater than 4°C, and particularly when the temperature is greater than 20°C, the concentration of HSA is 5%, the pH of the admixture is greater than 8.0, or both.
• the present invention provides a method for stabilizing an adenovirus vector at about 20°C by preparing an admixture of the adenovirus vector in an aqueous composition of Dulbecco's phosphate buffered saline, from about 5% to 15% glycerol, from about 0.25 to 2.0 mM CaCl 2 , and from about 0.1 to 1.0 mM MgCl 2 .
• the concentration of glycerol is 10%
• the concentration of CaCl 2 is about 1.0 mM
• the concentration of MgCl 2 is about 0.5 mM.
• the use of a formulation according to the invention makes it possible to preserve viral particles and viral vectors and to administer it directly into a subject, without a centrifugation or washing stage, with a good viability and/or without affecting its capacity to infect a susceptible cell of the organism.
• the present invention also relates to preparations containing the preservation formulation according to the invention and viral particles or viral vectors, as well as to a process for the storage of the viral particles or viral vectors.
• the viral particles or viral vectors may be packaged directly into the formulation according to the invention.
• viruses these are previously purified as described herein (e.g., by centrifugation on a cesium chloride gradient, column chromatography, plaque purification, and the like).
• the viral particles or viral vectors may then be packaged in the formulation according to the invention, in an appropriate container. It may an ampoule, a tube, especially a cryotube, a bag, a vial, a flask, and the like. The container is previously sterilized and the packaging operations are performed under sterile conditions.
• a medium or formulation according to the invention allows the storage and preservation of viral particles or viral vectors under conditions that preserve good viability.
• the formulation according to the invention may, in particular, allow the storage of the particles or vectors at a temperature above the freezing point of water.
• the media according to the invention may allow the storage of recombinant adenovirus vectors at a temperature above the freezing point of water or enhance a titer of the adenovirus vector compared to a titer in the absence of HSA, or both, in an aqueous buffer.
• Pharmaceutical Compositions for their use according to the present invention, the vectors, either in the form of a virus vector or virus particle are preferably combined with one or more pharmaceutically acceptable carriers for an injectable formulation.
• carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
• Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
• Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin.
• saline solutions monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts
• dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, allow the constitution of injectable solutions.
• the preferred sterile injectable preparations can be a solution or suspension in a nontoxic parenterally acceptable solvent or diluent.
• Examples of pharmaceutically acceptable carriers are saline, buffered saline, isotonic saline (e.g., monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, or mixtures of such salts), Ringer's solution, dextrose, water, sterile water, glycerol, ethanol, and combinations thereof.
• 1,3-butanediol and sterile fixed oils are conveniently employed as solvents or suspending media. Any bland fixed oil can be employed including synthetic mono- or di-glycerides. Fatty acids such as oleic acid also find use in the preparation of injectables.
• therapeutic ly effective amount is used herein to mean an amount sufficient to reduce by at least about 15 percent, preferably by at least 50 percent, more preferably by at least 90 percent, and most preferably prevent, a clinically significant deficit in the activity, function and response of the subject. Alternatively, a therapeutical ly effective amount is sufficient to cause an improvement in a clinically significant condition in the subject.
• the composition of the invention may be introduced parenterally or transmucosally, e.g., orally, nasally, or rectally, or transdermally.
• administration is parenteral, e.g., via intravenous injection, and also including, but is not limited to, intra-arteriole, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration.
• the administration of the composition may also be introduced by injection into a tumor or into tissues surrounding the tumor.
• the preferred route of administration to a tumor is by direct injection into the tumor.
• the tumor can be imaged using any of the techniques available in the art, such as magnetic resonance imaging or computer-assisted tomography, and the therapeutic composition administered by stereotactic injection, for example.
• a vector of the invention can be targeted to the antigen as described above, and administered systemically or subsystemically, as appropriate, e.g., intravenously, intraarterioally, intraperitoneally, intraventricularly, etc.
• the virus doses used for the administration may be adapted as a function of various parameters, and in particular as a function of the site of administration considered, the number of injections, the gene to be expressed or alternatively the desired duration of treatment.
• the recombinant adenoviruses according to the invention are formulated and administered in the form of doses of between 10 4 and 10 14 pfu, and preferably 10 5 to 10 u pfu.
• the term pfu (plaque forming unit) corresponds to the infectivity of a virus solution, and is determined by infecting an appropriate cell culture and measuring, generally within 15 days, the number of plaques of infected cells.
• compositions of the invention can be delivered by intravenous, intraarterial, intraperitoneal, intramuscular, pulmonary, or subcutaneous routes of administration.
• the compositions, properly formulated can be administered by nasal or oral administration.
• a constant supply of the viral particles or viral vectors can be ensured by providing a therapeutical ly effective dose (i.e., a dose effective to induce metabolic changes in a subject) at the necessary intervals, e.g., daily, weekly, monthly, etc.
• a therapeutical ly effective dose i.e., a dose effective to induce metabolic changes in a subject
• these parameters will depend on the severity of the disease condition being treated, other actions, such as diet modification, that are implemented, the weight, age, and sex of the subject, and other criteria, which can be readily determined according to standard good medical practice by those of skill in the art.
• a subject in whom administration of a viral particle or viral vector within the scope of the invention is administered is preferably a human, but can be any animal.
• the methods and pharmaceutical compositions of the present invention are particularly suited to administration to any animal, particularly a mammal, and including, but by no means limited to, domestic animals, such as feline or canine subjects, farm animals, such as but not limited to bovine, equine, caprine, ovine, and porcine subjects, wild animals (whether in the wild or in a zoological garden), research animals, such as mice, rats, rabbits, goats, sheep, pigs, dogs, cats, etc., avian species, such as chickens, turkeys, songbirds, etc., i.e., for veterinary medical use.
• the present invention may be better understood by reference to the following non-limiting Examples, which are provided as exemplary of the invention.
• EXAMPLE 1 Stability Testing of Adenovirus Formulations at Above Freezing Temperatures. Initially, the adenovirus preparations were stored at -70°C in buffered glycerol solutions to provide the best long term stability. Since -70°C storage may not be available in the clinical setting, a formulation which will preserve the virus at -20°C or higher temperatures is needed.
• This Example summarizes six month data for four Formulations tested at five storage temperatures. This study was designed to determine optimal formulation of an adenovirus vector to achieve at least one (1) year stability when stored frozen at -20°C, or as a liquid at +4°C or +20°C storage temperatures.
• a positive control consisted of the virus stored in Dulbecco's phosphate buffered saline (DPBS), 10% glycerol, OmM CaCl 2 and 0.5mM MgCl 2 at -70°C. Test samples for all four Formulations were passed though Sephadex G25 column for buffer exchange. Triplicate virus samples and positive controls were tested at one month intervals for 3 consecutive months, then every one and one-half months thereafter.
• DPBS Dulbecco's phosphate buffered saline
• Viral titer in plaque forming units was determined by plaque assay using 293 cells.
• An analytical HPLC method was used to determine viral particle/ml measurements by peak area of an optical density (OD) at 260nm.
• Viral particles were determined by UV measurements made on Formulations not containing HSA. The Bioactivity assay was used as an additional test for viral activity at selected time points.
• Virus Sample Preparation A type 5 adenovirus comprising the p53 gene under the control of the cytomegalovirus promoter, Ad5CMVp53 (as described in International Patent Application No. US95/04898), in DPBS + 10% glycerol, 0.2 ⁇ m filtered, in 10 ml vials was used in these experiments.
• the virus titer was estimated at 1.38x10" pfu/ml and 3.44xl0 12 particles/ml.
• thirteen vials of Ad5CMVp53 were thawed at room temperature and pooled. OD measurements were taken at 260nm on the pooled material to determine the original particles/ml.
• Form 1 DPBS, 10% glycerol, 1.0mM CaCl 2 and 0.5mM MgCl 2
• Form 2 DPBS, 5% HSA, 5% Sucrose, OmM CaCl 2 and 0.5mM MgCl 2
• Form 3 lOmM Tris-HCL, pH 7.5, 5% HSA, 5% Sucrose, 2.0mM MgCl 2 , 150mM NaCl
• Form 4 lOmM Tris-HCL, pH 8.2, 5% HSA, 5% Sucrose, 2.0mM MgCl 2 ,150mM NaCl buffer exchange. Post-buffer exchange OD measurements were taken at 260nm to determine the particle dilution that occurred during the exchange process. Particle dilution was reported as percent yield. After the buffer exchange and OD measurements were completed, HSA was added to the appropriate Formulations. Dilution of the samples by buffer exchange and addition of HSA was calculated by the following equation: (Column Dilution)X(% yield)X(HSA Dilution). All samples were diluted to a final dilution of 3.2 fold from the original pool concentration.
• All samples were stored overnight at +4°C and were aliquoted and tested the next day. All four virus Formulation samples were aliquoted into labeled glass amber vials (Kimble #203), 0.42 ml each and stored at the appropriate temperatures, -20°C (VWR Scientific freezer), -4°C (LabLine Ambi HiLo Chamber), +4°C (VWR Scientific 2-door deli case Model GDM-49, +20°C, and +37°C (Queue Systems, Inc., Cell Star Incubator Model No. QWJ8000ABA). The samples for -20°C storage were frozen to -40°C in a controlled rate freezer prior to storage at -20°C. The temperatures of -4°C, +20 and +37 were used as elevated temperatures with which to determine the "accelerated" stability of the virus formulations at -20°C.
• Positive Control Sample Preparation The remaining volume of original virus pool, after test samples had been removed for buffer exchange, was diluted 3.2 fold to match the dilution of the test samples described above.
• the control was diluted in DPBS, 10% glycerol, l .OmM CaCl 2 and 0.5mM MgCl 2 to mimic the current Ad5CMVp53 formulation.
• Control virus was aliquoted, 0.42 ml per cryovial (Nalgene No. 5000-0020, lot 072381) and stored at -70°C (BioFreezer, Forma Scientific Model No. 8328).
• Viral titer was determined by the plaque assay on 293 cells, human embryonic kidney cells transformed with sheared human Ad5 DNA (complement El region). At designated time points, triplicate test samples and triplicate positive controls were tested for plaque forming units. Two days prior to testing, 293 cells (ATCC® Catalog No. CRL 1573) were seeded onto 6 cm tissue culture dishes. On the day of testing, the virus samples and positive control were serially diluted in MEM+0.5% HEPES. Two or more dilutions which were expected to yield countable plaques were used to infect 293 cells. Three confluent 6 cm dishes of 293 cells were infected with 0.5 ml of each virus sample and positive control dilution.
• the dishes were incubated at 37°C, 5.0% C0 2 and 95% relative humidity for 2 hours with rocking every 15 minutes. After incubation, the samples were aspirated off and the dishes were overlaid with a 0.5% SeaKem Agarose, MEM, 7.5% FBS overlay. The dishes were then incubated at 37°C, 5.0% C0 2 and 95% Table 2: Summary of 6 Month Formulation Analysis.
• each dilution's titer was determined in plaque forming units per ml (pfu/ml) by the following equation: [(Average Count X Dilution)/ Volume]. A sample's pfu/ml titer was determined by averaging the pfu/ml of the dilutions. Triplicate sample averages were determined for each time point. Results were plotted in Microsoft EXCEL for further analysis.
• HPLC Analysis HPLC peak area comparisons were performed to determine the virus concentration in terms of viral particles between samples and positive controls. Each triplicate sample and positive control was tested on a Waters analytical HPLC system with Millennium software. Briefly, 100 ⁇ l of test sample and positive control were injected into a Resource Q 1ml, 6.4x30 mm column (Pharmacia Catalog No. 17-1 177-01), using a flow rate at 1 ml/minute. The samples are stored at 2-8°C in the Waters autosampler during the entire run.
• the running gradient consisted of line A: Buffer A (50mM Tris, pH 7.5), line B: Buffer B (50mM Tris + 1 M NaCl, pH 7.5), line C: Buffer C (0.5 N NaOH, cleaning solution, used to clean the column after each virus run), and line D: Buffer D (water).
• the intact adenovirus is eluted between 18-22 minutes after the start of the run.
• the peak area of every single peak eluted in the entire gradient is reported by the Millenium software. Only the adenovirus peak which is eluted as a single peak at the 18-22 minute range was used as an indication of the virus particle concentration.
• the peak area for the virus peak was integrated and reported by Millennium software. Data were plotted and analyzed in Microsoft EXCEL.
• the p53 transgene expression was determined by the inhibition of proliferation of Saos-LM2 cells, a human tumor cell line derived from a lung metastasis from osteogenic sarcoma.
• Saos-LM2 cells a human tumor cell line derived from a lung metastasis from osteogenic sarcoma.
• 3 x 10 3 Saos-LM2 cells/well were seeded onto 96 well titer plates in DMEM-high glucose (HG) + 10% FBS-heat inactivated (HI) 3 days prior to infection with the virus samples diluted to various Multiplicities of Infection (10, 20, 40, 80, and 160 MOI) in DMEM-HG + 10% FBS-HI.
• the infected cells were incubated at 37°C for 4 days and then stained with alamar blue, an indicator of cell growth, and incubated for 8 hours.
• OD Absorbance 570nm - Absorbance 595nm .
• the inhibition of proliferation was plotted as a percent reduction of alamar blue in Microsoft EXCEL for further analysis.
• Plaque Assay Results The results of the plaque assays for adenovirus stability at 4°C and 20°C are shown in Figures 1 and 2. At -20°C, all of the tested formulations were stable for six months (data not shown). At 4°C and 20°C, Formulation 4 demonstrated increased stability at all time points ( Figures 1 and 2). Su ⁇ risingly, at -4°C, Formulation 4 demonstrated the same pfu activity as the other formulations (data not shown). At 37°C, all of the formulations were unstable (data not shown). Storage at +4°C allowed the determination of the stability differences between the Formulations.
• Formulation 1 performed better than Formulations 2 and 3 following 1 month of storage at +4°C, however it was reduced significantly in pfu/ml by greater than 1 log at month 3 ( Figure 1). Formulation 1 did not perform as well as Formulation 4 when stored at +4°C at all tested time points ( Figure 1). Formulations 2 and 3 showed an initial decrease in titer following 1 month storage at +4°C ( Figure 1). At +4°C, Formulation 4 remained essentially unchanged up to 3 months and showed activity loss starting at 4.5 month ( Figure 1).
• Formulations 1 and 4 showed the best retention of viral activity at +20°C for 6 months ( Figure 2).
• Formulation 4 showed the lowest decrease over 6 months (Table 3) which is only 0.33 logs.
• Formulation 1 appears to be the second best formulation, as it only decreased by 2 logs.
• the "too numerous to count" (TNTC) data points in Figure 2 indicated that real titers should be higher than the reported values.
• Formulations 2 and 3 showed a reduction in titer when stored at +20°C ( Figure 2), which was supported by both the HPLC and Bioactivity assays.
• Formulations 2 and 3 were less favorable for long term stability than were Formulations 1 and 4.
• Table 3 Viral Titer Summary: Plaque Assay Results
• Formulation 4 represents a viral formulation that is preferable for preserving viral stability and activity (infectivity), especially at +20°C.
• HPLC peak area provides for the quantitation of viral particles.
• the HPLC assay variation has been determined to be + 3%. Due to the small standard deviations in the HPLC peak areas, any small variation between time points may be statistically significant.
• the HPLC peak area were plotted by temperature over time for all four Formulations and correlated well with the pfu/ml results described above.
• Formulation 4 At +4C, all four formulations except Formulation 4 showed significant loss in viral particles from 1-6 months. Formulation 4 maintained the viral particle concentration until 4.5 month, when its peak area was reduced similar to the level of Formulations 1, 2, and 3 (data not shown). At +20°C, only
• Formulations 2 and 3 showed consistent loss of viral particles from 1 to 6 months, while Formulations 1 and 4 maintained most of their viral particles (data not shown). Similar to the plaque assay data, for formulations stored at -4°C and +37°C, all four
• the HPLC results showed a similar trend in stability as the plaque assay results for each formulation. All four Formulations showed significant viral titer decreases along with significant viral particle decreases after just one month when stored at -4°C or +37°C.
• Bioactivity Assay Results The Formulations were plotted by temperature over time, at an MOI of 20. Results were reported as percent inhibition of cell growth. Increased cell death by viral infection caused an increase in inhibition of cell growth as determined by a reduction of alamar blue in infected cells compared to uninfected cells.
• bioactivity data also support the stability results obtained from the pfu and HPLC assays.
• Formulation 4 at +4°C for 6 months retained >50% of cell growth inhibition, which is the clinical specification for this product.
• -20°C all formulations performed similarly (data not shown).
• Formulation 1 performed poorly at 2 months, but not at 3 and 6 months, indicating that the 2 month data were anomalous (data not shown).
• Formulations 1, 2 and 3 did not perform as well with respect to preserving bioactivity as Formulation 4 for months 2 and 3 ( Figure 3).
• Formulations 1 , 2 and 3 decreased viral titer by more than 2 logs when stored at +4°C and, by more than 53% when measured by HPLC peak area.
• Formulation 4 was the most stable over the 3 month storage period, as evidenced by the plaque assay and HPLC peak area measurements of particle/ml which were reduced by only 14.8%.
• Formulation 4 lOmM Tris buffer at pH 8.2, 5% HSA, 5% sucrose, 2mM MgCl 2 and 150 mM NaCl, was the most favorable for long term viral storage.
• the results from the plaque assay, HPLC particle/ml measurement, and bioactivity assays correlated well with each other and suggested that Formulation 4 retains viral activity for 6 months when stored at -20°C and +20°C.
• These data also suggest that the original formulation of DPBS, 10% glycerol, l.OmM CaCl 2 and 0.5mM MgCl 2> (Formulation 1 ) retained most of its activity for 3 months at -20°C and +20°C.
• Formulation 1 resulted in a decline in both viral titer and particles/ml when stored at +4°C.
• Formulation 4 started to show the loss of activity and viral particles after 3 months.
• EXAMPLE 2 -20°C Screen of Formulations to Preserve Viral Titers.
• Virus Sample Formulations A type 5 adenovirus containing the p53 gene under the control of the cytomegalovirus (CMV) promoter, Ad5CMVp53, was used for these experiments. This material was diluted into various test formulations, frozen, thawed, and analyzed by plaque assay. The stock virus was diluted 100 fold into the various test formulations, which are listed in Table 4. The buffer components were either 10 mM phosphate buffered saline (PBS), pH 7.2, or 10 mM Tris buffered saline, pH 7.5.
• PBS phosphate buffered saline
• Tris buffered saline pH 7.5.
• Additional buffer excipients included 50mM, 150 mM, or 300mM NaCl, divalent cations (ImM CaCl 2 and 0.5mM MgCL), 5% sucrose, and 1%, 5%, or 10% HSA.
• the samples were aliquoted and frozen in vials using the controlled rate freezer, and transferred to a -20°C freezer for storage, as described in Example 1. At 1 day and 9 days after freezing, the samples were thawed and tested for virus activity by the standard plaque assay.
• Ad5CMVp53 used in the test formulation preparations above was diluted into the standard buffer (DPBS, 0.5 mM MgCl2, 1 mM CaCl2, and 10% glycerol, pH
• Plaque Assay The viral plaque assay was performed as described in Example 1. The duplicate plate counts for each dilution were averaged. The calculations for pfu/ml include the 100 fold dilution into test buffers and were calculated as described in Example 1.
• Formulations 1-12 were analyzed to determine the effects of buffer, salt concentration and/or divalent cation addition on viral titers.
• the results of the plaque assays after 1 day and 9 days of storage at -20°C for Formulations 1-12 are shown in Table 5.
• EXAMPLE 3 Formulation Screening for +2-10°C storage.
• Example 2 For future clinical studies, it will be preferred to store virus at temperatures of -20°C and above.
• These experiments are a continuation of those of Example 2, and are designed to screen a number of Formulations for storage at +2-10°C.
• Formulations with HSA preserved all of the virus activity when stored at +2-10°C, while those without HSA lost varying amounts of activity.
• the experiments described within this Example tested various buffers and excipients for their effects on virus titer when stored at +2-10°C.
• Example 2 The Ad5CMVp53 stock virus, dilutions and Formulations described in Example 2 were used in this study. The Formulations are listed in Table 4. The prepared Formulation samples for this study were kept at +2-10°C in a refrigerator. After 14 days, the samples were removed and tested for virus activity by the standard plaque assay. The viral plaque assay was performed and the viral titers were calculated as described above in Example 1.
• HSA significantly increased (p ⁇ 0.001) the plaque number observed in the assay.
• the reason for this is not known. It appears to be an actual increase in the infectivity of the virus, since it is also observed with a flow cytometer-based assay to determine viral protein expression.
• Example 2 Comparison to -20°C Reference Formulation Samples: The results of Example 2, in which identical Formulation samples were stored at -20°C for a similar length of time may be used for this comparison. These are compared in Table 7.
• samples without HSA (Formulations 1-12) showed a loss of viral titers when stored at +4°C compared to storage at -20°C.
• those Formulations with HSA 13-18 showed no loss of viral activity at +4°C.
• Formulation samples containing divalent cations #1,3,5,7,9) had reduced viral titers after storage at +4°C (27% of those kept at -20°C), than those without cations (#2,4,6,8, 10) (66% of those stored at -20°C).
• EXAMPLE 4 Ad5CMVp53 Formulation Screening: pH study.
• Virus Sample Preparation Ad5CMVp53, as described in Examples 1-3 above, was used in these experiments. This virus stock was diluted into formulations with different pH, treated for different storage conditions, and tested for viral titers by the plaque assay as described in Example 1. Control Virus Sample Preparation: The same starting virus stock was used as in the virus sample preparation above, except that this material was stored in PBS + glycerol at -70°C and thawed on the day of assay.
• Formulation Preparation In the first experiment, a number of lOmM Tris-buffered solutions with the same salt (150mM NaCl) and divalent cation concentrations (2mM MgCl 2 ) were prepared and adjusted to specific pH values, ranging from pH 5.0 to pH 9.0. Another set was made which contained 5% HSA and 5% sucrose in addition to the buffer. For one experiment, virus was diluted into the various formulations, and frozen to -40°C in the rate controlled freezer (length of storage at -40°C varies from 5 to 30 minutes). They were then thawed and incubated in a 37°C water bath for 1 hour prior to activity determination by the plaque assay.
• virus was prepared in Formulation 4 at each specific pH value, ranging from pH 6.6 to pH 8.8, stored for 1 week at room temperature or 37°C, and tested in the plaque assay.
• Ad5CMVp53 as used in other Examples, was also used for these experiments.
• the stock virus was diluted 1 : 100 into the test formulations, stored at various temperatures for different lengths of time, and were tested for viral titer (infectivity) using the plaque assay as described in Example 1.
• Formulation Preparation In the BSA study, three viral formulations were prepared: Control Formulation: DPBS + 10% glycerol + 0.5mM MgCl 2 + l.OmM CaCl 2 ; HSA Formulation: DPBS + 5% HSA + 0.5mM MgCl 2 + 1.OmM CaCl 2 ; and BSA Formulation: DPBS + 5% BSA + 0.5mM MgCl 2 + 1.OmM CaCl 2 .
• the BSA used in this study was a reagent grade material and in powder form. The virus stock was diluted 1:100 into each formulation and divided into two vials each.
• HSA Formulation lOmM Tris + 5% HSA (w/v) + 5% sucrose + 150 mM NaCl + 2.0mM MgCl 2 , pH 8.2; and RecombuminTM Formulation: lOmM Tris + 5% RecombuminTM (w/v) + 5% sucrose + 150 mM NaCl + 2.0mM MgCl 2 , pH 8.2.
• the virus stock was diluted 1 :100 into each formulation and divided into 3 vials each and stored at +37°C for 7 days. Viral plaque testing was performed as described in Example 1.
• EXAMPLE 6 Long Term Stability of Adenovirus in an HSA/Sucrose Formulation at -70°C, -20°C, +4°C. and +20°C.
• Examples 1-5 presented supra indicate that a liquid formulation containing human serum albumin (HSA) provides superior stabilization for adenoviral vectors compared to those containing other excipients.
• the pu ⁇ ose of this Example was to determine the stability of adenoviral vectors in a lOmM Tris-HCl + 5% HSA + 5% sucrose + 150mM NaCl + 2mM MgCl 2 , pH 8.4 formulation when stored at various temperatures up to 8.5 months.
• An adenoviral vector comprising the he ⁇ es simplex virus thymidine kinase gene (AV1.0HSVTK) was used in this Example.
• This Example summarizes the efficiency of a formulation comprising 1 OmM Tris-HCl + 5% HSA + 5% sucrose + 150mM NaCl + 2mM MgCl 2 , pH 8.4 (Formulation 19) to preserve adenoviral vectors at four storage temperatures for 0 (2 days), 1.5, 3.5, and 8.5 months.
• This study was designed to determine optimal formulation of an adenovirus vector to achieve at least one (1) year stability when stored frozen at -20°C, or as a liquid at +4°C or +20°C storage temperatures.
• the virus titer was estimated at 1.6 x 10 12 particles/ml.
• 22 vials of AV 1.0HSVTK each vial comprising about 200-250 ⁇ l/vial for a total of 4.5 ml, were thawed at room temperature and pooled.
• OD measurements were taken at 260nm on the pooled material to determine the original particles/ml.
• the AV1.0HSVTK virus pooled stocks were run through a Resource Q column (prepared with 8ml of Source Q15; Pharmacia Catalog No. 17-0947-01, lot 11/21/97 for buffer exchange and reformulated in lOmM Tris-HCl, 5% HSA, 5% sucrose, 150mM NaCl, 2mM MgCl 2 , pH 8.4 (Formulation 19). All solutions used in this buffer exchange process were degassed by an in-line degasser and the virus solution was purged with argon prior to filling.
• the material was aliquoted into polypropylene cryovials and flushed with argon to remove oxygen from the head space of vials.
• the samples were then placed at -20°C (VWR Scientific freezer), +4°C (VWR Scientific 2-door deli case Model GDM-49), room temperature (+20°C), and -70°C (control group).
• the samples for -20°C storage were frozen to -40°C in a controlled rate freezer prior to storage at -20°C.
• the temperature of +20°C was used as an elevated temperature with which to determine the "accelerated" stability of the virus formulation for +4°C.
• Triplicate virus samples and positive controls were tested at intervals of 0 (2 days), 1.5, 3.5, and 8.5 months at -20°C, +4°C, and +20°C with respect to viral particle integrity and viral infectivity.
• Viral titer in plaque forming units (pfu) was determined by plaque assay using 293 cells.
• An analytical HPLC method was used to determine viral particle/ml measurements by peak area of an optical density (OD) at 260nm.
• Positive Control Virus Preparation The control virus was diluted in lOmM Tris-HCl, 5% HSA, 5% sucrose, 150mM NaCl, 2mM MgCl 2 , pH 8.4 (Formulation 19) to mimic the test sample formulation and was aliquoted, 0.4 ml per cryovial (Nalgene No. 5000-0020, lot 072381), and stored at - 70°C (BioFreezer, Forma Scientific Model No. 8328). Triplicate virus samples and positive controls were tested at intervals of 0 (2 days), 1.5, 3.5, and 8.5 months at -70°C with respect to viral particle integrity and viral infectivity. Viral titer in plaque forming units (pfu) was determined by plaque assay using 293 cells. An analytical HPLC method was used to determine viral particle/ml measurements by peak area of an optical density (OD) at 260nm.
• OD optical density
• Viral titer was determined by the plaque assay on 293 cells, human embryonic kidney cells transformed with sheared human Ad5 DNA (complement El region). At designated time points, triplicate test samples and triplicate positive controls were tested for plaque forming units as described above in Example 1.
• HPLC Analysis HPLC peak area comparisons were performed to determine the virus concentration in terms of viral particles between samples and positive controls. Each triplicate sample and positive control was tested on a Waters analytical HPLC system with Millennium software as described above in Example 1.
• Plaque Assay Results The results of the plaque assays for adenovirus AVI .0HSVTK stability at -70°C, -20°C, +4°C, and +20°C are shown in Figure 1 1.
• the viral activity of AV1.0HSVTK was measured after 0 (2 days), 1.5, 3.5, and 8.5 months of storage using the plaque forming assay as described in Example 1.
• Both the +4°C and -20°C storage temperature samples maintained similar viral activities as compared to the -70°C positive control at all tested time points ( Figure 11).
• HPLC peak area provides for the quantitation of viral particles.
• the HPLC assay variation has been determined to be ⁇ 3%. Due to the small standard deviations in the HPLC peak areas, any small variation between time points may be statistically significant.
• the HPLC peak area was plotted by temperature over time for all four storage temperatures. Analysis by HPLC of particle integrity was performed following storage of the test and control samples for 0 (2 days), 1.5, 3.5, and 8.5 months. The results demonstrate that all storage conditions of -20°C, +4°C, and +20°C preserved viral particles as well as the -70°C condition over the course of 8.5 months (data not shown).
• the HPLC results showed a similar trend in stability as the plaque assay results for each storage temperature.
• Formulation 19 preserved adenoviral titer for at least 8.5 months when stored at -70°C, -20°C, +4°C, or +20°C.
• the HPLC result did not suggest a loss of activity in the room temperature condition at the 8.5 month time point as demonstrated by the plaque assay results (Figure 11).
• the results of this study clearly demonstrate that Formulation 19 (lOmM Tris-HCL, pH 8.4, 5%
• HSA 5% sucrose, 2.0mM MgCl 2 , and 150mM NaCl
• the plaque assay ( Figure 11) and HPLC data support this conclusion.
• the results from both particle integrity tests and activity titration show that viral stability is preserved up to at least 8.5 months under frozen (-20°C) and (+4°C) refrigerated conditions.
• viral activity, but not particle integrity was reduced when storage was performed in Formulation 19 at room temperature. Therefore, Formulation 19 comprising 5% human serum albumin and 5% sucrose at a pH of 8.4 efficiently preserves and stabilizes adenoviral particle integrity and viral activity for a period of at least 8.5 months at refrigerated conditions.

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