AU748523B2 - Adenovirus formulations for gene therapy - Google Patents

Description

WO 00/09675 PCT/US99/18515 ADENOVIRUS FORMULATIONS FOR GENE THERAPY This application claims the benefit of co-pending provisional application 60/096,600, which was filed August 14, 1998, the disclosure of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION 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.

BACKGROUND OF THE INVENTION In cell and gene therapy, as well as in blood transfusion or bone marrow transplantation, one of the principal problems encountered is that of the preservation of biological material. It is indeed important to be able to preserve the biological material, under good conditions of viability, for a sufficiently long period of time compatible with industrial scale production and storage and also to make it possible to carry out certain tests. The most commonly used method of preservation consists of freezing the material. However, during the freezing and thawing of biological material, loss of viability and/or infectivity may occur.

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, 1977). "Albumin structure, function and uses", Rosenoer, V.M. et al. Pergamon Press, Oxford, pp. 27-51). 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 nucleotides, 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 WO 00/09675 PCT/US99/18515 2 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. Furthermore, 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, and R.K. Gupta, 1996. Pharm. Sci., 85:129-132; Niemeijer, N. et al., 1996. Ann.

Allergy Asthma Immunol., 76:535-540; and Cannon, J.B. et al., 1995. PDA:J. Pharm. Sci. Tech., 49:77-82.) 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.

Preservation of adenovirus for clinical use has become a significant issue as clinical trials progress to phase II and, ultimately, regulatory approval. Presently, adenovirus formulations, such as formulation 1 (Example 1, infra), require storage at -70C to remain stable. The requirement to keep viral vector preparations at these temperatures necessitates acquisition by clinicians of freezers that maintain -70 0 C temperatures. Another complication arises during shipment of the vectors from the manufacturing site to the clinic. Until the present invention, there was little hope and even lower expectation that adenovirus vectors could be formulated for stable storage, with preservation of infectivity, at standard freezer (-20 0 refrigerator or room (20°C) temperatures.

As demonstrated in the Examples, infra, 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 citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

SUMMARY OF THE INVENTION 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. Thus, in a first embodiment, the invention provides a composition comprising a recombinant adenovirus vector and a WO 00/09675 PCT/US99/18515 3 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. In a specific embodiment, the serum albumin is human serum albumin (HSA). In a specific embodiment, the concentration of HSA is from about 0.01% to about 25% (wlv). Preferably, the concentration of HSA is from about 0.1% to about 15%. More preferably, the concentration of HSA is from about 1% to about Most preferably, the concentration of HSA is about 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.

In a further embodiment of the invention, the pH of the composition is greater than or equal to and less than or equal to 9.0. Preferably, the pH is greater than 7.5. Thus, the pH can be 7.6, 7.7, 7.8, or 7.9. More preferably, the pH is greater than 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, or 8.9. In a specific embodiment, the pH is 8.2. In another specific embodiment, the pH is 8.4. Preferably, when the pH of a composition of the invention is greater than 8.0, the concentration of HSA is 1% to 10%; more preferably In a specific embodiment, when the pH is 8.2, the concentration of HSA is In another specific embodiment, when the pH is 8.4, the concentration of HSA is The aqueous buffer of the invention comprises a physiological buffer, such as, but by no means, limited to phosphate buffer, Tris-HC1 buffer, Hepes buffer, and the like. A suitable buffer for use according to the invention is pharmaceutically acceptable, compatible with in vivo delivery. In an embodiment exemplified infra, the buffer is a Tris-HCI buffer. The buffer is set at a pH that stabilized adenovirus vectors. An aqueous buffer for use in the invention comprises salts, calcium chloride (CaCI 2 magnesium chloride (MgCI 2 and sodium chloride (NaCI). For example, the buffer may contain about 2.0 mM MgCI, and 150 mM NaCI. In a specific embodiment, the aqueous buffer contains a physiological concentration of salt.

In addition, a composition or formulation of the invention may contain additional components in addition to HSA to further stabilize the recombinant adenovirus. Examples of such components include, but are not limited to, carbohydrates and sugars, such as dextrose, sucrose, glucose, and the like, at a concentration; medium to long chain polyols, such as glycerol, polyethylene glycol, and the like, e.g., at 10% concentration; other proteins; amino acids; nucleic acids; chelators; proteolysis inhibitors; WO 00/09675 PCT/US99/18515 4 preservatives; and other components. Preferably, any constituent of a composition of the invention is pharmaceutically acceptable.

The compositions of the invention are particularly suitable for formulation of a recombinant adenovirus for gene therapy. Thus, in a preferred embodiment, the recombinant adenovirus expresses a heterologous protein. Examples of 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-4, glial-derived neurotrophic factor (GDNF), and ciliary neurotrophic factor (CNTF); etc. A more complete list of heterologous proteins for expression in a vector formulated in accordance with the present invention can be found infra. In a specific embodiment, the heterologous protein is p53. In another specific embodiment, the heterologous protein is HSV-TK.

Naturally, the present invention further provides a method for preparing a recombinant adenovirus vector formulation comprising preparing an admixture of a recombinant adenovirus 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. In one embodiment, the temperature is greater than or equal to 4 0 C and less than 37 0 C. In a further embodiment, the temperature is greater than or equal to 20 0 C. Preferably, when the temperature is greater than 4 0 C, and particularly when the temperature is greater than 20 0 C, the concentration of HSA is the pH of the admixture is greater than 8.0, or both.

In a further aspect, the present invention provides a method for stabilizing an adenovirus vector at about 20 0 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 CaCI 2 and from about 0.1 to 1.0 mM MgCI,. In a specific embodiment, the concentration of glycerol is 10%, the concentration of CaCI 2 is about 1.0 mM, and the concentration of MgCI, is about 0.5 mM.

The present invention will be further explained in the Drawings and Detailed Description.

DESCRIPTION OF THE DRAWINGS Figure 1. Plaque Assay +4 0 C Storage of 4 Formulations.

Figure 2. Plaque Assay +20 0 C Storage of 4 Formulations.

Figure 3. Bioactivity Assay +4 0 C Bioactivity for 4 Formulations.

Figure 4. Bioactivity Assay +20 0 C Bioactivity for 4 Formulations.

Figure 5. Effect of HSA and Sucrose on Viral Titers at -20 0

C.

Figure 6. Effect of Buffer, Salt and/or Cations on Viral Titers at +2-10 0

C.

WO 00/09675 PCT/US99/18515 Figure 7. Effect of HSA on Viral Titer at +2-10 0

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 0 C for 1 week in different formulations.

Figure 11. Stability of AV .OHSVTK Stored in Formulation 19 at -70 0 C, -20 0 C, +4 0 C, and +20°C for 3.5, and 8.5 months.

DETAILED DESCRIPTION OF THE INVENTION 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.

In specific examples, infra, various formulations were tested for their ability to stabilize adenovirus vector preparations for up to 8.5 months, relative to a control stored at -70 0 C. It was found that a formulation comprising 10 mM Tris-HC1, pH 8.2, 5% HSA, 5% sucrose, 2.0 mM MgCl, 150 mM NaCI was very stable, and preserved or even enhanced vector infectivity for up to 6 months at relatively high temperatures (4 0 C and 20 0 Additional experiments established that an optimum pH for adenovirus vector stability was above pH 8.0. It was further found that the presence of HSA in the solution increased the viral titer (as measured in a plaque assay). In addition, a second formulation comprising 10mM Tris-HCL, pH 8.4, 5% HSA, 5% sucrose, 2.0mM MgCI 2 150mM NaCI was tested for its ability to stabilize adenovirus vector preparations for at least 8.5 months, relative to a control stored at 0 C. It was found that this 10mM Tris-HC1, pH 8.4, 5% HSA, 5% sucrose, 2.0mM MgCl 2 150mM NaCI formulation was also very stable, preserving viral particle integrity and infectivity (titer) for at least months at +4 0 C and preserving viral particle integrity for at least 8.5 months at +20 0 C. Thus, the critical variables for a formulation to preserve adenovirus vectors were HSA and pH; sucrose was also found to enhance stability.

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. This formulation lacks any toxic agent and may be administered directly to an organism.

It may be used for preserving various virus preparations such as viral particles and viral vectors.

As noted above, 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 WO 00/09675 PCT/US99/18515 6 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. Preferably, 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. Further, as noted above, 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. In a specific embodiment, the pH is 8.2 and the concentration of HSA is In a second specific embodiment, the pH is 8.4 and the concentration of HSA is The various aspects of the invention will be set forth in greater detail in the following sections, directed to suitable media and formulations for preserving viral particles and viral vectors. This organization into various sections is intended to facilitate understanding of the invention, and is in no way intended to be limiting thereof.

Definitions The following defined terms are used throughout the present specification, and should be helpful in understanding the scope and practice of the present invention.

In a specific embodiment, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.

The term "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. Thus, the term "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 nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acids.

"Human serum albumin" or "HSA" refers to a non-glycosylated monomeric protein of 585 WO 00/09675 PCT/US99/18515 7 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, 1977). "Albumin structure, function and uses", Rosenoer, V.M. et al. Pergamon Press, Oxford, pp. 27-51). 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 nucleotides, from the supposed "capping" site up to the first site of addition of poly(A).

The phrase "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. Preferably, as used herein, the term "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, 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 plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, 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. In an expression vector, the coding region is operably associated with an expression control sequences, 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.).

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). Although the present invention is described primarily in the context of human serum albumin, it has been found infra that serum albumin from other WO 00/09675 PCT/US99/1 8515 8 species is equally effective. Thus, the term HSA should be considered to encompass any serum albumin, such as bovine serum albumin (BSA) and murine serum albumin (MSA). Naturally, for delivery of a formulation in vivo for gene therapy, it is preferable to use an autologous serum albumin. Thus, for human gene therapy, human serum albumin is desirable and preferred.

A recombinant or natural HSA is advantageously used which meets certain quality criteria homogenetic, purity, stability). Thus, 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). The use of an albumin corresponding to these criteria, although not essential, is particularly preferred.

Advantageously, the compositions according to the invention comprise a purified human plasma albumin or a recombinant human albumin, preferably produced in a eukaryotic host. In addition, the term 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- terminal fragments of HSA.

Purification of Natural 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 or by extraction from the human placenta, according to the technique described by J. Liautraud et al. (1973, 13Y" International lABS Conference, Budapest; A: "Purification of proteins. Development of biological standard", Karger Bale, 27:107 pp). Preferably, the purified albumin used within the framework of the present invention is a plasma albumin. Most particularly, a commercial plasma albumin may be used.

Recombinant Production of HSA. The development of genetic engineering and of new extraction and purification techniques has opened the possibility of obtaining, at a lower cost price, improved products of higher purity, of greater stability and without risk of contamination (for example, hepatitis B, hepatitis C, HIV, or infectious prions). Given the importance of the HSA market, the possibility of producing this protein by a recombinant route has been widely studied. Thus, numerous expression systems have been studied for the preparation of the recombinant HSA.

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 WO 00/09675 PCT/US99/18515 9 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. Thus, it has been possible to demonstrate the production of HSA under the control of the chelatin promoter in Saccharomyces cerevisiae (Etcheverry et al., 1986.

Bio/Technology, 4:726). The production of HSA has also been mentioned in the brewery yeast during the manufacture of beer, using a post-fermentative process (EP 201 239). Most recently, patent application EP 361 991 describes a particularly efficient system using the yeast Kluyveromyces as a host organism, transformed with vectors derived from the plasmid pKDI. Particularly high levels of HSA secreted into the culture medium were able to be obtained with this system. Finally, the production of recombinant HSA has also been described in Pichia pastoris, (EP 344 450). In addition, the purification of HSA has also been described (EP 319 067).

Various patents and scientific publications describe methods for expressing a heterologous gene, particularly HSA, in a transgenic animal, optimally in the mammary gland of a ruminant mammal. Such technology is useful for producing a heterologous protein in the milk of the mammal. Examples of production of human serum albumin in transgenic animals include US Patent No. 5,780,009, issued July 14, 1998 to Karatzas et al., directed to direct gene transfer into the ruminant mammary gland. US Patent No. 4,873,316, issued October 10, 1989 to Meade et al., directed to isolation of exogenous recombinant proteins from the milk of transgenic mammals, provides an expression system comprising the mammal's casein promoter, which when transgenically incorporated into a mammal permits the female of that mammal species to produce the desired recombinant protein in or along with its milk. A preferred construct for transgenic expression of HSA is described in US Patent No. 5,648,243, issued July 15, 1997 to Hurwitz et al., directed to a human serum albumin expression construct, the disclosure of which is incorporated herein by reference in its entirety. As disclosed in this patent, efficient expression of HSA is achieved when the human serum albumin sequence comprises at least one, but not all, of the introns in the naturally occurring gene encoding for the HSA protein; preferably the DNA constructs comprise a regulatory sequence which directs the expression and secretion of HSA protein in the milk of a transgenic animal. These patents refer to additional references from the scientific and patent literature for transgenic expression, particularly of HSA. Each of them is incorporated herein by reference it its entirety.

In a specific embodiment of the invention, 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. Recombumin T M is produced using a hybrid secretion leader sequence conjugated to a cDNA which encodes human serum albumin. The cDNA WO 00/09675 PCT/US99/18515 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).

HSA Formulations 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). Preferably, 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 respective proportions of the components of the media according to the invention may be adapted by persons skilled in the art according to the viral particle or viral vector considered. As illustrated in the Examples, although certain concentration ranges are preferred, the proportions may be modified.

The formulation according to the invention was discovered in connection with preservation of adenovirus vectors. However, this formulation may very well be useful for preserving other viral vectors.

Viral Particles and Vectors 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. Among these 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. However, 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 WO 00/09675 PCT/US99/18515 adenoviruses (Ad 2 or Ad 5) or adenoviruses of animal origin (see W094/26914). Those 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. Preferably, the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus Manhattan or A26/61 strain (ATCC VR-800), for example).

Preferably, 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 nucleotides 455 to 3329 in the sequence of the Ad5 adenovirus (PvuII-BglII fragment) or 382 to 3446 (Hinfll-Sau3A fragment). Other regions may also be modified, in particular the E3 region (W095/02697), the E2 region (W094/28938), the E4 region (W094/28152, W094/12649 and W095/02697), or in any of the late genes In a preferred embodiment, the adenoviral vector has a deletion in the El region (Ad 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 Examples of El/E4-deleted adenoviruses are disclosed in W095/02697 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 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 integrated into its genome, and cell lines which are able to complement the El and E4 functions, as described in applications W094/26914 and W095/02697. Recombinant adenoviruses are recovered and purified using standard molecular biological techniques, which are well known to one of ordinary skill in the art.

WO 00/09675 PCT/US99/18515 12 Other Vectors Other Viral Vectors. The adeno-associated viruses (AAV) 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, morphology or differentiation, and they do not appear to be involved in human pathologies. The AAV genome has been cloned, sequenced and characterized. It encompasses approximately 4700 bases and contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as an origin of replication for the virus. The remainder of the genome is divided into two essential regions which carry the encapsidation functions: the left-hand part of the genome, which contains the rep gene involved in viral replication and expression of the viral genes; and the right-hand part of the genome, which contains the cap gene encoding the capsid proteins of the virus.

The use of vectors derived from the AAVs for transferring genes in vitro and in vivo has been described (see WO 91/18088; WO 93/09239; US 4,797,368, US 5,139,941, EP 488 528). These publications describe various AAV-derived constructs in which the rep and/or cap genes are deleted and replaced by a gene of interest, and the use of these constructs for transferring the said gene of interest in vitro (into cultured cells) or in vivo, (directly into an organism). 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). The AAV recombinants which are produced are then purified by standard techniques.

In another embodiment the gene can be introduced in a retroviral vector, 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; McCormick, BioTechnology 3 (1985) 689; International Patent Publication No. WO 95/07358, published March 16, 1995, by Dougherty et al.; and Kuo et al., 1993, Blood 82:845. 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). In recombinant retroviral vectors, the gag, pol and env genes are generally deleted, in whole or in part, and replaced with a heterologous nucleic acid sequence of interest. These 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 W095/02697.

WO 00/09675 PCT/US99/18515 13 Regulatory Regions. Expression of a polypeptide from a vector of the invention may be controlled by any regulatory region, 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 EIA, MLP, CMV and RSV genes and the like.

In addition, 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). Moreover, when the nucleic acid does not contain promoter sequences, it may be inserted, such as into the virus genome downstream of such a sequence.

Some promoters useful for practice of this invention are ubiquitous promoters HPRT, vimentin, actin, tubulin), intermediate filament promoters desmin, neurofilaments, keratin, GFAP), therapeutic gene promoters MDR type, CFTR, factor VIII), tissue-specific promoters actin promoter in smooth muscle cells), promoters which are preferentially activated in dividing cells, promoters which respond to a stimulus steroid hormone receptor, retinoic acid receptor), tetracycline-regulated transcriptional modulators, cytomegalovirus immediate-early, retroviral LTR, metallothionein, SV-40, Ela, 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 incorporated herein by reference.

Thus, 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 herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42); 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. 80:21-25); see also "Useful proteins from WO 00/09675 PCT/US99/18515 14 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 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. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436- 1444), 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.

1:161-171), beta-globin gene control region which is active in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94), myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712), myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).

Therapeutic Genes. Examples of heterologous proteins expressed by vectors 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-4, glial-derived neurotrophic factor (GDNF), and ciliary neurotrophic factor (CNTF); etc. In a specific embodiment, the heterologous protein is p53. In a second specific embodiment, the heterologous protein is HSV-TK.

Naturally, the present invention further provides a method for preparing a recombinant adenovirus formulation comprising preparing an admixture of a recombinant adenovirus and a concentration of HSA effective to stabilize the adenovirus vector 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. In one embodiment, the temperature is greater than or equal to 4 0 C and less than 37 0 C. In a further embodiment, the temperature is greater than or equal to 20 0 C. Preferably, when the temperature is greater than 4 0 C, and particularly when the temperature is greater than 20 0 C, the WO 00/09675 PCT/US99/18515 concentration of HSA is the pH of the admixture is greater than 8.0, or both.

In a further aspect, 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 CaCI 2 and from about 0.1 to 1.0 mM MgCI,. In a specific embodiment, the concentration of glycerol is the concentration of CaCl2 is about 1.0 mM, and the concentration of MgCI, 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.

To this end, 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. As regards viruses, these are previously purified as described herein by centrifugation on a cesium chloride gradient, column chromatography, plaque purification, and the like). They may be packaged at the rate of 10' to 10' 5 particles per ml, preferably 10' to 10'0, or more preferably, 109-1013. 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. In a preferred embodiment, 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. The term "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. These may be in particular isotonic, sterile, saline solutions WO 00/09675 PCT/US99/18515 16 (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or 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 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.

The phrase "therapeutically 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 percent, and most preferably prevent, a clinically significant deficit in the activity, function and response of the subject. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in the subject.

Administration of Compositions. According to the invention, the composition of the invention may be introduced parenterally or transmucosally, orally, nasally, or rectally, or transdermally.

Preferably, administration is parenteral, via intravenous injection, and also including, but is not limited to, intra-arteriole, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. For gene therapy of a cancer, 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. Alternatively, if a tumor target is characterized by a particular antigen, a vector of the invention can be targeted to the antigen as described above, and administered systemically or subsystemically, as appropriate, 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. In general, the recombinant adenoviruses according to the invention are formulated and administered in the form of doses of between and 10 1 4 pfu, and preferably 10' to 10" 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, WO 00/09675 PCT/US99/18515 17 generally within 15 days, the number of plaques of infected cells. For p53 adenovirus and HSV-TK adenovirus, typically the number of plaques are counted at day 7. For other viruses, evaluation is made at day 12-14. The technique for determining the pfu titre of a viral solution are well documented in the literature.

Thus, the compositions of the invention can be delivered by intravenous, intraarterial, intraperitoneal, intramuscular, pulmonary, or subcutaneous routes of administration. Alternatively, 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 therapeutically effective dose a dose effective to induce metabolic changes in a subject) at the necessary intervals, daily, weekly, monthly, etc. 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. Thus, as can be readily appreciated by one of ordinary skill in the art, 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., 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.

EXAMPLES

General Molecular Biolov In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, Sambrook, Fritsch Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II Glover ed. 1985); Oligonucleotide Synthesis Gait ed. 1984); Nucleic Acid Hybridization Hames S.J. Higgins eds. (1985)]; Transcription And Translation Hamies S.J. Higgins, eds. (1984)]; Animal Cell Culture Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL WO 00/09675 PCT/US99/18515 18 Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley Sons, Inc. (1994).

EXAMPLE 1: Stability Testing of Adenovirus Formulations at Above Freezing Temperatures.

Initially, the adenovirus preparations were stored at -70 0 C in buffered glycerol solutions to provide the best long term stability. Since -70 0 C storage may not be available in the clinical setting, a formulation which will preserve the virus at -20 0 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 year stability when stored frozen at -20 0 C, or as a liquid at +4 0 C or +20 0

C

storage temperatures. A positive control consisted of the virus stored in Dulbecco's phosphate buffered saline (DPBS), 10% glycerol, 1.0mM CaCI, and 0.5mM MgCI 2 at -70 0 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. 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. 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.

Methods 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 tm filtered, in 10 ml vials was used in these experiments.

The virus titer was estimated at 1.38x10" pfu/ml and 3.44xl01 2 particles/ml. On the first day of the study, 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. Thirty ml of the pool were then run through a Sephadex G25 column (200ml; Pharmacia Catalog No. 17-0033-01, lot 241586) for buffer exchange into each of the formulations. Buffer exchange was performed to place the virus into appropriate formulations for testing. Four Formulations were tested and are defined in Table 1. Only 0.5% HSA was present in Formulations 2, 3 and 4 at the time of Table 1: Test Formulations.

Form 1: DPBS, 10% glycerol, 1.0mM CaCI, and 0.5mM MgCI 2 Form 2: DPBS, 5% HSA, 5% Sucrose, 1.0mM CaCI, and 0.5mM MgCI 2 Form 3: 10mM Tris-HCL, pH 7.5, 5% HSA, 5% Sucrose, 2.0mM MgCI 2 150mM NaCI Form 4: 10mM Tris-HCL, pH 8.2, 5% HSA, 5% Sucrose, 2.0mM MgCI1 ,150mM NaCI WO 00/09675 PCT/US99/18515 19 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. After the final dilution, all samples were stored overnight at +4 0 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 0 C (VWR Scientific freezer), -4°C (LabLine Ambi HiLo Chamber), +4 0 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 0 C storage were frozen to -40 0 C in a controlled rate freezer prior to storage at -20 0 C. The temperatures of -4 0 C, +20 and +37 were used as elevated temperatures with which to determine the "accelerated" stability of the virus formulations at -20 0

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, 1.0mM CaCI, and MgCI2 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 0 C (BioFreezer, Forma Scientific Model No.

8328).

Formulation Analysis: All four Formulations were analyzed after 0, 1, 2, 3, 4.5, and 6 months of storage at different temperatures with respect to several parameters as described in Table 2. However, all temperatures were not tested at each time point due to previous results which indicated that further analysis was not required.

Plaque Assay Analysis: 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 0 C, 5.0% CO, 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, WO 00/09675 PCT/US99/18515 MEM, 7.5% FBS overlay. The dishes were then incubated at 37 0 C, 5.0% CO, and Table 2: Summary of 6 Month Formulation Analysis.

Month Plaque Assay HPLC OD by UV Bioactivity Assay 0 Yes Yes Yes No 1 Yes Yes Yes No 2 Yes Yes Yes Yes 3 Yes Yes Yes Yes Yes Yes Yes No 6 Yes Yes Yes Yes relative humidity. After 5 days of incubation, the plates were stained with 0.1% neutral red in MEM overnight at 37 0 C. The next day, the stain was aspirated off and the plaques were counted. 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.

HPLCAnalysis: 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 tl of test sample and positive control were injected into a Resource Q Iml, 6.4x30 mm column (Pharmacia Catalog No. 17-1177-01), using a flow rate at 1 ml/minute. The samples are stored at 2-8 0

C

in the Waters autosampler during the entire run. The running gradient consisted of line A: Buffer A Tris, pH line B: Buffer B (50mM Tris 1 M NaCI, pH 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.

Optical Density by UVAnalysis: Viral particle per ml concentrations were determined for the positive control and Formulation 1 samples which do not contain HSA. HSA containing formulations were not tested for particles by OD because 5% HSA interfered with the reading (data not shown).

Briefly, 50pl of 1% SDS and 50pl of sample were incubated for 20 minutes at room temperature, then diluted 1:10 with deionized water. Samples were read at 260 nm and 280 nm. The ratio of 260/280 was calculated to determine the purity. The OD 26 0 extinction coefficient of I 1012 particles/ml was used to calculate the viral particles/ml of the test sample and positive control. Data were plotted and analyzed in WO 00/09675 PCT/US99/18515 21 Microsoft EXCEL.

Bioactivity Assay Analysis: 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. In this test, 3 x 103 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 0 C for 4 days and then stained with alamar blue, an indicator of cell growth, and incubated for 8 hours. The plates with alamar blue were then read at 570 nm and 595 nm to determine OD. Optical density was calculated using the following equation: OD Absorbance 7 Absorbance 5 9 The inhibition of proliferation was plotted as a percent reduction of alamar blue in Microsoft EXCEL for further analysis.

StatisticalAnalysis: Mean and standard deviations were made for triplicate samples.

Results Plaque Assay Results: The results of the plaque assays for adenovirus stability at 4 0 C and 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 0 C, Formulation 4 demonstrated increased stability at all time points (Figures I and Surprisingly, at -4 0 C, Formulation 4 demonstrated the same pfu activity as the other formulations (data not shown). At 37 0 C, all of the formulations were unstable (data not shown).

Storage at +4 0 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 0

C,

however it was reduced significantly in pfu/ml by greater than 1 log at month 3 (Figure Formulation I did not perform as well as Formulation 4 when stored at +4 0 C at all tested time points (Figure 1).

Formulations 2 and 3 showed an initial decrease in titer following 1 month storage at +4 0 C (Figure 1).

At +4 0 C, Formulation 4 remained essentially unchanged up to 3 months and showed activity loss starting at 4.5 month (Figure 1).

Storage at +20 0 C also permitted the determination of the stability differences between formulations. Formulations 1 and 4 showed the best retention of viral activity at +20 0 C for 6 months (Figure 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. Starting at one month, Formulations 2 and 3 showed a reduction in titer when stored at 0 C (Figure 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.

WO 00/09675 PCT/US99/18515 22 Table 3: Viral Titer Summary: Plaque Assay Results Log T=0 T=6M -70 0 C T=6M -20 0 C T= 6M +4 0 C T= 6M Delta: -20 0

C

pfu/ml pfu/ml Log pfu/ml Log pfu/ml Log pfu/ml Log 4.6E+10 3.9E+11 0.93 Control Form 1 5.5E+10 3.2E+11 0.76 1.4E+07 -3.59 7.0E+08 -1.90 Form 2 1.5E+12 2.6E+12 0.24 9.4E+04 -7.20 5.9E+05 -6.41 Form 3 1.0E+12 5.6E+12 0.75 2.4E+07 -4.62 2.3E+06 -5.64 Form 4 1.6E+12 4.5E+12 0.45 1.2E+09 -3.12 7.5E+11 -0.33 Note 1: Log is calculated by (Log T=6 pfu/ml) (Log T=0 pfu/ml). A result loss, a result gain.

Note 2: The samples stored at -4C and +37C samples lost more than 4 logs of activities at T= 1 month and were discontinued in the testing scheme.

Storage at -4 0 C and at +37 0 C for one month showed a drastic loss in activity for all four Formulations (data not shown). The decreased activity of the -4 0 C and +37 0 C samples were supported by results of HPLC and OD analyses. All samples stored at -4 0 C were liquid, not frozen. This may have been due to the large amounts of excipients in the formulations preventing complete freezing at -4 0

C.

Therefore, samples stored at -4 0 C may have been in a transition phase between liquid and solid where ice crystals may have caused damage. In addition, the -4 0 C samples cannot be used for accelerated shelf life calculations for -20 0 C frozen storage because they were not frozen. The -4 0 C and +37 0 C samples were discontinued at month 1.

In summary, the 6 month viral titer data indicated that Formulation 4 represents a viral formulation that is preferable for preserving viral stability and activity (infectivity), especially at +20 0

C.

HPLCResults: HPLC peak area provides for the quantitation of viral particles. The HPLC assay variation has been determined to be 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.

At time 0, all four Formulations have similar viral particles/ml. Although the peak area values showed a statistically significant variation, the difference between Formulations at time point 0 was less than This low percent variation indicates that a large variation in aliquoting and dilutions did not occur among the four formulations.

At -20 0 C, all four formulations were relatively unchanged over time (data not shown).

At +4C, all four formulations except Formulation 4 showed significant loss in viral particles WO 00/09675 PCT/US99/18515 23 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 +37C, all four Formulations showed a drastic decrease in HPLC peak area at 1 month (data not shown).

In summary, 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 0 C or +37 0

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.

The 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. At -20 0 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).

At Formulations 1, 2 and 3 did not perform as well with respect to preserving bioactivity as Formulation 4 for months 2 and 3 (Figure The data demonstrated that Formulation 4 retained bioactivity similar to the positive control at both 2 and 3 months, and lost the activity at 6-month. These data correlated well with the plaque assay and HPLC results presented above.

At +20'C, as in the plaque assay results, both Formulations 1 and 4 retained bioactivity similar to the positive control out to month 3, and started to show activity loss at 6-months (Figure 4).

Formulation 4 still gave the best bioactivity among all formulations. Formulations 2 and 3 did not perform well following 2 or 3 months storage (Figure 4).

Discussion The results of this study indicated that all four formulations analyzed did not significantly decrease viral titer (pfu/ml) over a 3 month period when stored at -20 0 C. The bioactivity data support this conclusion. All four formulations proved stable at -20 0 C for 6 months.

Formulations 1, 2 and 3 decreased viral titer by more than 2 logs when stored at +4 0 C and, by more than 53% when measured by HPLC peak area. At +4°C storage, Formulation 4 was the most stable over the 3 month storage period, as evidenced by the plaque assay and HPLC peak area measurements of WO 00/09675 PCT/US99/18515 24 particle/ml which were reduced by only 14.8%. These conclusions were supported by the bioactivity assay results. Thus, Formulation 4 proved most stable at +4 0 C for 3 months.

The viral titer for Formulations 1 and 4 remained stable, within assay variation, for 3 months when stored at +20 0 C, but the titer of Formulation 1 decreased by 2 logs and the titer of Formulation 4 decreased by 0.3 logs between 3-6 months. Viral particle measurement by HPLC peak area demonstrated a reduction of only 11.1% for Formulation 1 and 23.8% for Formulation 4 by month 3, but for Formulation 1 and 8% for Formulation 4 at 6 months. Virus stored in Formulations 2 and 3 were significantly reduced in viral titer: by more than 2 logs at month 3. The number of viral particles per ml, as measured by HPLC, decreased by more than 30%. The bioactivity decrease correlated with these conclusions. Thus, Formulation 4 proved most stable at +20 0 C for 6 months.

All four formulations were unstable when stored at resulting in a reduction in viral titer that was greater than 4 logs at one month, and greater than 71% loss in particles when measured by HPLC peak area. In addition, storage of all four formulations at 37°C for 1 month resulted in decreased viral titer that was greater than 5 logs, and a loss of more than 85% of particles when measured by HPLC peak area. Thus, none of the formulations analyzed proved stable at -4 0 C or +37 0

C.

According to this study, Formulation 4, 10mM Tris buffer at pH 8.2, 5% HSA, 5% sucrose, 2mM MgCI 2 and 150 mM NaCI, 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 0 C and +20 0 C. These data also suggest that the original formulation of DPBS, 10% glycerol, 1.0mM CaCI, and 0.5mM MgCI 2 (Formulation retained most of its activity for 3 months at -20 0 C and +20 0 C. Over time, Formulation 1, resulted in a decline in both viral titer and particles/ml when stored at +4 0 C. Formulation 4 started to show the loss of activity and viral particles after 3 months.

EXAMPLE 2: -20 0 C Screen of Formulations to Preserve Viral Titers.

These experiments screen several formulations for their ability to preserve virus activity as measured by plaque assay when stored at -20 0 C. These experiments tested various buffers and excipients for their effects on virus titer at -20 0 C. With regard to the type of buffer, salt concentration, presence of divalent cations, and length of time at -20 0 C (1 day versus 9 days), there were no differences observed in virus titer by the plaque assay. However, the addition of sucrose and/or human serum albumin (HSA) demonstrated an unexpected increase in the virus infectivity compared to the control.

WO 00/09675 PCT/US99/18515 Methods Preparation of 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. Additional buffer excipients included 50mM, 150 mM, or 300mM NaCI, divalent cations (ImM CaCI, and 0.5mM MgCl), 5% sucrose, and or 10% HSA. The samples were aliquoted and frozen in vials using the controlled rate freezer, and transferred to a -20 0 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.

Preparation of Control Virus Samples: Ad5CMVp53 used in the test formulation preparations above was diluted into the standard buffer (DPBS, 0.5 mM MgCl 2 1 mM CaCI 2 and 10% glycerol, pH and treated identically to the test samples as described above.

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.

Statistics: The data was transferred to Systat version 5 software for statistical analysis. Linear regression and t-tests were performed to determine differences between groups, using an alpha value of p=0.05.

WO 00/09675 PCT/US99/18515 Table 4. Test Formulations.

Form. ID buffer [NaCI] cations other 1 PBS 50 mM yes 10% glycerol 2 PBS 50 mM no 10% glycerol 3 PBS 150 mM yes 10% glycerol 4 PBS 150 mM no 10% glycerol PBS 300 mM yes 10% glycerol 6 PBS 300 mM no 10% glycerol 7 Tris 50 mM yes 10% glycerol 8 Tris 50 mM no 10% glycerol 9 Tris 150 mM yes 10% glycerol Tris 150 mM no 10% glycerol 11 Tris 300 mM yes 10% glycerol 12 Tris 300 mM no 10% glycerol 13 PBS 150 mM yes 5% sucrose, 1% HSA 14 PBS 150 mM yes 1%HSA PBS 150 mM yes 5% sucrose, 5% HSA 16 PBS 150 mM yes 5% HSA 17 PBS 150 mM yes 5% sucrose,

HSA

18 PBS 150 mM yes 10% HSA Results and Discussion Effect of Buffer, Salt and Divalent Cations on Viral Titer at -20 0 C: 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 0 C for Formulations 1-12 are shown in Table 5. The samples from the frost-free freezer were used for day 1 testing.

It is apparent from the data that there are no great differences to be seen. In fact, the overall confidence of variation (CV (SD/mean)x00%) for each day obtained by combining all formulations is about the same as that for replicates within an experiment for results of intra-assay variation.

A multiple regression analysis was performed for viral titer (pfu/ml) with respect to buffer, WO 00/09675 PCT/US99/18515 Table 5. Plaque Assay Results (pfu/ml) at -20 0

C.

Formulation Day 1 Day 9 1 4.40 x10 9 4.62 xl0 9 2 4.34 x10 9 3.88 xl0 9 3 2.56 xl0 9 3.44 xl0 9 4 3.06 x10 9 4.50 xl0 9 4.62 x10 9 3.94 xl0 9 6 3.86 x10 9 3.56 xl0 9 7 3.62 xl0 9 3.90 x10 9 8 4.58 x10 9 3.62 xl0 9 9 5.12 x10 9 4.04 x10 9 3.78 x10 9 5.06 xl0 9 11 4.60 xl0 9 2.86 xl0 9 12 3.56 xl0 9 3.02 x10 9 mean 4.01 x10 9 3.87 xl0 9 SD 7.37 xl0 8 6.39 CV 18% 17% salt concentration, presence of divalent cations, and 1 versus 9 days of-20 0 C exposure, but no significant differences were found (data not shown). With respect to the PBS and Tris buffers, no difference was found 0.784). There was no relationship for the effect of salt concentration, from 50 mM to 150 mM (p=0.

3 22) and the presence of divalent cations had no effect (p=0.801). In addition, the length of storage time at -20 0 C, 1 day versus 9 days, also showed no difference (p=0.643).

Effect of HSA and Sucrose at -20 0 C: Formulations 13-18 were analyzed to determine the effects of the addition of HSA and sucrose to PBS on viral titer. The results of the day 9 plaque assay are shown in Table 6. The combined effects of HSA and sucrose on viral titer are shown in Figure There was an increase in the viral titer with the addition of HSA alone and with HSA sucrose.

When the HSA sucrose Formulation was compared to the control, a significant difference was seen A significant difference was also seen for all samples with HSA as compared to the control (p=0.004). The best effects in this single data point experiment were observed with 5% HSA, at either or 0% sucrose. Good results were also found for 5% sucrose, 1% HSA.

WO 00/09675 PCT/US99/18515 28 Table 6. The Effect of HSA and Sucrose on Viral Titers at -20 0

C.

Form. ID sucrose HSA pfu/ml 13 5 1 6.10x10 9 14 0 1 3.76 xl0 9 5 5 6.66 xl0 9 16 0 5 6.46 xl0 9 17 5 10 5.44 xl0 9 18 0 10 5.92 x10 9 EXAMPLE 3: Formulation Screening for +2-10°C storage.

For future clinical studies, it will be preferred to store virus at temperatures of-20 0 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 0 C. As described in Example 1 and as determined within this Example, Formulations with HSA preserved all of the virus activity when stored at +2-10C, 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 0

C.

Methods Preparation of Virus Sample Formulations: 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.

Statistics: For this experiment, the plaque values from only one dilution (3.3x10 8 were used for analysis, as plaque counts were low, and only this dilution consistently produced plaque numbers in the desired range. The data was transferred to Systat version 5 software for statistical analysis. Linear regression and t-tests were performed to determine differences between groups, using an alpha value of p= 0 0 5 Results and Discussion Effects of Storage at +2-10 0 C: The results of the plaque assay after 14 days of storage at +2- 0 C are shown in Figure 6. The values in Figure 6 were taken from a single assay dilution for each sample. No error bars are shown as there were no replicates for each condition.

WO 00/09675 PCT/US99/18515 29 As shown in Figure 6, there were distinct groups of virus activity. The viral titers of Formulations 1-10 were fairly consistent, while Formulations 11 and 12 (which contained Tris buffer with 300 mM NaCI) were considerably lower. Formulations 13-18, which contained HSA, showed increased viral activity compared to those without HSA.

Effects of HSA: The effects of HSA can be seen more clearly in Figure 7. The "no protein" group comprised a sample size of n=8 formulations and the HSA group comprised a sample size on n=6 formulations. Those formulations with 300mM salt were not included in this comparison. The error bar equals 1 Standard deviation.

As demonstrated above, the addition of 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. Three formulations (#14,16,18) tested increasing HSA concentrations: and These three results are all within the intra-assay variation, so the optimum HSA concentration varies over a broad range.

Effects of Divalent Cations: Samples with less than 300mM NaCI and no added HSA were analyzed for the effect of divalent cations on viral titer after storage at +2-10 0 C for 14 days. Although there are few replicates, when the samples with divalent cations were compared to those without cations, a non-significant difference was observed. For the limited number of samples tested at +2-10 0 C (n=4 each group), the addition of divalent cations appeared to result in the loss of virus activity (p=0.09; data not shown). This is an unexpected finding, as many literature sources call for the use of divalent cations in adenovirus formulations (Huyghe et al., Human Gene Therapy 6: 1403-1416, 1995).

Comparison to -20 0 C Reference Formulation Samples: The results of Example 2, in which identical Formulation samples were stored at -20 0 C for a similar length of time may be used for this comparison. These are compared in Table 7. In general, samples without HSA (Formulations 1-12) showed a loss of viral titers when stored at +4°C compared to storage at -20 0 C. In contrast, those Formulations with HSA (13-18) showed no loss of viral activity at +4 0 C. In addition, Formulation samples containing divalent cations had reduced viral titers after storage at +4 0 C (27% of those kept at -20 0 than those without cations 10) (66% of those stored at -20 0

C).

When the adenovirus formulations were stored at +2-10°C for 14 days, the choice of buffer (Tris or PBS) did not have an effect on viral titers, although high NaCI (300mM) concentrations adversely affected virus titer.

WO 00/09675 WO 0009675PCTIUS99/18515 Table 7. Comparison of Formulations 1- 18 at -20'C and +4'C.

form. pfu/m1I: 4'C pfulml: -20"C ratio 4*/-20'C 1 2.09 x 10 9 4.62 x 10 9 2 1.97 x10 9 3.88 x10 9 51% 3 1.00 x10 9 3.44 x10 9 29% 4 2.3 x10 9 4.5 x10 9 51% 4.55 x10 8 3.94 x10 9 12% 6 1.82 x10 9 3.56 x10 9 51% 7 6.06 x10' 3.9 x10 9 16% 8 2.52 x10 9 3.62 x10 9 69% 9 1.42 x 10 9 4.04 x 10 9 5.52 x 10 9 5.06 x 10 9 109% 11 9.09 X10 7 2.86 x10 9 3% 12 6.06 X10 7 3.02 x10 9 2% 13 9.55 x10 9 6.1 x10 9 156% 14 9.15 x10 9 3.76 x10 9 243% 7.03 x10 9 6.66 x10 9 106% 16 6.76 x10 9 6.46 x10 9 105% 17 8.7 x10 9 5.44 x10 9 160% 18 6.09 x10 9 5.92 x10 9 103% EXAMPLE 4: Ad5CMVp53 Formulation Screening: jpH study.

The above Examples with Ad5CMVp53 formulations were all performed at physiological pH, except Formulation 4 shown in Example 1. The experiments within this Example were performed as a short-term study to examine the effects of pH on virus activity over a wide range at -40'C, +201C, and +37*C. The addition of other excipients was also analyzed for protective effects against extremes of pH.

For buffered salt solutions at the extremes of pH 5.0 and pH 9.0, there was a significant loss of virus activity. Viral stability was enhanced at pH values between 6 and about 8, with optimum stability around pH 8.2. In the presence of HSA sucrose, this effect was negated. In addition, as observed previously, the presence of HSA sucrose appeared to increase the infectivity of the virus in the plaque assay.

WO 00/09675 PCT/US99/18515 31 Methods 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 0 C and thawed on the day of assay.

Formulation Preparation: In the first experiment, a number of 10mM Tris-buffered solutions with the same salt (150mM NaCI) and divalent cation concentrations (2mM MgCI 2 were prepared and adjusted to specific pH values, ranging from pH 5.0 to pH 9.0. Another set was made which contained 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 0 C varies from 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. In a second experiment, virus was diluted into Formulation 4 (see Table and the pH was adjusted to specific pH values, ranging from pH 6.6 to pH 8.8. These virus formulations were then stored at either room temperature (RT 20 0 C) or at 37°C for 1 week prior to activity determination by plaque assay. The viral plaque assays were performed as described in Example 1.

Statistics. Plaque assay data from each of two dilutions and two replicates were combined.

Means, standard deviations and CV were calculated. A Student's t-test was performed using Excel Results and Discussion In the first experiment, two replicates of each pH, ranging from pH 5.0 to pH 9.0, were tested in the plaque assay, following short-term storage at -40 0 C and 1 hour incubation at 37°C. A second set of formulations with HSA sucrose added was also tested in this experiment. The standard deviation from the mean was averaged from two dilutions of two replicates. In the first group without HSA sucrose, the upper and lower extremes ofpH resulted in reduced viral titers. The pH 5.0 and pH 9.0 formulation samples were significantly decreased from the mean of the control group: control, pH 6.0, pH 7.0, and pH 8.0 (p<0.001 and p=0.008, respectively) (data not shown). When HSA sucrose were present at the various pH levels, no effects ofpH were seen (data not shown). In addition, the mean of the group with HSA sucrose was significantly increased as compared to the mean of the control group without those excipients (p<0.001). Thus, the presence of HSA sucrose protected viral titers from extremes of pH and appeared to enhance the infectivity of the virus in the assay as seen in previous studies (Examples 1- Therefore, for buffered salt formulations, the optimum pH range for Ad5CMVp53 is pH 6.0-pH WO 00/09675 PCTIUS99/18515 32 The addition of HSA sucrose is able to protect the virus from pH values above and below this range.

In a second experiment, virus was prepared in Formulation 4 at each specific pH value, ranging from pH 6.6 to pH 8.8, stored for I week at room temperature or 37 0 C, and tested in the plaque assay.

The results are shown in Figure 8.

In general, the formulations with pH from 8.0 to 8.6 maintained the highest levelof activity following storage at room temperature for one week, with the virus sample at pH 8.2 retaining the most activity (Figure The "too numerous to count" (tntc) data designation indicates that the plaque counts for these samples were above the upper limit for counting. Therefore, a value of 300 plaques, which is the limit for counting, was assigned to these "tntc" samples and used to calculate the titer reported in Figure 8. The real titer is slightly higher than this assigned value. Following storage at 37 0 C for one week, Formulation 4 having a pH value of pH 8.0, pH 8.4, and pH 8.6 resulted in the best preservation of virus stability (Figure Taken together, the stability data obtained from the room temperature and 37°C short term pH study indicates that Formulation 4, with an adjusted pH value, ranging from pH to pH 8.6 is the most preferable formulation to preserve virus activity (infectivity).

EXAMPLE 5: Bovine serum albumin and recombinant human albumin provide similar effect as natural

HSA.

The purpose of the studies within this Example was to determine whether albumin from other sources can provide the same stability and viral titer enhancement observed with HSA in Example 2.

These studies were also performed to investigate the possibility that the other components in the commercial HSA (0.02M acetyltryptophane and 0.02M sodium caprylate; Miles, Inc.) contribute to stability and titer enhancement in HSA-based viral formulations. In these studies bovine serum albumin (BSA, reagent grade material, powder form) and recombinant human albumin (rHA) were tested in different studies under different conditions. The recombinant human albumin used is RecombuminTM which is produced in a yeast expression system by Centeon and is described supra.

Methods Virus Sample Preparation: Ad5CMVp53, as used in other Examples, was also used for these experiments. For both the BSA and the RecombuminTM studies, 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 MgCI, 1.0mM CaCI 2 HSA Formulation: DPBS HSA 0.5mM MgC 2 1.0mM CaC 2 I; and BSA Formulation: DPBS 5% BSA 0.5mM MgCI, WO 00/09675 PCT/US99/18515 33 CaCI 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. One vial was stored at RT 0 C) for 2 hours and the other vial was frozen to -40 0 C in a rate controlled freezer then thawed and stored at +37 0 C for 2 hours. Viral plaque testing was performed as described in Example 1.

In the Recombumin T M study, three formulations were prepared: Control Formulation: DPBS glycerol; HSA Formulation: 10mM Tris 5% HSA 5% sucrose 150 mM NaCI MgCI 2 pH 8.2; and RecombuminTM Formulation: 10mM Tris 5% RecombuminTM 5% sucrose 150 mM NaCI 2.0mM MgCIl, pH 8.2. The virus stock was diluted 1:100 into each formulation and divided into 3 vials each and stored at +37 0 C for 7 days. Viral plaque testing was performed as described in Example 1.

Results and Discussion The results of the BSA study showed that the BSA Formulation enhanced viral titer, as observed for the HSA Formulation, when compared to the viral titer of the Control Formulation (Figure In addition, similar results were observed when murine serum albumin was used in the storage formulation instead of BSA or HSA (data not shown). The data also indicated that HSA, BSA, and the Control Formulations appear to have a protective effect on the virus undergoing a freeze-thaw and up to 2 hour storage at +37 0 C (Figure 9).

The results of the RecombuminTM study demonstrated that the RecombuminTM and HSA Formulations provide similar protective effects on viral titer for viruses stored at +37 0 C for 1 week (Figure 10). This protective effect was not observed with the Control Formulation in which a viral titer decrease of 4 logs was observed (Figure The results of this Example demonstrate that formulations containing albumin from other sources (bovine and recombinant human) can provide the same stability and viral titer enhancement as that observed with the natural HSA Formulation. These studies also indicate that the other components in the commercial HSA (0.02M acetyltryptophane and 0.02M sodium caprylate) do not contribute to the stability and titer enhancement in HSA-based viral formulations.

EXAMPLE 6: Long Term Stability of Adenovirus in an HSA/Sucrose Formulation at -70 0 C, -20 0

C,

+4 0 C, and The results of 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 purpose of this Example was to determine the stability of adenoviral vectors in a 10mM Tris-HCI 5% HSA 5% sucrose 150mM NaCI 2mM MgCl 2 pH 8.4 WO 00/09675 PCT/US99/18515 34 formulation when stored at various temperatures up to 8.5 months. An adenoviral vector comprising the herpes simplex virus thymidine kinase gene (AV 1.0HSVTK) was used in this Example.

This Example summarizes the efficiency of a formulation comprising 10mM Tris-HCI HSA 5% sucrose 150mM NaCI 2mM MgCI,, 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 year stability when stored frozen at -20 0 C, or as a liquid at +4 0 C or +20 0 C storage temperatures.

Methods Virus Sample Preparation: A type 5 adenovirus comprising the HSV-TK gene under the control of the cytomegalovirus promoter, AVI .OHSVTK (as described in French Patent Application No.

FR93/13772), in DPBS 10% glycerol, 0.2 pm filtered, was used in these experiments. The virus titer was estimated at 1.6 x 1012 particles/mi. On the first day of the study, 22 vials of AVI.OHSVTK, each vial comprising about 200-250 pl/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/mi.

The AV I.OHSVTK virus pooled stocks were run through a Resource Q column (prepared with 8ml of Source Q 15; Pharmacia Catalog No. 17-0947-01, lot 11/21/97 for buffer exchange and reformulated in 10mM Tris-HCl, 5% HSA, 5% sucrose, 150mM NaCI, 2mM MgCI,, 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 0 C (VWR Scientific freezer), +4 0 C (VWR Scientific 2-door deli case Model GDM-49), room temperature (+20 0 and -70 0 C (control group). Triplicate vials were tested for particle integrity by HPLC and activity by plaque assay at specified time points of up to 8.5 months.

After the final dilution and filling, all samples were stored at each desired temperature for 2 days and then tested as T=0 time point to give a baseline information. The samples for -20 0 C storage were frozen to -40 0 C in a controlled rate freezer prior to storage at -20 0 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 0 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 0 C, and +20 0 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.

WO 00/09675 PCT/US99/18515 Positive Control Virus Preparation: The control virus was diluted in 10mM Tris-HCI, HSA, 5% sucrose, 150mM NaCI, 2mM MgCI,, 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 0 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 0 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.

Plaque Assay Analysis: 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.

HPLCAnalysis: 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.

Results and Discussion Plaque Assay Results: The results of the plaque assays for adenovirus AV1.OHSVTK stability at -70 0 C, -20 0 C, +4 0 C, and +20 0 C are shown in Figure 11. The viral activity of AV .OHSVTK 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 I. Both the +4°C and -20 0 C storage temperature samples maintained similar viral activities as compared to the -70 0 C positive control at all tested time points (Figure 11). The results showed a significant drop (p=0.02) of approximately 2 logs for the room temperature condition. All other storage conditions preserved viral activity to an amount within the error limit of the assay logs).

HPLC Results: HPLC peak area provides for the quantitation of viral particles. The HPLC assay variation has been determined to be 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 months. The results demonstrate that all storage conditions of-20 0 C, +4 0 C, and +20 0 C preserved viral particles as well as the -70 0 C condition over the course of 8.5 months (data not shown).

In summary, the HPLC results showed a similar trend in stability as the plaque assay results for 36 each storage temperature. Formulation 19 preserved adenoviral titer for at least months when stored at -70 0 C, -20 0 C, +4 0 C, or +20 0 C. Interestingly, the HPLC result did not suggest a loss of activity in the room temperature condition at the month time point as demonstrated by the plaque assay results (Figure 11).

The results of this study clearly demonstrate that Formulation 19 Tris-HCL, pH 8.4, 5% HSA, 5% sucrose, 2.0mM MgCI 2 and 150mM NaCI) is effective for stably preserving viral activity and particle integrity at all four storage temperatures analyzed over an 8.5 month time period. 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 (-200C) and (+4 0 C) refrigerated conditions.

However, 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 15 preserves and stabilizes adenoviral particle integrity and viral activity for a period of at least 8.5 months at refrigerated conditions.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art 20 from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

oo..

It is furthe" to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description.

Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.

The term "comprises/comprising" when used in this specification is taken to specify the presence of stated features, integers, steps or components but does not preclude the -presence or addition of one or more other features, integers, steps, components or groups thereof.

Claims (27)

1. A composition 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.

2. The composition of claim 1, wherein the concentration of HSA is from about 0.01% to about

3. The composition of claim 2, wherein the concentration of HSA is from about 0.1% to about

4. The composition of claim 3, wherein the concentration of HSA is from about 1% to about The composition of claim 4, wherein the concentration of HSA is about

6. The composition of claim 1, wherein the pH is greater than or equal to 5.0 and less than or equal to

7. The composition of claim 6, wherein the pH is greater than

8. The composition of claim 7, wherein the pH is greater than

9. The composition of claim 8, wherein the pH is 8.2. The composition of claim 8, wherein the pH is 8.4.

11. The composition of claim 4, wherein the pH is greater than

12. The composition of claim 5, wherein the pH is 8.2.

13. The composition of claim 5, wherein the pH is 8.4.

14. The composition of claim 1, wherein the buffer is a Tris-HCI buffer. The composition of claim 11, wherein the buffer is a Tris-HCI buffer.

16. The composition of claim 12, wherein the buffer is a Tris-HCI buffer. WO 00/09675 PCT/US99/18515 38

17. The composition of claim 13, wherein the buffer is a Tris-HCI buffer.

18. The composition of claim 1, further comprising about 5% sucrose, about 2.0 mM MgCI 2 and about 150 mM NaCI.

19. The composition of claim 15, further comprising about 5% sucrose, about 2.0 mM MgCI, and about 150 mM NaCI. The composition of claim 16, further comprising about 5% sucrose, about 2.0 mM MgCI, and 150 mM NaCI.

21. The composition of claim 17, further comprising about 5% sucrose, about 2.0 mM MgCI 2 and 150 mM NaCI.

22. The composition of claim 1, wherein the recombinant adenovirus expresses a heterologous protein.

23. The composition of claim 22, wherein the heterologous protein is p53.

24. The composition of claim 22, wherein the heterologous protein is HSV-TK. A method for preparing a stabilized recombinant adenovirus formulation comprising suspending a recombinant adenovirus in an aqueous buffer comprising a concentration of human serum albumin (HSA) effective to stabilize the adenovirus vector 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.

26. The method according to claim 25, wherein the temperature is greater than or equal to 4 0 C and less than 37 0 C.

27. The method according to claim 25, wherein the temperature is greater than or equal to 20 0 C.

28. The method according to claim 26, wherein the concentration of HSA is

29. The method according to claim 26, wherein the pH of the admixture is greater than The method according to claim 26, wherein the pH of the admixture is 8.2.

31. The method according to claim 26, wherein the pH of the admixture is 8.4.

32. A method for stabilizing an adenovirus vector at about 20 0 C, which comprises preparing an WO 00/09675 PCT/US99/18515 39 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 CaClI, and from about 0.1 to 1.0 mM MgCI,.

33. The method according to claim 32, wherein the concentration of glycerol is about 10%, the concentration of CaCl2 is about 1.0 mM, and the concentration of MgCI, is about 0.5 mM.