+

WO2003033029A1 - Replication competent dual-ad vectors for vaccine and immunotherapy applications - Google Patents

Replication competent dual-ad vectors for vaccine and immunotherapy applications Download PDF

Info

Publication number
WO2003033029A1
WO2003033029A1 PCT/US2002/033162 US0233162W WO03033029A1 WO 2003033029 A1 WO2003033029 A1 WO 2003033029A1 US 0233162 W US0233162 W US 0233162W WO 03033029 A1 WO03033029 A1 WO 03033029A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
vector
vectors
cell
replication
Prior art date
Application number
PCT/US2002/033162
Other languages
French (fr)
Inventor
Sybille Sauter
Xiangming Fang
Amena Rahman
Nathan P. Englund
Scott Gallichan
Mark I. Fitchmun
Robert E. Sobol
Original Assignee
Magnum Therapeutics, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Magnum Therapeutics, Inc. filed Critical Magnum Therapeutics, Inc.
Priority to JP2003535831A priority Critical patent/JP2005511534A/en
Priority to CA 2463996 priority patent/CA2463996A1/en
Priority to EP02770603A priority patent/EP1450862A4/en
Publication of WO2003033029A1 publication Critical patent/WO2003033029A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/235—Adenoviridae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10345—Special targeting system for viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/38—Vector systems having a special element relevant for transcription being a stuffer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron

Definitions

  • This invention is related to adenoviral (Ad) vectors and their use in the fields of vaccine and immunotherapy applications. More specifically, this invention is related to complementary Ad vectors that comprise and express exogenous sequences and yet retain the ability to replicate when introduced into an appropriate host organism or cell. Also contemplated are prophylactic and therapeutic formulations/compositions comprising the Ad vectors as well as prophylactic and therapeutic treatments using the Ad vectors.
  • Ad adenoviral
  • Adenoviruses consist of nonenveloped icosahedral (20 facets and 12 vertices) protein capsids with a diameter of 60-90 nm and inner DNA/protein cores (Horwitz, M.S. (1990) "Adenovindae and their replication.” In: Fundamental Virology (2nd Ed.) Field, B.N. ⁇ t al., Eds., pp. 771-813, Raven Press: NY, NY).
  • the outer capsid is composed of 252 capsomers arranged geometrically to form 240 hexons (12 hexons per facet) and 12 penton bases; the latter are located at each vertex from which protrude the antenna-like fibers. This structure is responsible for attachment of Ad to cells during infection. Wild-type Ad contain 87% protein and 13% DNA and have a density of 1.34 g/ml in CsCI.
  • the double-stranded linear DNA genome of Ad is approximately 36 kb, and is conventionally divided into 100 map units (mu). Each end of the viral genome has a 100-150 bp repeated DNA sequence, called the inverted terminal repeats (ITR). The left end (194-385 bp) contains the signal for encapsidation (packaging signal). Both the ITRs and the packaging signal are c/s-acting elements necessary for adenoviral DNA replication and packaging (Sussenbach, J.S. (1984) "The structure of the genome.” In: The Adenoviruses, Ginsberg, H.S. Ed., pp. 35-124, Plenum Publishing Corp.: NY, NY; Philipson, L. (1984) "Adenovirus assembly.” In: The Adenoviruses, Ginsberg, H.S., Ed., pp. 309-337, Plenum Publishing Corp.: NY, NY).
  • FIG. 1 A simplified map of the adenovirus type 5 (Ad5) genome with a few key landmarks is diagrammed in Figure 1 (Stratford-Perricaudet, L. & Perricaudet, M. (1991) "Gene transfer into animals: the promise of adenovirus.” In: Human Gene Transfer, Cohen-Haguenauer, O. & Boiron, M., Eds., p. 51-61, John Libbey Eurotext: France.; Graham, F.L., & Prevec, L. (1991) "Manipulation of adenovirus vectors.” In: Methods in Molecular Biology (Vol.
  • E1A and E1B encodes proteins responsible for the regulation of transcription of the viral genome as well as a few cellular genes (Nevins, J.R. (1990) "Adenovirus E1A-dependent trans-activation of transcription.” Semin. Cancer Biol., vol. 1 , no. 1, pp. 59-68; Nevins, J.R. (1993) "Transcriptional activation by the adenovirus E1A proteins.” Semin. Virol., vol.
  • E2A and E2B The expression of the E2 region leads to the synthesis of the proteins needed for viral DNA replication (Pettersson, U. & Roberts, R.J. (1986) "Adenovirus gene expression and replication: a historical review.” In: Cancer Cells (Vol. 4): DNA Tumor Viruses (Botchan, M. et al., Eds., pp. 37-57, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY).
  • the proteins from the E3 region prevent cytolysis by cytotoxic T cells and tumor necrosis factor (Wold, W.S.M. & Gooding, L.R.
  • Ad vectors for expression of heterologous genes began soon after the observation of hybrids between Ad and simian virus 40 (SV40) during the 1960s. Since then, Ad vectors have gradually developed into one of the major viral vectors in the current field of gene therapy, because: (a) Ad have been widely studied and well characterized as a model system for eukaryotic gene regulation, which served as a solid base for vector development; (b) the vectors are easy to generate and manipulate; (c) Ad exhibits a broad host range in vitro and in vivo with high infectivity, including non-dividing cells; (d) Ad particles are relatively stable and can be obtained in high titers (e.g., 10 10 -10 12 plaque-forming unit (PFU)/ml); (e) the life cycle of adenovirus does not require integration into the host cell genome, and, therefore, the foreign genes delivered by Ad vectors are expressed episomally, thus having low genotoxicity if applied in vivo; (f) side effects have not been reported following vaccination
  • Ad vectors have been successfully used in eukaryotic gene expression (Levrero, M. et al. (1991 ) "Defective and nondefective adenovirus vectors for expressing foreign genes in vitro and in vivo.” Gene, vol. 101 , pp. 195-202; Ghosh-Choudhury, G. et al. (1986) "Human adenovirus cloning vectors based on infectious bacterial plasmids.” Gene, vol. 50, pp. 161-171), vaccine development (Grunhaus, A. & Horwitz, M.S.
  • a typical approach to vaccine therapy involves alteration of the infectious agent-host relationship and facilitation of recognition and destruction of infectious agents by the host immune system.
  • cytokine genes have been isolated, cloned and characterized. Significant toxicity has accompanied the use of many of these biologies owing to the high concentrations needed to generate clinical effects.
  • the combination of significant undesired effects and marginal therapeutic outcomes from systemic administration has stimulated efforts to genetically engineer target cells to produce the cytokines themselves (Rosenberg, et al. (1989) "Experience with the use of high-dose interleukin-2 in the treatment of 652 cancer patients.” Ann. Surg., vol. 210, pp. 474-484).
  • Antigens expressed on infectious cells in combination with high local concentrations of cytokine(s), may be exploited to create an immunological micro-environment virtually impossible to reproduce with exogenous cytokine administration.
  • This immunological micro-environment created by such cytokine-producing target cells may result in generation of cytotoxic T lymphocytes.
  • cytokine-producing target cells have been shown to be effective in decreasing the destructive potential of particular target cells and increasing the expression of immunologically important molecules (Miller et al. (1994) supra; Dranoff, G. & Mulligan, R.C. (1995) supra.
  • the initial target cell rejection appears to be accompanied by a nonspecific inflammatory response.
  • rejection of cytokine-secreting target cells has in most instances led to the generation of systemic, target cell-specific immunity that is T cell-dependent.
  • Class I MHC has been shown to activate target cell-specific CTL in vitro.
  • Early work on target cell vaccination included transfection of MHC class I genes and resulted in suppression of the target cells (Hui, K. et al.
  • MHC class II genes were shown to be involved in activation of target cell-specific T-helper cells, and the introduction of Class II genes into target cells resulted in a decrease in the disease potential of the target cell and generated a systemic immune response against the target cell (Ostrand-Rosenberg, S. et al. (1990) "Rejection of mouse sarcoma cells after transfection of MHC class II gene.” J. Immunol., vol. 144, pp. 4068-4071 ).
  • Interferon gamma is a pleiotropic cytokine that, for example, activates macrophages and plays an important role in the inflammatory response (Billiau, A. (1996) "Interferon- ⁇ : biology and role in pathogenesis.” Adv. Immunol., vol. 62, pp. 61-131).
  • This pleiotropic cytokine is also a potent inducer of MHC class I and class II antigens and thus is capable of enhancing immune responses (Wallach, D. et al. (1982) "Preferential effect of interferon- ⁇ on the synthesis of HLA-antigens and their mRNAs in human cells.” Nature, vol. 299. pp.
  • the cDNA for human IFN- ⁇ has also been introduced into human target cells (Gansbacher, B. et al. (1992) "Retroviral gene transfer induced constitutive expression of interleukin-2 or interferon- ⁇ in irradiated human melanoma cells.” Blood, vol. 80, pp. 2817-2825; Gastl, G. et al. (1992) "Retroviral vector-mediated lymphokine gene transfer into human renal cancer cells.” Cancer Res., vol. 52, pp. 6229-6236).
  • Such cells secreting IFN- ⁇ showed increased expression of MHC class I antigen, ⁇ 2-microglobulin, and intracellular adhesion molecule I, as well as induction of MHC class II antigen expression.
  • neoplasia formation by a human target cell line transplanted into nu/nu mice was not affected by IFN- ⁇ secretion, whereas IL-2 production inhibited growth of the neoplasia.
  • adenoviruses and adenovirus-based vectors make attractive candidates for the basis upon which a variety of vaccines may be made (Fooks, A.R. (2000) "Development of oral vaccines for human use.” Curr. Opin. Mol. Ther., vol. 2, no. 1 , pp. 80-86; Babiuk, L.A. & Tikoo, S.K. (2000) "Adenoviruses as vectors for delivering vaccines to mucosal surfaces.” J. Biotechnol, vol. 83, nos. 1-2, pp. 105-113; Hofling, K. et al.
  • U.S. Patents Nos. 5,643,579 and 4,920,209 describe oral vaccines using recombinant adenovirus coding for heterologous antigens of other infectious organisms.
  • the vaccines disclosed therein are contained within enteric-coated formulations so that the adenovirus infects the intestinal tract and expresses the heterologous antigen as well as the adenovirus antigens.
  • the invention disclosed herein improves upon the adenovirus vaccines known in the art by providing a complementary dual-Ad system as the basis for vaccines.
  • a complementary dual-Ad system as the basis for vaccines.
  • Such a system has a larger gene delivery capacity permitting the incorporation of multiple antigen and immune stimulatory DNA beyond other viral systems.
  • Such a system allows the separation of functions into the component parts and yet remains fully functional upon introduction into a patient or subject in need of treatment.
  • the present invention provides novel vectors, compositions and methods for treating disease or other conditions.
  • the invention provides a composition.
  • first and second adenoviral vectors are provided having complementary function and being mutually dependent on each other for replication in a host organism or target cell.
  • one of said adenoviral vectors comprises a promoter that regulates expression of an early gene that controls propagation of the adenoviral vector in a host organism or target cell.
  • one of said adenoviral vectors comprises a host organism or target cell activated promoter and limits propagation of the adenoviral vectors to specific host organisms or target cells.
  • another adenoviral vector of the composition comprises a partial Ad genome which can support the Ad replication cycle in the host organism or target cell.
  • the replication of these vectors in the host organism or target cell directly or indirectly induces one or more immune response(s) that may alter the growth, function or other activity of the host organism, target cells, pathogens and/or infectious agents.
  • the host organism, target cells, pathogens and/or infectious agents may be involved in a disease state, including, but not limited to, infection, cancer, autoimmune disease, cardiovascular disorder or other condition(s) known to those skilled in the art.
  • the invention is related to the invention described in the following published International Applications (and to any applications from which the
  • Ad5 genome and transcription units The length Ad5 genome is about 36 kb, divided into 100 map units (mu).
  • the dotted arrows represent early (E) transcription and the solid arrows represent late (L) transcription.
  • the directions of transcription are indicated by arrows. Gaps between arrows indicate intervening sequences.
  • the box represents location of the major later promoter and tripartite leader sequences (MLP).
  • MLP tripartite leader sequences
  • the solid triangle at 1 mu represents the location of the packaging signal.
  • Figure 2. Principle of a complementary-Ad vector system. Shown are two major components of the system: the supplemental-Ad, and the controlled-Ad vector. With the E1-transactivation from the helper cell by activation of the specific promoter/enhancer carried by the controlled-Ad, the supplemental-Ad replicates itself and produces the late proteins to form capsids. However, in the embodiment shown in this figure, packaging of the supplemental-Ad genome into the capsid is inefficient due to the packaging attenuation associated with the supplemental-Ad.
  • the supplemental-Ad In the presence of the controlled-Ad vector genome, the supplemental-Ad also supports the DNA replication of the controlled-Ad vector genome, which is preferentially packaged due to its wild-type packaging signal that has high affinity to the limiting amount of the packaging proteins. Further purification of the Ad vectors can be achieved by a biochemical or physical method, such as ultracentrifugation.
  • FIG. 3 Prototype supplemental-Ad and controlled-Ad vectors. Shown is the general structure of the helper virus and the controlled-Ad vectors. The latter have up to 36-kb gene delivery capacity that can be quipped with single or multiple gene expression cassettes.
  • FIG. 4 Basic composition of an complementary dual-Ad vector system.
  • the basic elements of the system are the supplemental-Ad vector and the controlled-Ad vector.
  • the supplemental-Ad is similar to the E1 -substituted first generation Ad and, in this embodiment, has a partial deletion of its packaging signal. The E1 region is replaced by a reporter gene and, in this embodiment, lacZ gene for ⁇ -gal protein.
  • the controlled-Ad containing only the minimal cis- element of Ad genome (two ITRs with the packaging signal), carries a promoter/enhancer to drive the Ad E1 gene (Ad5E1 ). The promoter/enhancer is activated specifically in target cell, such as a cancerous or transformed cell.
  • the AdE1 gene under the control of this element is specifically transcribed in tumor cells.
  • the E1 protein then trans-activates the genome of the supplemental virus that has the E1 region substituted by a reporter gene and also has a manipulated packaging signal such that packaging of the controlled-Ad is preferred in the cell.
  • the supplemental virus, activated by the E1 proteins is able to replicate in the tumor cells, resulting in lysis of those cells.
  • the critical aspect of the variation is that the two vectors complement each other in Ad genome function such as E1 genes and/or other early genes.
  • the complementation elements of the two vectors are listed in the Table (C).
  • the transgene and supporting elements were mainly included in the controlled-Ad vectors, but this does not exclude the requirement for the supplemental-Ad for those elements in certain circumstances.
  • Deletion of an early Ad gene that encodes the cell cycling inducer will abrogate the replicative capability of adenovirus in the normal resting cells, but allow the controlled-Ad specifically drive replication and propagation of the complementary-Ad vectors in tumor cells that have defect in the counterpart of the deleted viral gene product.
  • Two examples are the viral proteins E1b-p55 and E1 b that are dispensible for virus propagation in p53-deleted (or mutated) or retinoblastoma-deleted (or mutated) tumor cells, respectively.
  • FIG. 6 Methods for generation of the complementary-Ad vector system.
  • two different complementation protocols may be used that give similar yields.
  • the controlled-Ad plasmid is co-transfected with vDNA from AdH ⁇ , and the helper cells are cultured until CPE is observed.
  • AdH ⁇ is added as supplemental virus, and the cells are cultured until CPE is observed.
  • Controlled-Ad are generated by co-transfection of two plasmids in a helper cell line:
  • the controlled-Ad plasmid may contain one or more Ad genes regulated by a cell-activated promoter/enhancer, the supplemental-Ad plasmid may contain the remainder of the Ad genes, and the helper cell line is a tumor- derived cell line in which the cell-activated promoter of the controlled-Ad is functional.
  • the controlled-Ad and the supplemental-Ad complement each other in the supplemental cell line and propagate as a virus mixture.
  • the vector mixture can be purified through CsCI gradients and injected locally or systemically into the tumor mass or the tumor bed following surgical debulking.
  • the cell-activated promoter/enhancer specifically transcribes the AdE1 genes in target cells to produce E1A and E1 B proteins that transactivate transcription and replication of the supplemental-Ad.
  • the controlled-Ad vector also replicates with the propagation of the supplemental-Ad.
  • the replication of the controlled-Ad produces high copy numbers of the controlled-Ad genome, which supports high level expression of the immunomodulatory genes to induce specific anti-cancer immunity.
  • Propagation of the supplemental-Ad in the target cells results in lysis of the target cells. This system may generate a local target cell destructive effect and a systemic target cell destructive response that results in rejection of distant target cells.
  • FIG 8. A MaxAd vector comprising 4 (four) expression cassettes. Shown is a schematic view of GTV8053, an exemplary, non-limiting vector according to the present invention.
  • Figure 9. Dual Ad replication in permissive cells. Shown is representative dual Ad replication in 13 cell types, including LNCaP cells, A549 cells, Chang cells, Saos-2 cells, H358 cells, Huh-7 cells, DU145 cells, SKHep-1 cells, 2237 cells, U2OS cells, 293 cells, Hep-2 cells, and HepG2 cells.
  • FIG. 1 Interferon-gamma (IFNgamma or IFN ⁇ ) expression in PSA Dual Ad infected cells.
  • FIG. 12 Serum IgG antibody levels to GFP following MaxAd/GFP immunization of mice. Mice were immunized twice by the intranasal (i.n.) or intraperitoneal (i.p.) route (5 x 10 9 vp) and the serum examined for IgG antibodies to GFP. Control animals were immunized intranasally with a MaxAd virus expressing FVIII protein.
  • FIG 13 Induction of IgG and IgA antibodies in the genital tract of MaxAd/GFP immunized mice. Mice were immunized twice by the intranasal (i.n.) or intraperitoneal (i.p.) route (5 x 10 9 vp) and genital tracts washes were taken 3 weeks later and examined for IgA and IgG antibodies to GFP. Control animals were immunized intranasally with a MaxAd virus expressing FVIII protein (5 x 10 9 vp).
  • FIG. 14 Lysis of GFP expressing and control cells by splenocytes from individual mice immunized intranasally or intraperitoneally twice with MaxAd/GFP (5 x 10 9 vp).
  • FIG. 15 Anti-HIV p24 (capsid) Western blot of 293 cells transfected with HIV-1 DEN(3) or the safety-modified HIV-1 immunogen in pcDNA3.1.
  • the primary antibody is a 1 :2,000 dil. mouse anti-p24 antibody (Cell Sciences) followed by the 1 :10,000 dil. secondary anti-mouse HRP- labeled antibody (Jackson ImmunoResearch).
  • the present invention provides reagents and methodologies that substantially improve current vaccine and immunotherapy technologies and applications useful for the treatment of infectious diseases and other conditions.
  • the present invention provides mutually-dependent complementary, replicable adenoviral (Ad) vector system useful for the treatment of infectious diseases and other conditions.
  • the system comprises a "controlled Ad” vector and a "supplemental Ad” vector.
  • the controlled-Ad has minimal Ad c/s-elements (inverted terminal repeats (ITR) and a packaging signal) and a transcriptional regulatory sequence that a target cell is capable of activating or is activated ubiquitously through a generalized, "house-keeping" gene promoter.
  • the transcriptional regulatory region may be a cell- or tissue-specific transcriptional regulatory region driving expression of the Ad E1 genes.
  • the transcriptional regulatory region may be a ubiquitous, general "house- keeping" transcriptional regulatory region driving expression of the Ad E1 genes.
  • the controlled-Ad vector further comprises an expression cassette providing for expression of a gene of interest such as an immunomodulatory gene or an Ad early-region gene.
  • the supplemental Ad may comprise a packaging signal, a deletion of the E1 region, and the remainder of the Ad genome with or without substitution.
  • Ad vectors thus produced using this system comprise a pair of recombinant adenoviruses that are mutually dependent, and are termed "complementary Ad vectors.” It is also possible that a composition comprising more than one type of complementary Ad with a single type of supplemental Ad vector may be utilized in practicing the present invention.
  • the system is useful for the treatment of conditions in which particular infectious agents or target cells are known to be involved.
  • the vectors upon local or systemic injection to treat an infectious agent or target cell population, the vectors will replicate in the host organism or target cell and express an effector protein, such as an antigen, immunogen, epitope or immunomodulatory protein, resulting in a local and/or systemic prophylactic and/or therapeutic effect and induction of a local and/or systemic prophylactic and/or therapeutic immune response.
  • an effector protein such as an antigen, immunogen, epitope or immunomodulatory protein
  • the complementary Ad system provides Ad vectors that replicate locally and/or systemically, such as within a host organism or in the direct vicinity of an infectious agent or target cell (e.g., including, but not limited to, dendritic cells) capable of utilizing the E1 expression cassette of the controlled Ad vector.
  • an infectious agent or target cell e.g., including, but not limited to, dendritic cells
  • a "nucleic acid molecule” is defined a plasmid, virus, autonomously replicating sequence, phage or linear segment of a single- or double-stranded DNA or RNA derived from any source.
  • a "transcriptional regulatory region” may comprise a promoter, enhancer, silencer or repressor element and is functionally associated with a nucleic acid of the present invention. Unless otherwise stated, the transcriptional regulatory region may alternatively be referred to as a promoter or an enhancer. Preferably, the transcriptional regulatory region drives high level gene expression in the target cell.
  • Exemplary transcriptional regulatory regions suitable for use in the present invention include, but are not limited to, the human cytomegalovirus (CMV) immediate-early enhancer/promoter, the SV40 early enhancer/promoter, the JC polyomavirus promoter and the chicken ⁇ -actin promoter coupled to the CMV enhancer (Doll, R.F. et al. (1996) "Comparison of promoter strengths on gene delivery into mammalian brain cells using AAV vectors.” Gene Ther., vol. 3, no. 5, pp.
  • CMV human cytomegalovirus
  • the Rous sarcoma virus (RSV) promoter the Rous sarcoma virus (RSV) promoter, the tyrosine kinase (TK) promoter, the Rous Sarcoma Virus (RSV) promoter, the ⁇ -fetoprotein (AFP) promoter, the DF-3 mucin enhancer, the tyrosinase promoter, the carcinoembryonic (CEA) promoter, the tyrosinase promoter, the prostate specific antigen (PSA) promoter, the Hi parvovirus promoter or other suitable promoter.
  • RSV Rous sarcoma virus
  • TK tyrosine kinase
  • AFP Rous Sarcoma Virus
  • DF-3 mucin enhancer the tyrosinase promoter
  • CEA carcinoembryonic
  • PSA prostate specific antigen
  • An "expression cassette” may be a DNA fragment comprising a coding sequence for one or more reporter gene(s) and/or one or more effector gene(s) operably linked to a transcriptional regulatory region or a transcriptional control region sufficient for expression of the encoded protein in an appropriate cell type.
  • a “reporter gene” or “reporter construct” may be a subchromosomal and purified DNA molecule comprising a gene encoding an assayable product.
  • An “effector gene” refers to any gene that, upon expression of the polypeptide encoded by the gene, confers an effect on an organism, tissue or cell.
  • a “transgene” refers to a gene that has been inserted into the genome of an organism other than that normally present in the genome of the organism.
  • a gene expressed in a "tissue-specific” or “tumor-specific” manner is that which demonstrates a greater amount of expression in one tissue as opposed to one or more second tissues in an organism (i.e., preferential expression in one tissue as compared to another cell or tissue).
  • a tissue-, cell- or tumor- specific promoter demonstrates greater activity in one tissue, cell or tumor, respectively, as compared to another tissue, cell or tumor.
  • an "antigen,” “epitope,” or “immunogen” refers to any molecule capable of inducing an immune response. Such a molecule includes, but is not limited to, cell-mediated antigens, those that induce a humoral and/or cellular immune response, those that interact with T-cell and/or B-cell receptors; those that induce an innate immune response, and the like.
  • an “immunomodulatory gene” refers to any gene that, upon expression of its nucleic acid or protein product, serves to alter an immune reaction, including both activation and repression or suppression of an immune response.
  • a “tumor suppressor gene” refers to a gene that, upon expression of its protein product, serves to suppress the development of a tumor including but not limited to growth suppression or induction of cell death.
  • a “growth suppressor gene” is defined as a gene that, upon expression of its protein product, serves to suppress the growth of a cell.
  • An “oncogene” is defined as a cancer-causing gene.
  • Addenoviral particle refers to an infectious adenovirus, including both wild type or recombinant.
  • the adenovirus includes but is not limited to a DNA molecule encapsidated by a protein coat encoded within an adenoviral genome.
  • Heterologous DNA refers to DNA introduced into an adenoviral construct that was isolated from a source other than an adenoviral genome or other DNA, either in its natural state or as a recombinant, that is foreign to a host cell.
  • recombinant adenoviral particle is defined as an infectious adenovirus having at least one portion of its genome derived from at least one other source, including both adenoviral genetic material as well as genetic material other than adenoviral genetic material.
  • a “treatable condition” refers to a condition of an organism that may be altered by administration of a form of treatment including but not limited to those treatments commonly defined as being of medicinal origin.
  • a “genetic condition” is defined in this application as a condition of an organism that is at least partially the result of expression or suppression of at least one specific gene including but not limited to the wild-type form of that gene and any mutant form of that gene.
  • adenoviral vectors currently in use by those skilled in the art are deleted in the E1 region and propagated in a permissive cell line that may provide the missing E1 functions (Graham, F.L. et al. (1977) "Characteristics of a human cell line transformed by DNA from human adenovirus type 5.” J. Gen. Virol, vol. 36, pp. 59-72).
  • the E1 function(s) may be provided in trans through some other component of the system (e.g., including, but not limited to, the helper-Ad).
  • helper viruses have been used to package vectors containing large deletions of the viral genome.
  • helper virus similar to, in the present invention, the supplemental-Ad.
  • Applicants have previously demonstrated, (see e.g., Related Applications, supra) complementation of large deletions in the Ad vector genome by a helper vector having a partial deletion of the packaging signal.
  • the supplemental vector may have the E1 region substituted by a ⁇ -gal expression cassette or other cassette and may be propagated in a cell line that expresses E1 , such as A549 cells, during preparation for use in the complementary Ad system.
  • the supplemental vector complements the controlled-Ad vectors having those the adenoviral genes deleted.
  • controlled-Ad vector refers to a vector in which, with the exception of the E1 gene region, adenoviral coding sequences are deleted. Further, the controlled Ad vector retains the minimal genetic elements necessary for replication and packaging.
  • the "supplemental-Ad vector” provides proteins required to maintain replication and packaging of both the controlled-Ad vector and the supplemental-Ad vector.
  • a controlled-Ad vector comprising one or more early Ad genes (such as E1, E4 or E2) not present within the genome of the supplemental-Ad vector, is provided.
  • the controlled-Ad may also support replication of the supplemental-Ad (acting as a helper of the helper). Therefore, the two vectors, the supplemental- Ad and the controlled-Ad, are mutually dependent upon one another for propagation.
  • Expression of E1 from the complementary Ad vector may be driven by a constitutively active or "general" promoter that is active in many cell types, such as the CMV promoter/enhancer, SV40 early enhancer/promoter, the JC polyomavirus promoter, the chicken ⁇ -actin promoter coupled to the CMV enhancer (Doll, R.F. et al. (1996) supra), the Rous sarcoma virus (RSV) promoter, and the tyrosine kinase (TK) promoter, for example.
  • the complementary Ad system may be used to treat conditions in which are particular population of target cells are either difficult to specifically identify or target.
  • Conditions such as these would include autoimmune conditions including systemic lupus erythematosis (SLE or "lupus”), arthritis, or infectious disease.
  • SLE systemic lupus erythematosis
  • the constitutive promoter in the controlled Ad allows for expression of a beneficial immunosuppressive cytokine in the local environment of an arthritic "flare-up" (ie, the areas in which the patient is experiencing pain).
  • a beneficial immunosuppressive cytokine in the local environment of an arthritic "flare-up" (ie, the areas in which the patient is experiencing pain).
  • the complementary Ad system may still provide for some replication in the local area, thus providing expression of the immunomodulatory proteins for a substantial period of time.
  • expression of E1 may be limited to certain types of cells or tissues.
  • the promoter of one or more of the early genes necessary for adenoviral replication is operably linked to a cell or tissue-specific promoter. Any adenoviral gene necessary for replication may be operably linked to the target cell-specific promoter such as E1, E2, and E4.
  • any promoter showing activity in the target cell is suitable while cell-specific activity such as that provided by promoters for ⁇ -fetoprotein, carcinoembryonic antigen (CEA), melanotransferrin, Erb-B2, tyrosinase, MUC1, prostate specific antigen (PSA, osteocalcin (sarcomas, prostate cancer), tyrosinase promoter, DF3 promoter, chromogranin A (neuroendocrine tumors, SMLC), prostate specific membrane antigen (PSMA) promoter, or the glial fibrillary protein (GFP) promoter, for example, are preferred.
  • the present invention may comprise a controlled-Ad vector containing the E1 region with the E1A promoter substituted by the ⁇ -fetoprotein (AFP) promoter. Only cells that are able to utilize the ⁇ - fetoprotein (AFP) promoter and that have been co-infected with both the controlled-Ad and the supplemental-Ad (i.e., at the site of injection) support propagation of the controlled-Ad and the supplemental-Ad.
  • AFP ⁇ -fetoprotein
  • both viruses are produced in similar amounts (due to the effect of the partial deletion or modification in the packaging signal of the supplemental-Ad), neighboring cells will be co-infected and, if those cells are able drive E1 expression from the AFP promoter, such as hepatocellular carcinoma cells, both viruses will continue to propagate.
  • Another advantage of this invention over currently available vectors is that the combination of two defective vectors provides added capacity for therapeutic heterologous DNA.
  • the capacity of the controlled-Ad vector for exogenous non-viral DNA is up to 36 Kb. Any gene with an antigenic potential may be incorporated into the controlled-Ad.
  • the immuno-stimulatory genes encoding B7.1 (a co-stimulatory molecule) and the interferon (IFN- ⁇ ) are incorporated into a single controlled-Ad as two separate expression cassettes. Many other expression cassettes would be suitable for use in this system.
  • the complementary-Ad vector system consists of two major parts: (1) a packaging-attenuated supplemental-Ad vector which is E1 deleted or otherwise substituted and (2) the controlled Ad vector including only minimal c/s-elements of the Ad viral genome such as E1 or other early stage genes such as E2 or E4.
  • E1 function may be provide by a component of the system other than the cell line.
  • the supplemental-Ad has all of the viral genes and elements required to replicate and frans-complement the controlled-Ad vectors, except that the supplemental Ad vector has an E1 deletion or substitution.
  • the modified supplemental- Ad vector may further comprise a mutated (less efficient) packaging signal that selects for preferential packaging of the controlled-Ad vector.
  • the controlled-Ad vector on the other hand, has the inverted terminal repeats (ITRs) and wild-type packaging signal (the c/s-elements for Ad DNA replication and packaging).
  • the controlled-Ad may be controlled using at least two different methods: promoter replacement and/or functional deletion. Other suitable methods of control would be understood by the skilled artisan.
  • the natural viral promoter of AdE1A is substituted with a constitutive promoter that drives expression of AdE1 in the host cell, such as the cytomegalovirus (CMV) promoter/enhancer.
  • a constitutive promoter that drives expression of AdE1 in the host cell such as the cytomegalovirus (CMV) promoter/enhancer.
  • CMV cytomegalovirus
  • suitable promoters include SV40 early enhancer/promoter, the JC polyomavirus promoter and the chicken ⁇ -actin promoter coupled to the CMV enhancer (Doll, R.F. et al. (1996) supra), the Rous sarcoma virus (RSV) promoter, and the tyrosine kinase (TK) promoter, for example.
  • TK tyrosine kinase
  • the natural viral promoter of the early gene for control of viral replication may be substituted by a heterologous promoter, which is active or inducible only in target cells or production cells.
  • a heterologous promoter which is active or inducible only in target cells or production cells.
  • the E1a promoter of adenovirus is the initiator of the viral replication cycle.
  • the E1a promoter may be replaced by the ⁇ -fetoprotein promoter, which is active mainly in hepatocarcinoma cells.
  • Other examples of promoters that are lineage-specific or may be specifically activated in tumor cells are described herein.
  • An adenoviral gene that is not essential for the viral replicative cycle in the target cells can be deleted, which can differentiate the viral specific propagation in the target cells from that of non-permissive in non-target cells. For example, tumor cells are more likely to be cycling than non- tumor cells. As such, the typical adenoviral functions that induce cycling of normal resting of cells are not required. Deletion of the adenoviral gene encoding an Ad cell protein that induces the cell cycle will abrogate the replicative capability of Ad in normal resting cells.
  • Ad E1 b-p55 and E1b are dispensible for virus propagation in p53-deleted (or mutated to decrease function) and retinoblastoma-deleted (or mutated to decrease function) tumor cells, respectively.
  • a supplemental Ad vector may be designed by deleting E1b-p55 or E1b.
  • Ad helper cell lines similar to A549 cells may also be utilized to trans-activate supplemental- Ad transcription and replication. These cells may be used to propagate the supplemental-Ad without the aid of the controlled-Ad. Cell lines may also contain control mechanisms for the packaging attenuation of the supplemental-Ad.
  • Such mechanisms may include expression of proteins that bind the supplemental Ad packaging signal or interfere with other such functions.
  • b. Mechanism of operation of the system This system is designed to limit Ad replication locally to either a specific area of the body or a particular target cell or tissue, or both. For treatment of conditions for which targeting specific cells or tissues is either not desired or feasible, replication may still be limited using complementary Ad and a constitutive promoter by virtue of the complementary Ad system.
  • the complementary Ad vector mixture is locally injected where particular target cells are available, replication can be limited through the use of target cell-specific promoters and either local or systemic administration. Systemic administration of transcriptionally targeted vectors provides for replication in only those cells capable of expressing E1 from the target cell specific promoter.
  • this system comprises a tissue-specific or tumor-activated promoter activated in the helper cells or target tumor cells.
  • the controlled Ad comprise a promoter driving transcription of the E1 gene.
  • the E1 gene products may in turn drive the transcription and replication of the supplemental-Ad genome and controlled-Ad genomes simultaneously.
  • the packaging protein of Ad is a frans-acting factor present in low amount in the infected cells and is the rate-limiting step for the packaging of Ad.
  • the wild- type packaging signal is recognized by the packaging protein with higher affinity than the engineered signal, packaging of the supplemental viral genomes with mutated packaging signal is partially or completely suppressed in the presence of the controlled-Ad viral genomes having a wild-type packaging signal. This provides for preferential packaging of the controlled-Ad vector.
  • the two viral vectors are mutually dependent.
  • supplemental-Ad in the other early regions may also be performed. Incorporation of the corresponding deleted genes in the controlled-Ad increases the packaging and titer of the controlled-Ad and provides for dependence of the supplemental-Ad on the controlled-Ad. This is particularly useful for development of, for example, anti-tumor complementary-Ad vectors.
  • standard biochemical techniques may be utilized.
  • the complementary-Ad vectors may be further purified from supplemental Ad vectors through biological, biochemical, or physical methods such as ultracentrifugation through CsCI gradient, if purification of the Ad vectors is required for application.
  • High gene-delivery capacity is one of the major features of this complementary-Ad vector system.
  • Both the complementary and the supplemental Ad vectors may be utilized for delivery of heterologous DNA to cells or tissues, or to increase or decrease express of genes in cells or tissues.
  • Nucleic acids encoding various effector or reporter gene sequences may be incorporated into either the controlled Ad or the supplemental Ad, thus providing for expression of the gene sequence in an infected cell.
  • the maximal packaging capacity of Ad is about 105% of the genome, or approximately 38 kb (Ghosh-Choudhury, G. et al.
  • the size of the viral c/s-element in the controlled-Ad vector may comprise less than 1 kb. Deletion of E1 and E3 genes in the supplemental-Ad provides an additional 8 kb capacity. As such, the total capacity of the complementary-Ad vector system for heterologous DNA is up to 42 kb.
  • the heterologous DNA can be either transgene expression cassettes or regulatory elements.
  • the expression cassettes can be single or multiple, bicistronic or polycistronic.
  • the regulatory elements can be DNA sequences for controlling transgene retention, transcription, and vector targeting.
  • complementary Ad vectors Multiple genes may be incorporated into the complementary Ad vectors to provide one or more cytokines, chemokines, interleukins, and/or immuno- modulating agents to optimize the systemic anti-target cell immune responses resulting from infection with the complementary Ad vectors. It is also possible to construct a complementary Ad system that delivers one or more cytotoxic and/or immunostimulatory genes singly or in combination to enhance a cytotoxic effect.
  • the complementary Ad system may be utilized to shift the immune response toward a T helper 1 type may avoid the antibody blockage of viral spread, thereby assisting in rejection of metastases.
  • a transgene such as the antigen costimulatory molecule B7 may serve to elicit optimal cytolytic immune responses.
  • target cells transduced by one of the complementary vectors i.e., either the controlled Ad or the supplemental Ad
  • target cells transduced by one of the complementary vectors may not be lysed until co-infected by the complementary vector, providing expression and presentation of immunomodulatory molecules. It is important to compare infection by single and complementary replication-competent vectors for safety and efficacy in immunocompetent models cells that to identify those complementary Ad vectors with the greatest potential as therapeutic reagents.
  • IFN- ⁇ gene expression has been shown to sensitize human and other mammalian target cells to the effects of secondary treatments (see e.g., Gruninger, L. et al.
  • the B7.1 (CD80) co-stimulatory molecule has a dramatic role in T cell- antigen presenting cell interactions and in generating effective T cell responses (see e.g., Lenschow, D.J. et al. (1996) "CD28/B7 system of T cell co-stimulation.” Annu. Rev. Immunol, vol. 14, pp. 233-258).
  • CD80 is expressed on dendritic cells and is induced on activated B cells, T cells, NK cells and macrophages (Azuma, M. et al. (1993) "Functional expression of B7/BB1 on activated T lymphocytes.” J. Exp. Med., vol. 177, pp. 845-850; Freeman, G.J. et al. (1989) "B7, a new member of the Ig superfamily with unique expression on activated and neoplastic B cells.” J. Immunol, vol. 143, pp. 2714-2722).
  • nucleic acids encoding immunomodulatory proteins including, but not limited to, IFN ⁇ y and CD80, into the complementary Ad system (i.e., by insertion of an expression cassette into a complementary Ad) to induce anti-target cell immune response(s).
  • the complementary Ad system is useful for lysis or destruction of cells, where, for example, the controlled Ad comprises E1 under the transcriptional control of a cell-specific promoter, and one or more nucleotide sequences encoding an immunomodulatory protein, such as IFN ⁇ y and/or CD80.
  • the controlled Ad comprises E1 under the transcriptional control of a cell-specific promoter, and one or more nucleotide sequences encoding an immunomodulatory protein, such as IFN ⁇ y and/or CD80.
  • the complementary Ad system may be complete as a system for lysis of target cells by incorporating into the controlled Ad vector a cell- or tissue-specific or non-specific E1 expression cassette, an immunomodulatory effector gene expression cassette, and a supplemental Ad vector for complementation of Ad proteins that are not encoded by the controlled Ad.
  • the complementary Ad system may also be utilized to modify in vivo target cell function or regulate target cell growth.
  • Ad vectors have distinct advantages over other viral vectors (i.e., retroviruses, HSV) in that high titers may be produced, which is useful in preparing vectors for in vivo gene therapy.
  • the complementary-Ad vectors may be useful for transiently suppressing cell- or tissue-specific proliferation following local administration. Therefore, the complementary Ad system may be useful for modifying in vivo target cell function or regulating target cell growth.
  • the complementary Ad system may also be useful for selective delivery of transgenes to target cells or tissues in vivo by surface modification of the vectors.
  • the capsid proteins (i.e., hexons and fibers) encoded by the supplemental Ad vector may be modified to increase the affinity for target cell surface proteins, thereby targeting replication to a particular target cell or tissue.
  • a hexon or fiber may be engineered to bind certain epitopes or ligands (i.e., protein A for binding to an Fc portion of an IgG molecules).
  • modified hexon and fiber genes may be incorporated into the recombinant viral genome for generation of complementary Ad preparations having surface sites that interact with particular ligands as targeting agents.
  • the viral particles thus produced will provide tissue or cell recognition capabilities (see e.g., Curiel, D.T. (1999) "Strategies to adapt adenoviral vectors for targeted delivery.” Ann. NY. Acad. Sci.; vol. 886, pp. 158-171).
  • the complementary Ad system may be used for Ad-mediated vaccination via direct in vivo approaches.
  • the immunogenicity of the E1 -substituted Ad vectors may be beneficial, and this has been taken advantage of in the development of Ad-based recombinant vaccines.
  • the contribution of the controlled-Ad vectors for this type of application may be realized by using the E1 -substituted Ad vectors as helper virus and co-delivering genes encoding antigens and immuno-enhancing proteins.
  • complementary Ad system may be used in combination with other gene transfer and ⁇ or gene therapy methodologies.
  • Conventional Ad vectors have been used in combination with polylysine, liposome, and other conjugation materials as gene delivery complexes.
  • the complementary-Ad vectors may also be used in these types of combinations. Many such combinations are known by those of skill in the art and would be applicable to the present invention.
  • the complementary Ad system may be utilized to deliver nucleic acids encoding one or more immunomodulatory gene(s) (e.g., including, but not limited to, interieukin (e.g., IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 , IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, or IL-18, for example), interferon (e.g., IFN- ⁇ , - ⁇ , - ⁇ ), a co-stimulatory molecule (e.g., B7.1 (CD80), B7.2), chemokine (CCR5), suicide gene (e.g.., HSV-TK, CDA, among others); a cell cycle gene (e.g., a gene having an effect on progession of the cell cycle such as p16, p15, WAF1/CIP1/p21 , p27, ataxi
  • nucleotide and amino acid sequences of each of the nucleic acids listed above, as well as the many other suitable sequences, are well known in the art and widely available (e.g., GenBank) to the skilled artisan.
  • Methods for generating complementary Ad (e.g., controlled Ad or supplemental Ad) constructs using these sequences, using the reagents and methodologies provided herein, is well within the capabilities of the skilled artisan.
  • the complementary Ad system may also provide a nucleotide sequence providing a sequence-specific ribozyme, a sequence-specific RNase P, or an antisense construct.
  • a suitable effector sequence would inhibit the expression or function of a target sequence encoding a protein or the protein itself, where expression or function of such a protein causes a detrimental effect on the generation of an immune response in a patient or subject (e.g., including, but not limited to, TNFbeta (TNF ⁇ ), other immuno-responsive proteins and the like).
  • TNFbeta TNFbeta
  • the nucleotide and amino acid sequences of each of the targets described herein, as well as the many other suitable target sequences, are well known in the art and widely available to the skilled artisan. Methods for generating ribozymes (Perlman, H.
  • the basic structure of the supplemental-Ad provides the proteins required for replication of the controlled Ad vector, which is deleted for the DNA encoding Ad structural and other proteins required for replication. It is possible to utilize a supplemental Ad vector having either a wild-type or an engineered packaging signal.
  • the supplemental-Ad vector has two main structural features: a partial wild-type Ad genome and engineered packaging signal.
  • the supplemental-Ad is preferably defective in replication, such as by deletion or substitution of E1.
  • the supplemental is preferably attenuated in packaging.
  • the general structure of the supplemental-Ad may be summarized as an Ad vector comprising a wild-type Ad genome with the exception of an altered E1 region and packaging signal.
  • Ad vector comprising a wild-type Ad genome with the exception of an altered E1 region and packaging signal.
  • the viral genome may also be fragmented and incorporated into separate supplemental Ad vectors to further inhibit the replication competence of the supplemental-Ad or to reduce the size of the supplemental-Ad genome. It is important that these manipulations do not significantly alter the titer of the supplemental-Ad during preparation of stock reagents. With this proviso, modifications affecting both supplemental-Ad replication and packaging attenuation of the supplemental-Ad should be considered in designing the vector.
  • the primary function of the supplemental-Ad is to supply capsids for packaging of the controlled-Ad vectors and lyse the target cells through viral propagation.
  • the supplemental-Ad must be able to reproduce itself, although it is dependent on the activation of a promoter by a target cell driving expression of E1 , provided by the controlled-Ad. It is preferred that DNA replication and transcription of the supplemental Ad genome is sufficient to yield functional quantities of the late gene products (the capsid proteins) such that the titer of the controlled-Ad vectors remains high.
  • a supplemental-Ad vector having a wild-type or only slightly attenuated packaging signal it is also possible to utilize a supplemental-Ad vector having a wild-type or only slightly attenuated packaging signal.
  • the supplemental Ad will be more useful for co-expression of effector genes, along with those express from the controlled-Ad.
  • the controlled Ad vector contains DNA encoding E1 along with several effector proteins such that it is close to maximum capacity
  • the designs for packaging attenuation One purpose for attenuating packaging the supplemental-Ad is to reduce the potential for overgrowth of the supplemental-Ad as compared to the controlled-Ad vectors. This is important when relatively high titer of the controlled-Ad vectors is required for particular applications.
  • the packaging function of the supplemental-Ad is designed to be defective but not completely disabled, as the supplemental-Ad is preferably maintained in cunjunction with the controlled-Ad vector, thus forming the complementary-Ad vector system.
  • the Ad5 packaging signal is composed of a repeated element that is functionally redundant (Hearing, et al. (1987)
  • Partial deletions of the packaging signal elements have been shown to reduce the yield of mutant Ad from several fold to approximately a hundred fold that of the Ad with wild-type packaging signal (Grable, M. & Hearing, P. (1992) "cis and trans requirements for the selective packaging of adenovirus type 5 DNA.” J. Virol, vol. 6, pp. 723-731).
  • the packaging signal is partially deleted in one or more A-repeats of the wild-type Ad packaging signal.
  • the Ad5 packaging signal has a consensus A (adenosine) enriched motif (e.g., A-repeat: TAAATTTG). Therefore, incorporation of an array of tandem repeats of a selected A-repeat or any other synthetic DNA motif may alter the affinity of the artificial packaging signal to the packaging proteins and thus packaging of the supplemental-Ad.
  • A-repeat TAAATTTG
  • Packaging signal interference It is also possible to design mechanisms for interfering with the packaging signal. In order to interfere with the effective binding of the packaging proteins to the signal, DNA binding sequences can be incorporated into or adjacent to the A-repeat array of the packaging signal of the supplemental-Ad. The inserted binding sites provide for high-affinity binding by the respective DNA binding proteins resulting in positional competition for the Ad packaging proteins to the Ad packaging signal. In certain embodiments, binding sites may be incorporated into packaging signal structures that have been modified to reduce functionality.
  • Packaging signal relocation In its native orientation, the Ad packaging signal is positioned at the left end of the wild-type Ad genome. It has been reported that the packaging signal may be positioned at the right end and retain functionality. As such, the packaging signal is relocatable. In one embodiment, then, an engineered packaging signal may be inserted at a non-wild-type location, further attenuating the packaging efficiency of the supplemental-Ad. This may assist in minimizing reversion of the supplemental-Ad to wild-type Ad by homologous recombination between the engineered packaging signal of the supplemental-Ad and the wild-type packaging signal of the controlled-Ad vectors.
  • c/s-elements and frans-acting factors.
  • useful designs may wither of these two basic aspects or some combination of these two configurations.
  • the c/s-elements of the packaging signal are considered to be the A-repeats and the fra ⁇ s-acting factors are the packaging proteins.
  • the skilled artisan would understand that many other methods are suitable for disabling the supplemental Ad packaging signal, and such methods are contemplated as part of the present invention.
  • Supplemental-Ad vectors with the wild-type packaging signal As described above, it may be preferable to incorporate a packaging signal in the supplemental Ad that is only mildly disabled.
  • the supplemental-Ad vector may comprise a wild-type packaging signal. The final outcome of the process depends upon the relative propagation speed of the complementary vectors versus the tumor cells. The rate of propagation of the supplemental Ad vector limited by the number of viruses produced per infected cell and may be increased using a wild-type packaging signal in the supplemental vector.
  • the use of a wild-type packaging signal for both the controlled and supplemental vectors in this system is advantageous, because supplemental vector propagation depends upon the controlled-Ad vector and wee versa.
  • the use of wild-type packaging signal should increase the propagation efficiency of the complementary vectors and therefore the potency of treatments using the complementary Ad system.
  • the controlled Ad vector is utilized to deliver effector genes (e.g., including, but not limited to, E1 ) that limit expression of the complementary Ad vectors to cells that are capable of expressing an effector gene coding sequence carried by the controlled Ad.
  • the controlled- Ad may provide a construct comprising a tissue-specific promoter such as a ubiquitous, generalized "house-keeping" promoter or a cell- or tissue-specific promoter operably linked to an E1 coding sequence.
  • E1 will only be expressed in cells capable of utilizing the ubiquitous, generalized "housekeeping" promoter or the cell- or tissue-specific promoter.
  • the supplemental Ad depends upon E1 for replication, and therefore upon the complementary Ad to provide E1.
  • the supplemental Ad will not replicate in the absence of E1 and the complementary Ad will not replicate in the absence of proteins expressed only by the supplemental Ad vector. Therefore, replication will only occur in cells capable of utilizing the AFP promoter.
  • the controlled Ad comprises DNA encoding effector proteins.
  • Ad vectors are typically utilized as a circularized plasmid form through the fusion of ITRs (Graham, F. L. (1984) "Covalently closed circles of human adenovirus DNA.” EMBO J., vol. 3, pp. 2917-2922.). Ad vectors may also be employed as linear DNA.
  • a basic form of a controlled-Ad vector is a circular DNA containing an ITR fusion, a plasmid DNA replication origin, the Ad E1 gene, and one or more polycloning sites.
  • the ITR fusion preferably contains the left end of the wild-type Ad from map unit 0 to 1 and the right end from map unit 99 to 100, in which the DNA replication origins of Ad are located in the both ITRs and the wild-type packaging signal located adjacent to the left ITR.
  • the controlled-Ad vector may be controlled by at least two basic mechanisms: promoter replacement and functional deletion.
  • the Ad E1 -region genes are incorporated into a controlled-Ad within an expression cassette comprising a promoter sequence.
  • the promoter may be cell-specific, tissue-specific, tumor-activated or otherwise active in a particular cell to control expression of the E1 genes.
  • the promoter may be a ubiquitous, generalized "house-keeping" gene promoter active in many cell and/or tissue types.
  • the promoter may be an adenoviral promoter.
  • the structural and functional possibilities of the controlled-Ad vectors It is also possible to modify the structure of the controlled Ad vector. Additional DNA sequences and elements may be modified or included as described below:
  • Expression cassettes of transgenes As understood in the art, an expression cassette is a basic transcription unit. A basic expression cassette is
  • the expression cassette may comprise two or more genes of interest arranged as a bi- or poly-cistronic structure, provided additional elements for translation or splicing of RNA are provided between the genes.
  • multiple expression cassettes may be joined using an internal ribosome entry site (IRES) construct (Martinez-Salas, E. (1999) "Internal ribosome entry site biology and its use in expression vectors.” Curr. Opin. Biotechnol, vol. 10, no. 5, pp. 458-464).
  • controlled-Ad vectors may comprise one or multiple transgene expression cassettes.
  • a broad range of gene sequences may be useful for practicing the present invention. Such gene sequences may be incorporated into the complementary Ad vector as part of expression cassettes, thus providing for expression in cells following infection by the vectors of the complementary Ad vector system.
  • Functional elements for vector DNA retention It is also possible to include elements that assist in integrating the expression cassette into the target cell genome such as, for example, an adeno-associate virus inverted terminal repeat (AAV-ITR), homologous regions of DNA that stimulate recombinatinon of the expression cassette into the host cell genome. It is a further possibility to include elements that retain the controlled-Ad vectors as an episomal form in target cells such as origin of replication.
  • AAV-ITR adeno-associate virus inverted terminal repeat
  • DNA elements having transcriptional regulation functions such as enhancers, repressors or activator-binding sites, introns, 5' or 3'-untranslated regions (e.g., constitutive promoter) may also be incorporated into the complementary Ad vector.
  • the regulatory elements may drive expression in multiple cell types or may restrict expression to particular cell types (i.e., cell-or tissue-specific promoter) .
  • Various regulatory elements, such as weak promoters, silencers and the like may also be utilized to regulate expression in a particular cell type.
  • the regulatory elements may also restrict expression of, for example, E1 from the controlled-Ad vector due to the presence of DNA sequences capable of binding a ligand that interferes with transcription.
  • E1 may also restrict expression of, for example, E1 from the controlled-Ad vector due to the presence of DNA sequences capable of binding a ligand that interferes with transcription.
  • Expression of large amounts of E1 can be toxic to certain host cells; a method for regulating the amount of E1 expressed in a cell is desireable.
  • such a system would be useful for limiting the spread of Ad particles in vivo.
  • the CMV promoter may be engineered to comprise tetO sequences to which may bind the tetR/KRAB protein expressed in a producer cell line.
  • Tetracycline binds to tetR KRAB. Therefore, addition of tetracycline to the cell culture would result in an inactive tetR/KRAB protein, and transcription from the CMV promoter would occur. Removal of tetracylcine from the cell culture results in free tetR/KRAB, which binds to the tetO sequences in the promoter and inhibits transcription.
  • expression of coding sequences of the controlled-Ad may be regulated using the "tetracycline-reversed repression system".
  • An exemplary controlled-Ad construct for use in a tetracycline-reversed repression system may comprise the Ad 5'ITR/packaging signal, stuffer DNA, a tefO/CMV promoter containing 1-10, preferably 7, repeats of the tetO sequence operably linked to E1a/E1b coding sequences, an antibiotic selection marker for propagation of the plasmid in prokaryote host cells, and an expression cassette.
  • One such expression cassette comprises human IL-3 under the transcriptional control of the Rous sarcoma virus (RSV) promoter.
  • RSV Rous sarcoma virus
  • Propogation of this vector would be accomplished using a producer cell line that constituitively expresses tetR/KRAB, which binds to the tefO/CMV promoter and represses transcription of the E1a/E1b transcripts.
  • tetR/KRAB binds to the tefO/CMV promoter and represses transcription of the E1a/E1b transcripts.
  • tetracycline prevents the binding of TetR/KRAB to the tetOCMV promoter, and the sequences under the transcriptional control of the tefO/CMV promoter are expressed.
  • Targeting can be achieved at least at two levels: vector surface modification and tissue-specific expression. Tissue specific promoters can be utilized to avoid expression in any cell type but that targeted for delivery in vivo.
  • Additional elements may comprise DNA replication origins of prokaryotic or eukaryotic cells, plasmid or vector selection markers, and backbones of the vectors, and the like. Also contemplated herein is the incorporation of of non-mammalian nucleic acid sequences that influence one or more immunes (e.g., including, but not limited to, CpG islands and the like)
  • High- titer production of the controlled-Ad vectors is contemplated by the present invention.
  • Advantages of the complementary Ad vector system over conventional vectors include the ability to obtain high-titer preparation This is mainly due to the large quantity of viral capsid protein provided by the supplemental Ad and the high copy number of the controlled Ad viral genome.
  • the following provide examples of methods for generating high-titer controlled- Ad vectors:
  • Enhanced DNA replication Ad has a unique enzymatic system for DNA replication.
  • the E2 region proteins are the major frans-acting elements responsible for viral DNA replication.
  • the replication origins are the cis-elements located at both ends of the viral genome.
  • a sufficient amount of E2 protein expressed from the supplemental virus is preferably provided.
  • High-level expression of E2 region proteins may also be achieved by including the E2 gene within the controlled-Ad genome in addition to that in the supplemental virus.
  • Other mechanisms for increase in copy numbers of the controlled-Ad genome would be understood by the skilled artisan, and may include the following: 2.
  • Enhanced packaging signal Additional or enhanced packaging sequences may be utilized to enhance packaging by either adding more tandem repeats at one end or both, or generating synthetic packaging signals.
  • Enhanced packaging process The packaging processes of Ad are not yet completely understood. Whether proteins binding to DNA at sites other than the packaging signal of Ad play synergistic roles for packaging is not yet certain. If so, the sequences for DNA-binding proteins, refereed to anchorage points for packaging, naturally existing in the Ad genome may need to put back to the controlled-Ad genome.
  • controlled Ad and the supplemental Ad may also comprise DNA encoding effector proteins that are expressed in cells where viral replication results from co-infection of cells by the controlled and the supplemental Ad vectors. Infection of cells separately by the controlled Ad or the supplemental Ad still results in cellular expression of encoded antigens and contributes to the. generation of immunity against the expressed antigenic proteins.
  • a dual vector vaccine system has several advantageous features for generating immune responses to encoded antigens.
  • such a system has a very large capacity for expressing multiple antigens and immunomodulatory proteins that exceeds the capabilities of standard adenoviral vector systems.
  • Expression of multiple antigens and immunomodulatory genes is desirable to increase immune responses in a variety of clinical circumstances where immune responses to multiple antigens is desirable to better protect against or treat an infectious pathogen or to destroy tumor cells that comprise multiple antigenic targets.
  • the vector system is capable of replication and it is generally appreciated that "live" replicating vaccines are typically more efficacious than those that do not replicate. This beneficial effect is likely due in part to the amplification of antigen expression provided by replicating vectors.
  • the replication capability may be adjusted to provide desirable degrees of attenuated replication.
  • each of the vectors may function as separate adenoviral vectors to express antigens providing an additional source of antigens to generate immune responses.
  • Cells infected by just one of the vectors provides for several days to several weeks of antigen expression to further the development of immunity.
  • the dual Ad vectors contain all of the genes required for their replication, it is not necessary to genetically engineer producer cell lines containing adenoviral genes for manufacturing the vectors and they may be readily adapted for production in cell lines accepted for vaccine production including, but not limited to, those that have been well characterized with safety histories for vaccine production purposes (e.g., including, but not limited to, WI38 cells, MRC5 cells, VERO cells, embryonic cells and cell lines, and the like).
  • these dual ad vector systems still retain the advantageous features of standard adenoviral vectors including, but not limited to, highly efficient gene transfer and expression, broad host range of infectable human cell types, readily formulated for oral, nasal, mucosal, intradermal, intramuscular, intraperitoneal and systemic administration.
  • the vectors will be complementary and will together contain the complete complement of genes required for the replication of one or more of the component vectors.
  • the complementing vectors may be of the same or different types as long as they together provide the complete gene functions required to propagate one or more of the vectors.
  • the deleted regions of the vectors that are complemented by the supplemental vectors may be substituted to deliver expression cassettes for a variety of medicinal purposes.
  • the controlled Ad and supplemental vectors may be propagated in any suitable cell line.
  • Ad helper cell lines are used to efficiently produce complementary Ad vectors.
  • the supplemental vector are propagated standard cell lines for adenovirus production, including, but not limited to, A549 cells, VIRO cells and the like. Alternatively, the cells may be of the type that do not express E1.
  • the complementary Ad vectors may be produced in a helper cell line. Suitable cell lines are also described in the published applications referred to in the Related Applications section, supra.
  • the helper cell lines may trans-activate transcription of the AdE1 genes of the controlled-Ad, resulting in activation of transcription and replication of the supplemental-Ad genome.
  • the helper cells are unique from 293 cells in that the E1 fragment is carried within the controlled-Ad genome which has no overlapping sequence with the supplemental-Ad genome.
  • the helper cell will preferably have the ability to activate the promoter controlling the Ad E1 genes in the controlled-Ad for the complementary-Ad system to function properly.
  • the cell line utilized for production may be LNCaP, a cell line that produces PSA and supports oncolytic adenovirus production.
  • helper cells may also be modified by inclusion of genes that support high copy-number production of the controlled-Ad vector, enhancing packaging of the controlled-Ad vector, and attenuation of packaging of the supplemental-Ad.
  • a cell line may be engineered to express tetR/KRAB or other protein capable of interfering with transcription from a promoter construct. It would be understood by the skilled artisan that other suitable helper cell lines may be constructed using standard techniques in the art.
  • the cell lines are preferably characterized to rule out the presence of adventitious agents, mycoplasma and other viral or infectious contaminants.
  • Cell banking is preferably done using serum free and antibiotic free medium. The cells are grown to sufficient quantities in either suspension culture (spinner flask or suspension bioreactor) or as adherent cells in large capacity bioreactors (e.g., the CellCube system in a GMP manufacturing environment).
  • a master cell bank may be made and a working cell bank derived therefrom.
  • a master virus bank may then be made from viruses propagated in cells from the working bank.
  • the method of virus production depends on whether adherent or non- adherent cell cultures are used.
  • the type of culture will depend on the amount of virus production that can be obtained (burst size).
  • conventional suspension bioreactors are utilized for scalable production.
  • the virus preparations should also be tested for expression of IL-3 and specific activity determined by antigen and functional assays in vitro appropriate for detecting expression of a particular reporter or effector gene. Assays may also be performed to determined the amount of replication competent adenovirus (RCA) in a particular preparation. It is preferred that RCA is not detected at all in the preparations.
  • the virus may also be tested for genome integrity. For clinical applications, efficacy and toxicity studies should be performed using animal model systems for each lot. Stability tests should also be performed on the specified formulation for a clinical trial, for example.
  • Example 1 Replication-competent adenoviral vectors with deletions other than
  • E1 for vaccine and immunotherapy applications Contemplated herein are adenoviral vectors, constructed using techniques known in the art, as well as derived adenoviral particles with one or more deletions in the adenoviral genome other than functional deletions in the E1 gene. Such deletions will not significantly affect the ability of the virus to replicate or to form viral progeny.
  • E3 represses an essential part of antigen presentation to the immune system, namely MHC class I expression (Burgert, H.G. & Blusch, J.H. (2000) "Immunomodulatory functions encoded by the E3 transcription unit of adenoviruses.” Virus Genes, vol. 21, nos. 1-2, pp. 13-25; Sparer, T.E. & Gooding, L.R. (1998) "Suppression of MHC class I antigen presentation by human adenoviruses.” Curr. Top. Microbiol. Immunol, vol. 232, pp. 135-147).
  • the lack of functional E3 may allow better immune recognition of the immunogen(s) (i.e., antigen(s)) inserted into the adenoviral genome. Such insertions may be at the site of E3 deletion. Full deletion of the E3 gene would generate roughly 2 Kb of space in which to insert heterologous DNA sequence(s).
  • E4 deletion would not affect the ability of the virus to replicate or generate viral progeny and may disable all or part of the oncogenic potential reported for E4 (Nevels, M. et al. (2000) "Two distinct activities contribute to the oncogenic potential of the adenovirus type 5 E4orf6 protein.” J. Virol, vol. 74, no. 11 , pp. 5168-5181). Full deletion of the E4 region would generate about 2 Kb of space in which to insert heterologous DNA sequence(s).
  • Another possibility for generating replication-competent adenoviral vectors would be to insert heterologous sequence(s) into a wild-type adenoviral genome (a so-called "add-Ad"). This is possible since adenoviral replication and packaging are known to accommodate up to about 105% of the size of the wild- type adenoviral genome (see above). Thus, about 2 Kb of heterologous sequence(s) may be added to the wild-type adenovirus genome.
  • the transduction efficiency (i.e., infectivity) of the replication-competent adenoviral vectors may be tested in appropriate primary cells and cell lines, prior to in vivo animal tests. It is possible to test for transduction efficiency as well as viability of the transduced cell or cell line using techniques known in the art. Viability may be assessed using trypan blue exclusion, for example. Any of the cells and/or cell lines that adenoviruses are known to infect may be used for these analyses. Also, it is possible to use known techniques to test for expression of the heterologous DNA sequence(s) that are inserted into the adenoviral vector(s).
  • ELISA immunoprecipitation and gel electrophoresis are techniques known in the art for analyzing gene expression and protein quantification.
  • the effects of the expressed heterologous DNA sequences may be assessed by noting morphological changes of the transduced cell or cell line.
  • adenovirus vectors Once particular replication-competent adenovirus vectors have been generated and characterized, they will be tested in vivo using appropriate host organisms (e.g., mice, rabbits and/or non-human primates).
  • the host organism may be exposed to the adenovirus by any route previously mentioned (e.g., orally, mucosally, intramuscularly, sub-cutaneously, etc.).
  • the exposed host may then be analyzed for adenovirus infection and heterologous DNA sequence expression by any number of techniques known in the art.
  • the ability of the heterologous DNA sequence expression to protect the host organism from subsequent infection by the disease agent from which the heterologous DNA sequence was derived may be assessed by exposing the host organism to the intact disease agent and observing the course of infection (if any) of the disease agent in the host organism.
  • Immunological compositions and formulations comprising the replication- competent adenoviral vectors may be prepared using techniques and methods well known in the art (see e.g., Remington's Pharmaceutical Sciences (Martin, E. W. (Ed.) latest edition, Mack Publishing Co., Easton, PA; and the like).
  • Adenoviral vaccines may be prepared using techniques known in the art (see e.g., Hilleman, M.R. (1958) "Efficacy of and indications for use of adenovirus vaccine.” Am. J. Public Health, vol. 48, pp. 153-158; Gaydos, C.A. & J.C. Gaydos (1995) "Adenovirus vaccines in the U.S. military.” Mil. /Wed, vol.
  • adenoviral vaccines may also be assessed using techniques known in the art (see e.g., Simpson, S. & Marshall, E. (2001 ) "Immune control, memory, and vaccines.” Science, vol. 293, p. 233; Enserink, M. (2001) "Driving a stake into resurgent TB.” Science, vol. 293, pp.
  • the invention comprises a mixture of two complementary adenoviral vector constructs and derived adenoviruses dependent on each other for replication. These two complementing adenoviral vector particles are capable to deliver one or multiple immunogens and/or immunomodulatory genes to cells.
  • the first vector is the helper vector in form of a wildtype adenovirus construct deleted for functional E1 expression. Additional modifications may be generated in the helper virus with some modifications described in US08/658.961.
  • the second vector consists of the "gutless" adenoviral vector deleted in all functional 1 adenoviral proteins except for E1 while retaining both ITRs and the packaging signal.
  • E1 can be driven either by its own promoter, by any heterologous constitutive promoter, by any inducible/repressable or tissue-specific promoter.
  • the immunogen(s) and/or immunomodulatory gene(s) may be coded for by the gutless adenovirus or by the helper virus in the E1 -deleted region or in any other region not necessary for replication once E1 is provided.
  • This dual-Ad system is replication-competent in those cells that harbor both adenoviral constructs or derived viruses and thus turn into dual-Ad producing cells leading to an in vivo amplification of these vectors.
  • the dual-Ad vectors can become a powerful gene delivery system for immunogens and other immunomodulatory genes for vaccines and immunotherapy applications.
  • Current adenoviral vectors for vaccines are usually single adenoviral systems.
  • replicating vectors are generally much more powerful when it comes to the induction of strong immune responses.
  • a lower dose of replication-competent dual-Ad vectors will most likely achieve comparable results seen with higher doses of a replication-incompetent Ad vectors, which in turn helps reduce costs.
  • certain adenoviral proteins e.g., capsid proteins
  • capsid proteins are immunogenic and are known to induce an innate immune response (Molec. Ther.3:708, 2001 ; Molec. Ther.3:697, 2001 ; Molec. Ther. 3:757, 2001).
  • the de novo generation of these adenoviral proteins may induce a general nonspecific immune response, which may help in the immune recognition of the delivered immunogen(s).
  • this vaccine/immunotherapeutic can be generated in any mammalian cell line suitable for the propagation of adenoviruses and the production of vaccines/immunotherapeutics without the need to express E1 sequences.
  • E1 is driven by an inducible/repressable promoter
  • producer lines modified to induce/repress E1 are necessary.
  • E1 is driven by a tissue-specific promoter
  • the producer line must be able to provide the component that allows for expression of E1 driven by the tissue-specific promoter.
  • Another advantage of such a system would be reduced toxicity due to the complementary nature of the system and the divided functions between the component parts.
  • Still another advantage of such a system would be a combined benefit of vector replication coupled with long-term antigen expression and/or immunomodulatory gene expression.
  • the immune responses to immunogens delivered by replication- incompetent adenoviral vectors may be increased by the use of Dual-Ad replication-competent adenoviral vectors.
  • HIV antigen targets contemplated by the present invention are a variety of heterologous genes including those coded by the HIV virus, namely HIVgag, HIVpol, HIVtat, HIVrev, HIVenv, HIVvif, HIVnef, HIVvpu and HIVvpr genes as well as the HIV RRE sequence and combinations thereof. Also modifications of these genes to disable specific functions, such as deleting the transactivating function of tat (see e.g., Caputo et al. (1999) J. Immunol, vol. 162, pp. :5631-, Singer, D. et al. (1998) J.Acquir. Immune Defic. Syndr. Hum. Retrovirol. vol. 17, pp.
  • HIVpol enzymes coded for by HIVpol such as reverse transcriptase, integrase and polymerase and the like.
  • HIVpol enzymes coded for by HIVpol
  • enzymes coded for by HIVpol such as reverse transcriptase, integrase and polymerase and the like.
  • partial gene sequences epitopopes, peptides
  • fusion proteins thereof as well as fusion proteins thereof and the like.
  • heterologous genes include genes from the various HPV strains (eg HPV-16 with E1. E2, E4, E5, E6, E7, L1, L2), HSV (eg glycoproteins gB, gC, gD, gE, gG, gH, gl, gK, gL, gM plus capsid proteins ), RSV (proteins: NS1 , NS2, N, P, M, SH, G, F, M2, L), HCV (protein/glycoproteins: C, E1 , E2, p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B) and chiamydia (MOMP, 60 Kda cystein-rich membrane protein, protein E, OMP, Npt2cp, IpdA, OMPB).
  • HSV eg glycoproteins gB, gC, gD, gE, gG, gH, gl,
  • the invention disclosed herein contemplates modifications of any of the aforementioned heterologous genes, as well as others known to those skilled in the art, such that the genes express increased amounts via optimization of the codon usage as described by Pavlakis et al. (see e.g., U.S. Patent No. 5,965,726; U.S. Patent No. 6,174,666; U.S. Patent No. 6,291 ,664; and the like) and Seed, B. et al. (see e.g., U.S. Patent No. 5,786,464; U.S. Patent No. 5,795,737; U.S. Patent No. 6,114,148; and the like). Also, any known oncogenic properties or activities of these genes not necessary for immune induction may be eliminated by mutations in the respective areas of the genes.
  • Figure 8 depicts a dual Ad system where in the MaxAd controlled vector: 1) the PSA promoter is utilized to drive E1 expression, 2) the RSV promoter is utilized to drive expression of the immunomodulatory gene, CD80 (b7.1), 3) the EF2 promoter is utilized to drive the expression of the immunomodulatory gene, interferon-gamma (IFNgamma or IFN ⁇ ) and 4) the SV40 promoter is utilized to drive the expression of the green fluorescent protein (GFP) antigen (Fig. 8).
  • GFP green fluorescent protein
  • LNCaP cells are PSA positive and are permissive for E1 expression from a PSA promoter
  • 293 control cells that constitutively express E1.
  • Higher levels of interferon-gamma are observed in PSA positive LNCaP cells (Fig. 10) where the viruses can replicate compared to DU145 cells that are PSA negative and do not support viral replication.
  • interferon-gamma expression is still observed in the PSA negative DU145 cell line indicating the ability of the Max-Ad to express target genes in the absence of replication.
  • Expression of the immunomodulatory gene CD80 is depicted in the LNCaP cell line (Fig. 11).
  • Example 7 In vivo Murine Studies Utilizing a MaxAd/GFP Vector
  • mice were immunized and boosted 2 weeks later by the intranasal (i.n.) or intraperitoneal (i.p.) route with MaxAd/GFP.
  • Control animals were immunized intranasally (i.n.) with a MaxAd virus expressing an FVIII protein.
  • the serum was examined for anti-GFP IgG antibodies by ELISA (Fig. 12). Both intranasal and intraperitoneal immunization resulted in the generation of high levels of serum IgG antibodies to GFP.
  • splenocytes were isolated 5 weeks post primary and 3 weeks post secondary immunization and expanded in vitro. Both routes of immunization were found to induce strong CTL responses as seen by the high level of lysis of GFP expressing but not control targets (Fig. 14).
  • the ability to induce strong GFP- specific CTL responses following mucosal immunization is proof-of-principle for a dual Ad system vaccine platform (e.g., including, but not limited to, a system comprising MaxAd as the controlled Ad).
  • the above studies demonstrate the ability of a dual Ad system to replicate in permissive cells, to express multiple target antigens and immunomodulatory genes and to induce immune responses to the target antigen carried by the controlled Ad (e.g., Max-Ad) component of the vector system.
  • the controlled Ad e.g., Max-Ad
  • Table 1 Phenotype of antigen-presenting cells (APC). Splenocytes were transduced with MaxAd/GFP and double stained with PE-conjugated macrophage, B and dendritic cell surface molecules and allophycocyanin-labeled antibodies to the cell surface molecules as shown above. The expression of cell surface markers on APC (B cells, macrophages and dendritic cells) was determined by flow cytometry.
  • Example 8 Controlled Ad Vector (e.g., MaxAd) Construction for HIV Vaccines
  • HIV-1 immunogen derived from strain NL4-3 (Genbank accession #M19921) is safe and elicits HIV-specific CTL responses in vitro.
  • the HIV-1 derived immunogen HIVgag/pol/tat nf /rev is driven by an EF1a promoter, generates viral particles, mimicking attenuated virus without the risk of infection.
  • Safety regarding the emergence of an infectious virus is assured by deleting the accessory genes vif, vpr, vpu and nef as well as the envelope gene env. Additionally, all cis acting elements such as both HIV LTRs and the packaging signal are completely deleted.
  • the HIV-1 immunogen codes for the regulatory protein HIV Rev, which ensures export of HIV mRNA into the cytoplasm and itself can elicit immune responses to help combat HIV infection.
  • the second regulatory protein, the transactivator Tat is known to be a powerful immunogen as well and, hence, is retained in MaxAd/HIV. However, some undesired effects of Tat's transactivating potential were avoided by expressing a nonfunctional form of Tat that still retains its immunogenic potential.
  • the cDNA for the HIV-1 gag/pol/tat/rev was isolated from the previously described HIV-1 based lentiviral vector HIV-1 DEN(3) (Blood, vol. 96, pp. 1327- 1333, (2000)) and cloned into a derivative of the eukaryotic expression vector pcDNA3.1 For safety purposes, the maximum amount of non-functional HIV sequence was deleted including areas in the env, vif, vpr and vpu sequence while retaining the full gag/pol/tat/rev sequence.
  • Group A GTV8063 injected into a single site at 5 x 10 vp/tumor in 100 ⁇ l PBS + 10% glycerol.
  • Group B AdTK injected into a single site at 5 x 10 vp/tumor in 100 ⁇ l PBS + 10% glycerol.
  • Group C 100 ⁇ l PBS + 10% glycerol injected into a single site.
  • LNCaP "fast” cells a human prostate cancer cell line, passage 38; grown in RPMI1640 + 10% fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • mice 15 nude mice received from Harlan on 3-27-01, DOB 2-26-01 prebleeds done on all mice on 4-3-01 LNCaP cells were split on 4-3-01, 1 :4 and grown in 225 cm flasks in RPMI1640 + 10% FBS.
  • LNCaP cells were harvested for injection using 5 ml trypsin-EDTA/flask, stopped with 10 ml DME + 10% FBS.
  • LNCaP cells were pooled into one 50 ml conical tube, spun at 1000 rpm for 10 minutes in the Megafuge tabletop centrifuge, room temperature. Media was aspirated off, then cells washed in 25 ml PBS 1 time by tituration. Aliquot taken for counting. Spun rest of cells again at 1000 rpm for 10 minutes. Cells were counted on hemacytometer after making a 1/10 dilution of cells.
  • Tumors were grown in mice for 5 weeks prior to treatment with virus or control. Tumor sizes were measured weekly with calipers. Volumes were calculated by taking length x width x depth x 0.52. At week 5, the 15 mice were randomized into 3 groups of 5 mice each based upon their average tumor volumes. The randomization attempted to have the average tumor sizes of each group be the same. Tumors were treated with virus or control by making the appropriate dilution of virus in 100 ⁇ l vehicle control (PBS + 10% glycerol). Virus or control were injected intratumorally in one site per tumor (groups A, B, C).
  • GTV8063 is a dual adenovirus (Dual Ad virus) that contains a therapeutic vector expressing human IL-3 plus the Adenovirus El gene under control of the prostate specific antigen (PSA) promoter. GTV8063 is also composed of a second vector, the helper virus AdTK deleted for the El gene.
  • PSA prostate specific antigen
  • mice palpable, treatable tumors developed within 5 weeks after tumor cell injection into the flanks of male nude mice. Twelve weeks from the time of tumor cell injection, which was 7 weeks after virus treatment, the mice treated intratumorally with PBS vehicle control or AdTK helper virus either had died or were terminated due to their tumor burden. In contrast, the mice that received the GTV8063 Dual Ad virus survived, with tumors that were significantly smaller than in the AdTK or PBS treated control groups. GTV8063-treated tumors decreased in size during the first 3 weeks post virus treatment, unlike the control groups which increased in size and also in growth rate.
  • FIG. 1 represents the volume of the tumors injected for groups A, B and C over the course of the experiment.
  • the arrow indicates point of treatment with virus or control.
  • GTV8063 Dual Ad demonstrated its oncolytic efficacy by preventing unchecked tumor growth in the LNCaP xenograft model in nude mice. Neither the helper virus AdTK alone nor the buffer was effective at reducing tumor size. There was a difference in tumor sizes between the GTV8063 and PBS treated group as early as 1 week following virus treatment. A significant difference became evident between the GTV8063-treated group and both of the control groups from the third week following virus treatment until the termination of the experiment. Tumors in the groups treated with AdTK or PBS continued to grow during the course of the experiment, increasing in size rapidly up to the termination of the experiment at week 12. However, GTV8063 treated tumors diminished in size for 3 weeks following virus treatment and remained small through week 12.
  • AdTK helper virus could not replicate on its own due to a lack of El, treating tumors with AdTK helper virus had no effect upon their growth, similar to the PBS treated control group. Therefore it was not the presence of a non-replicating adenovirus that exerted an oncolytic effect, but rather the Dual Ad virus that curtailed tumor growth in the GTV8063 treated group. This is attributed to viral replication within the tumors.
  • Group A GTV8063 injected into a single site at 5 x 10 vp/tumor in 100 ⁇ l formulation buffer.
  • Group B AdTK injected into a single site at 2.5 x 10 vp/tumor in 100 ⁇ l formulation buffer.
  • Group C 100 ⁇ l formulation buffer injected into a single site.
  • LNCaP "fast” cells a human prostate cancer cell line, passage 37; grown in RPMI1640 + 10% fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • Formulation buffer 40mg/ml Mannitol, lOmg/ml Sucrose, lOmM Tris, 0.001% Lutrol F-68, pH 7.2. Tracker number 111-184-001. 18 gauge needles 25 gauge needles 1 ml syringes METHODS:
  • mice 15 nude male mice received from Harlan on 12-11-01, DOB 11-16-01. prebleeds done on all mice on 12-12-01 LNCaP cells were split on 1-16-02, 1 :5 and grown in 225 cm flasks in RPMI1640 + 10% FBS.
  • LNCaP cells were pooled into one 50 ml conical tube, spun at 1200 for 7 minutes in the Megafuge tabletop centrifuge, room temperature. Media was aspirated off, then cells washed in 25 ml PBS 1 time by tituration. Aliquot taken for counting.
  • Tumors were established in 15 nude mice by injecting subcutaneously (S.Q.) in both legs, 100 ⁇ l per leg, on 1-22-02 with 1.5 x 10 cells per leg LNCaP "fast" cells, psg 37. Cells were injected S.Q. near the anterior region of the rectus femoris muscle.
  • Tumors were grown in mice for 3 weeks prior to treatment with virus or control. Tumor sizes were measured weekly with calipers. Volumes were calculated by taking length x width x depth x 0.52. At week 3, the 15 mice were randomized into 3 groups of 5 mice each based upon their average tumor volumes.
  • Tumors were treated with virus or control by making the appropriate dilution of virus in 100 ⁇ l formulation buffer vehicle control. Virus or control were injected intratumorally in one site per tumor (groups A, B, C).
  • GTV8063 is a dual adenovirus (Dual Ad virus) that contains a therapeutic vector expressing human IL-3 plus the Adenovirus El gene under control of the prostate specific antigen (PSA) promoter. GTV8063 is also composed of a second vector, the helper virus AdTK deleted for the El gene.
  • PSA prostate specific antigen
  • GTV8063 is also composed of a second vector, the helper virus AdTK deleted for the El gene.
  • the LNCaP xenograft tumor model palpable, treatable tumors developed within 3 weeks after tumor cell injection into the flanks of male nude mice. Twelve weeks from the time of tumor cell injection, which was nine weeks after virus treatment, the mice treated intratumorally with formulation buffer vehicle control or AdTK helper virus either had died or were terminated due to their tumor burden.
  • mice that received the GTV8063 Dual Ad virus survived, with tumors that were significantly smaller than in the AdTK or vehicle treated control groups.
  • GTV8063 -treated tumors decreased in size during the first 3 weeks post virus treatment, unlike the control groups which increased in size and also in growth rate.
  • the GTV8063 Dual Ad treated mice survived for the duration of the experiment, 12 weeks following tumor cell injection, without the tumor burden experienced by the control groups.
  • Figure 1 represents the volume of the tumors for the groups A, B and C over the course of the experiment.
  • Figure 1 Graph of tumor sizes. Arrow indicates time of treatment with viruses or control at week 3.
  • GTV8063 group B AdTK group
  • GTV8063 (group A) was statistically different from AdTK (group B) and PBS (group C) from week 8 on.
  • GTV8063 Dual Ad demonstrated its oncolytic efficacy by preventing unchecked tumor growth in the LNCaP xenograft model in nude mice. Neither the helper virus AdTK alone nor the formulation buffer was effective at reducing tumor size. There was a difference in tumor sizes between the GTV8063 and vehicle treated group as early as the second week following virus treatment until the termination of the experiment. This difference became statistically significant five weeks after injection of the tumors. Tumors in the groups treated with AdTK or vehicle continued to grow during the course of the experiment, increasing in size rapidly up to the termination of the animals at week 12 or 13 due to morbidity from their tumor burden. However, the mice with GTV8063- treated tumors were alive at the study end with much smaller tumor burden.
  • AdTK helper virus could not replicate on its own due to a lack of El, treating tumors with AdTK helper virus had no effect upon their growth, similar to the vehicle treated control group. Therefore, it was not the presence of a non-replicating adenovirus that exerted an oncolytic effect, but the combination Dual Ad virus replication that curtailed tumor growth in the GTV8063 group.
  • HIV Human immunodeficiency virus
  • Gallichan, W. S., and K. L. Rosenthal. 1996 Long-lived cytotoxic T lymphocyte memory in mucosal tissues after mucosal but not systemic immunization. J Exp Med 184:1879. Gallichan, W. S., and K. L. Rosenthal. 1998. Long-term immunity and protection against herpes simplex virus type 2 in the murine female genital tract after mucosal but not systemic immunization. J Infect Dis 177:1155.
  • Trillo-Pazos G., E. McFariane-Abdulla, I. C. Campbell, G. J. Pilkington, and I. P. Everall. 2000. Recombinant nef HIV-IIIB protein is toxic to human neurons in culture. Brain Res 864:315.
  • Extracellular HIV-1 virus protein R causes a large inward current and cell death in cultured hippocampal neurons: implications for AIDS pathology. Proc Natl Acad Sci U S A 95:4595.
  • Tat protein of human immunodeficiency virus type-1 promotes vascular cell growth and locomotion by engaging the alpha ⁇ betal and alphabeta3 integrins and by mobilizing sequestered basic fibroblast growth factor. Blood 94:663.
  • Ensoli B., L. Buonaguro, G. Barillari, V. Fiorelli, R. Gendelman, R. A. Morgan, P. Wingfield, and R. C. Gallo. 1993. Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat protein on cell growth and viral transactivation. J Virol 67:277.
  • HIV- 1 Tat protein exits from cells via a leaderless secretory pathway and binds to extracellular matrix-associated heparan sulfate proteoglycans through its basic region. Aids 11:1421. Helland, D. E., J. L. Welles, A. Caputo, and W. A. Haseltine. 1991 . Transcellular transactivation by the human immunodeficiency virus type 1 tat protein. J Virol 65:4547.
  • Extracellular HIV-1 Tat protein induces the rapid Ser133 phosphorylation and activation of CREB transcription factor in both Jurkat lymphoblastoid T cells and primary peripheral blood mononuclear cells. J Immunol 160:3891.
  • Tat protein induces human immunodeficiency virus type 1 (HIV-1 ) coreceptors and promotes infection with both macrophage-tropic and T- lymphotropic HIV-1 strains. J Virol 72:8952.
  • HIV-1 human immunodeficiency virus type 1
  • Dendritic cells transduced by multiply deleted HIV-1 vectors exhibit normal phenotypes and functions and elicit an HIV-specific cytotoxic T- lymphocyte response in vitro. Blood 96:1327.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Epidemiology (AREA)
  • Biotechnology (AREA)
  • Communicable Diseases (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Mycology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Oncology (AREA)
  • Plant Pathology (AREA)
  • Cardiology (AREA)
  • AIDS & HIV (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Transplantation (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

A composition for targeting one or more infectious agent(s) or target cell(s) comprising a complementary set of adenoviral vectors, wherein each vector is dependent upon the other for replication. The first vector of the pair may comprise an E1 expression cassette where E1 is expressed within one or more infectious agent(s) or target cell(s) from a promoter abd does not expres other adenoviral proteins. The secondvector provides adenoviral proteins required for packaging and replication. As such, the composition is useful for the delivery and expression of prophylactic and/or therapeutic genes, some of which demonstrate direct cytotoxic effects, indirect cytotoxic effects, or sensitize target cells to the effects of other treatments. The composition may encode one or more antigen(s), with or without immunomodulatory genes, to induce one or more immune response(s) to the encoded antigen or antigens.

Description

REPLICATION COMPETENT DUAL-AD VECTORS FOR VACCINE AND IMMUNOTHERAPY APPLICATIONS
CROSS-REFERENCES TO RELATED APPLICATIONS
This application claims priority from U.S. Patent App. No. 60/329,798, filed 16 October 2001. All patents, published and unpublished patent applications as well as any other scientific, technical and general writings referred to herein are incorporated by reference to the extent that they are not contradictory.
BACKGROUND OF THE INVENTION
Field Of The Invention
This invention is related to adenoviral (Ad) vectors and their use in the fields of vaccine and immunotherapy applications. More specifically, this invention is related to complementary Ad vectors that comprise and express exogenous sequences and yet retain the ability to replicate when introduced into an appropriate host organism or cell. Also contemplated are prophylactic and therapeutic formulations/compositions comprising the Ad vectors as well as prophylactic and therapeutic treatments using the Ad vectors.
Description Of The Related Art
Basic Adenoviral Vector Technology
Adenoviruses (Ad) consist of nonenveloped icosahedral (20 facets and 12 vertices) protein capsids with a diameter of 60-90 nm and inner DNA/protein cores (Horwitz, M.S. (1990) "Adenovindae and their replication." In: Fundamental Virology (2nd Ed.) Field, B.N. θt al., Eds., pp. 771-813, Raven Press: NY, NY). The outer capsid is composed of 252 capsomers arranged geometrically to form 240 hexons (12 hexons per facet) and 12 penton bases; the latter are located at each vertex from which protrude the antenna-like fibers. This structure is responsible for attachment of Ad to cells during infection. Wild-type Ad contain 87% protein and 13% DNA and have a density of 1.34 g/ml in CsCI.
The double-stranded linear DNA genome of Ad is approximately 36 kb, and is conventionally divided into 100 map units (mu). Each end of the viral genome has a 100-150 bp repeated DNA sequence, called the inverted terminal repeats (ITR). The left end (194-385 bp) contains the signal for encapsidation (packaging signal). Both the ITRs and the packaging signal are c/s-acting elements necessary for adenoviral DNA replication and packaging (Sussenbach, J.S. (1984) "The structure of the genome." In: The Adenoviruses, Ginsberg, H.S. Ed., pp. 35-124, Plenum Publishing Corp.: NY, NY; Philipson, L. (1984) "Adenovirus assembly." In: The Adenoviruses, Ginsberg, H.S., Ed., pp. 309-337, Plenum Publishing Corp.: NY, NY).
A simplified map of the adenovirus type 5 (Ad5) genome with a few key landmarks is diagrammed in Figure 1 (Stratford-Perricaudet, L. & Perricaudet, M. (1991) "Gene transfer into animals: the promise of adenovirus." In: Human Gene Transfer, Cohen-Haguenauer, O. & Boiron, M., Eds., p. 51-61, John Libbey Eurotext: France.; Graham, F.L., & Prevec, L. (1991) "Manipulation of adenovirus vectors." In: Methods in Molecular Biology (Vol. 7): Gene Transfer and Expression Protocols, Murray, E.J., Ed., The Humana Press Inc.: Clifton, NJ). The early (E) and late (L) regions of the genome contain several transcription units and are divided according to the onset of viral DNA replication. The E1 region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome as well as a few cellular genes (Nevins, J.R. (1990) "Adenovirus E1A-dependent trans-activation of transcription." Semin. Cancer Biol., vol. 1 , no. 1, pp. 59-68; Nevins, J.R. (1993) "Transcriptional activation by the adenovirus E1A proteins." Semin. Virol., vol. 4, pp. 25-31). The expression of the E2 region (E2A and E2B) leads to the synthesis of the proteins needed for viral DNA replication (Pettersson, U. & Roberts, R.J. (1986) "Adenovirus gene expression and replication: a historical review." In: Cancer Cells (Vol. 4): DNA Tumor Viruses (Botchan, M. et al., Eds., pp. 37-57, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY). The proteins from the E3 region prevent cytolysis by cytotoxic T cells and tumor necrosis factor (Wold, W.S.M. & Gooding, L.R. (1991) "Region E3 of adenovirus: a cassette of genes involved in host immunosurveillance and virus-cell interactions." Virology, vol. 184, pp. 1-8). The E4 proteins are involved in DNA replication, late gene expression and splicing, and host cell shut-off (Halbert, D.N. et al. (1985) "Adenovirus early region 4 encodes functions required for efficient DNA replication, late gene expression, and host cell shutoff." J. Virol., vol. 56, pp. 250- 257). The products of the late genes, including the majority of the viral capsid proteins, are expressed after processing of a 20-kb primary transcript driven by the major late promoter (MLP) (Shaw, A.R. & Ziff, E.B. (1980) "Transcriptions from the adenovirus-2 major later promoter yield a single early family of 31 coterminal mRNA and five late families." Cell, vol. 22, pp. 905-916). The MLP (located at 16.8 mu) is particularly efficient during the late phase of infection, and the mRNAs issued from this promoter possess a 5' tripartite leader (TL) sequence, which increases the preference of the host cell for such transcripts as opposed to host cell mRNAs.
The use of Ad as vectors for expression of heterologous genes began soon after the observation of hybrids between Ad and simian virus 40 (SV40) during the 1960s. Since then, Ad vectors have gradually developed into one of the major viral vectors in the current field of gene therapy, because: (a) Ad have been widely studied and well characterized as a model system for eukaryotic gene regulation, which served as a solid base for vector development; (b) the vectors are easy to generate and manipulate; (c) Ad exhibits a broad host range in vitro and in vivo with high infectivity, including non-dividing cells; (d) Ad particles are relatively stable and can be obtained in high titers (e.g., 1010-1012 plaque-forming unit (PFU)/ml); (e) the life cycle of adenovirus does not require integration into the host cell genome, and, therefore, the foreign genes delivered by Ad vectors are expressed episomally, thus having low genotoxicity if applied in vivo; (f) side effects have not been reported following vaccination of U. S. recruits with wild-type Ad, demonstrating their safety for in vivo gene transfer. Furthermore, Ad vectors have been successfully used in eukaryotic gene expression (Levrero, M. et al. (1991 ) "Defective and nondefective adenovirus vectors for expressing foreign genes in vitro and in vivo." Gene, vol. 101 , pp. 195-202; Ghosh-Choudhury, G. et al. (1986) "Human adenovirus cloning vectors based on infectious bacterial plasmids." Gene, vol. 50, pp. 161-171), vaccine development (Grunhaus, A. & Horwitz, M.S. (1992) "Adenoviruses as cloning vectors." Semin. Virol., vol. 3, pp. 237-252; Graham, F. L. & Prevec, L. (1992) "Adenovirus-based expression vectors and recombinant vaccines." In Vaccines: New Approaches to Immunological Problems, Ellis, R. V., Ed., pp. 363-390, Butterworth-heinemann: Boston, MA), and gene transfer in animal models (Stratford-Perricaudet, L. & Perricaudet, M. (1991) "Gene transfer into animals: the promise of adenovirus." In: Human Gene Transfer, Cohen-Haguenauer, O. & Boiron, M., Eds, pp. 51-61, John Libbey Eurotext: France; Stratford-Perricaudet, L. et al. (1992) "Widespread long-term gene transfer to mouse skeletal muscles and heart." J. Clin. Invest, vol. 90, pp. 626-630; Rich, D.P. et al. (1993) "Development and analysis of recombinant adenoviruses for gene therapy of cystic fibrosis." Human Gene Ther., vol. 4, pp. 461-476). Experimental routes for administrating recombinant Ad to different tissues in vivo include, but are not limited to, intratracheal instillation (Rosenfeld, M. A. et al. (1992) "In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium." Cell, vol. 68, pp. 143-155), muscle injection (Quantin, B. et al. (1992) "Adenovirus as an expression vector in muscle cells in vivo." Proc. Natl. Acad. Sci. USA, vol. 89, pp. 2581-2584), peripheral intravenous injection (Herz, J. & R.D. Gerard (1993) "Adenovirus-mediated transfer of low density lipoprotein receptor gene acutely accelerates cholesterol clearance in normal mice." Proc. Natl. Acad. Sci. USA, vol. 90, pp. 2812-2816), stereotactic inoculation to brain (Le Gal La Salle, G. et al. (1993) "An adenovirus vector for gene transfer into neurons and glia in the brain." Science, vol. 259, pp. 988-990) and orally (Hilleman, M.R. (1958) "Efficacy of and indications for use of adenovirus vaccine." Am. J. Public Health, vol. 48, pp. 153-158; Gaydos, CA. & J.C. Gaydos (1995) "Adenovirus vaccines in the U.S. military." Mil. Med., vol. 160, , no. 6, pp. 300-304; Gray, G.C. et al. (1999) "Respiratory diseases among U.S. military personnel: countering emerging threats." Emβrg. Infect. Dis., vol. 5, no. 3, pp. 379-387). The first trial of Ad-mediated gene therapy in humans was the transfer of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to lung (Crystal, R. G. et al. (1994) "Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis." Nature Genetics, vol. 8, no. 1, pp. 42-51).
A typical approach to vaccine therapy involves alteration of the infectious agent-host relationship and facilitation of recognition and destruction of infectious agents by the host immune system. In recent years, a number of cytokine genes have been isolated, cloned and characterized. Significant toxicity has accompanied the use of many of these biologies owing to the high concentrations needed to generate clinical effects. The combination of significant undesired effects and marginal therapeutic outcomes from systemic administration has stimulated efforts to genetically engineer target cells to produce the cytokines themselves (Rosenberg, et al. (1989) "Experience with the use of high-dose interleukin-2 in the treatment of 652 cancer patients." Ann. Surg., vol. 210, pp. 474-484).
In animal models, gene-modified target cells have been used as vaccines to stimulate anti-target cell response (Miller, A.R. et al. (1994) "Cytokine- mediated gene therapy for cancer." Ann. of Surg. One, vol. 1 , pp. 436-450; Dranoff, G. & Mulligan, R.C. (1995) "Gene transfer as cancer therapy." Adv. Immunol., vol. 58, pp. 417-454). The appeal of target cell-directed cytokine gene transfer is that the cytokine, produced locally, is immunologically more efficient and does not cause systemic toxicity. Antigens expressed on infectious cells in combination with high local concentrations of cytokine(s), may be exploited to create an immunological micro-environment virtually impossible to reproduce with exogenous cytokine administration. This immunological micro-environment created by such cytokine-producing target cells may result in generation of cytotoxic T lymphocytes. In a number of different animal models, cytokine- producing target cells have been shown to be effective in decreasing the destructive potential of particular target cells and increasing the expression of immunologically important molecules (Miller et al. (1994) supra; Dranoff, G. & Mulligan, R.C. (1995) supra. The initial target cell rejection appears to be accompanied by a nonspecific inflammatory response. However, rejection of cytokine-secreting target cells has in most instances led to the generation of systemic, target cell-specific immunity that is T cell-dependent.
In addition, new evidence indicates that co-stimulation of T cells by the co- stimulatory molecule B7 has both a positive and negative effect on T cell activation (Leach, D.R. et al. (1996) "Enhancement of antitumor immunity by CTLA-4 blockade." Science, vol. 271 , pp. 1734-1736). Other co-stimulatory molecules for T cells such as ICAM-I, LFA-3 and VCAM-I have also been implicated in the induction of an anti-target cell response (Springer, T.A. (1990) "Adhesion receptors of the immune system." Nature, vol. 346, pp. 425-434). The most powerful of these co-stimulatory signals is provided by the interaction of CD28 on a T cell with either or both of its primary ligands, B7-1 (CD80) and B7-2 (CD86) on the surface of an antigen presenting cell (Lenschow, D.J. et al. (1996) "CD28/B7 system of T cell costimulation." Annu. Rev. Immunol., vol. 14, pp. 233- 58). In a variety of model systems, target cells transfected with the B7 cDNA induced potent anti-target cell responses against both modified and unmodified target cells (Townsend, S. & Allison, J. (1993) "Tumor rejection after direct co- stimulation of CD8+ cells by B7-transfected melanoma cells." Science, vol. 259, pp. 268-370).
It has long been known that both Class I and Class II MHC molecules are involved in antigen presentation, although different pathways are utilized by the two classes of molecules. Class I MHC has been shown to activate target cell- specific CTL in vitro. Early work on target cell vaccination included transfection of MHC class I genes and resulted in suppression of the target cells (Hui, K. et al.
(1984) "Rejection of transplantable AKR leukemia cells following MHC DNA- mediated cell transformation." Nature, vol. 311 , pp. 750-752; Wallich, R. et al.
(1985) "Abrogation of metastatic properties of tumor cells by de novo expression of H-2K antigens following H-2 gene transfection." Nature, vol. 315, pp. 301-305). MHC class II genes were shown to be involved in activation of target cell-specific T-helper cells, and the introduction of Class II genes into target cells resulted in a decrease in the disease potential of the target cell and generated a systemic immune response against the target cell (Ostrand-Rosenberg, S. et al. (1990) "Rejection of mouse sarcoma cells after transfection of MHC class II gene." J. Immunol., vol. 144, pp. 4068-4071 ). Despite these positive results, the relationship between levels of MHC expression and immunogenicity is inconsistent among various models. Researchers have recently postulated that the inconsistency is caused by other cofactors, such as the B7 co-stimulatory molecule, which affects the antigen presentation by MHC/peptide complexes.
Interferon gamma (IFN-γ) is a pleiotropic cytokine that, for example, activates macrophages and plays an important role in the inflammatory response (Billiau, A. (1996) "Interferon-γ: biology and role in pathogenesis." Adv. Immunol., vol. 62, pp. 61-131). This pleiotropic cytokine is also a potent inducer of MHC class I and class II antigens and thus is capable of enhancing immune responses (Wallach, D. et al. (1982) "Preferential effect of interferon-γ on the synthesis of HLA-antigens and their mRNAs in human cells." Nature, vol. 299. pp. 833-836; Chen, L.K. et al. (1986) "Interferon: a cytotoxic T-lymphocyte differentiation signal." Eur. J. Immunol., vol. 16, pp. 767-770). Retroviral transduction of a cDNA encoding murine IFN-γ into a non-immunogenic murine target cell line that expresses low levels of MHC Class I only weakly induced upregulation of MHC class I antigen expression and generated anti-tumor CD8+ TIL. Following target cell rejection, long-lasting protection from rechallenge with parental cells was induced (Restifo, N.P. et al. (1992) "A nonimmunogenic sarcoma transduced with cDNA for interferon-γ elicits CD8+ T cells against the wild-type tumor: correlation with antigen presentation capability." J. Exp. Med., vol. 175, pp. 1423-1431). Moreover, innoculation of mice having micro-metastases with target cells producing large amounts of IFN-γ almost completely cured these mice by inducing CTL (Porgador, A. et al. (1993) "Antimetastatic vaccination of tumor- bearing mice with two types of IFN-γ gene-inserted tumor cells." J. Immunol., vol. 150, pp. 1458-1470). The cDNA for human IFN-γ has also been introduced into human target cells (Gansbacher, B. et al. (1992) "Retroviral gene transfer induced constitutive expression of interleukin-2 or interferon-γ in irradiated human melanoma cells." Blood, vol. 80, pp. 2817-2825; Gastl, G. et al. (1992) "Retroviral vector-mediated lymphokine gene transfer into human renal cancer cells." Cancer Res., vol. 52, pp. 6229-6236). Such cells secreting IFN-γ showed increased expression of MHC class I antigen, β2-microglobulin, and intracellular adhesion molecule I, as well as induction of MHC class II antigen expression. However, neoplasia formation by a human target cell line transplanted into nu/nu mice was not affected by IFN-γ secretion, whereas IL-2 production inhibited growth of the neoplasia.
Many other cytokines, chemokines, and intercrines have been shown to play several different roles in eliciting anti-target cell immunity (Allione, A. et al. (1994) "Immunizing and curative potential of replicating and nonreplicating murine mammary adenocarcinoma cells engineered with interieukin (IL)-2, IL-4, IL-6, IL-7, IL-10, tumor necrosis factor α, granulocyte-macrophage colony- stimulating factor, and γ-interferon gene or admixed with conventional adjuvants." Cancer Res., vol. 54, pp. 6022-6026; Plata-Salaman, C. R. & Borkoski, J. P. (1994) "Chemokines/ intercrines and central regulation of feeding." Am. J. Physiol, vol. 266, pp. R1711-1715; Zhang, W.-W. & Fang, X. (1995) "Gene therapy strategies for cancer." Exp. Opin. Invest. Drugs, vol. 4, pp. 487-514). Further studies using gene transfer of multiple cytokine or immuno-stimulatory genes have obtained induction of stronger anti-target cell immunity (Vagliani, M. et al. (1996) "Interieukin 12 potentates the curative effect of a vaccine based on interieukin 2-transduced tumor cells." Cancer Res., vol. 56, pp. 467-470). The cytokine or immune modulatory genes have also been used in combination with other gene transfer for development of more effective approaches to gene therapy against particular target cells (Zhang, W.-W. & Fang, X. (1995) supra).
Utilization of Adenovirus in Vaccines
Due to their size and capacity for carrying large segments of heterologous DNA (see supra), adenoviruses and adenovirus-based vectors make attractive candidates for the basis upon which a variety of vaccines may be made (Fooks, A.R. (2000) "Development of oral vaccines for human use." Curr. Opin. Mol. Ther., vol. 2, no. 1 , pp. 80-86; Babiuk, L.A. & Tikoo, S.K. (2000) "Adenoviruses as vectors for delivering vaccines to mucosal surfaces." J. Biotechnol, vol. 83, nos. 1-2, pp. 105-113; Hofling, K. et al. (2000) "Progress toward vaccines against viruses that cause heart disease." Hen., vol. 25, no. 3, pp. 286-290; Paolazzi, C.C. et al. (1999) "Rabies vaccine. Developments employing molecular biology methods." Mol. Biotechnol., vol. 11 , no. 2, pp. 137-147; Stephenson, J. (1998) "Defective adenoviruses as novel vaccines for the Flaviviridae." Clin. Diagn. Virol, vol. 10, nos. 2-3, pp. 187-194; Yamanouchi, K. et al. (1998) "New approaches to the development of virus vaccines for veterinary use." Rev. Sci. Tech., vol. 17, no. 3, pp. 641-653; Rolph, M.S. & Ramshaw, I.A. (1997) "Recombinant viruses as vaccines and immunological tools." Curr. Opin. Immunol, vol. 9, no. 4, pp. 517-524; Babiuk, L.A. et al. (1996) "Novel viral vaccines for livestock." Vet. Immunol. ImmunopathoL, vol. 54, nos. 1-4, pp. 355- 363; Ertl, H.C. & Xiang, Z. (1996) "Novel vaccine approaches." J. Immunol, vol. 156, no. 10, pp. 3579-3582; Imler, J.L. (1995) "Adenovirus vectors as recombinant viral vaccines." Vaccine, vol. 13, no. 13, pp. 1143-1151 ; Randrianarison-Jewtoukoff, V. & Perricaudet, M. (1995) "Recombinant adenovirus as vaccines." Biologicals, vol. 23, no. 2, pp. 145-157; Wilkinson, G.W. & Borysiewicz, L.K. (1995) "Gene therapy and viral vaccination: the interface." Br. Med. Bull, vol. 51, no. 1, pp. 205-216; Connell, N. et al. (1992) "Old microbes with new faces: molecular biology and the design of new vaccines." Curr. Opin. Immunol, vol. 4, no. 4, pp. 442-448; Graham, F.L. & Prevec, L. (1992) "Adenovirus-based expression vectors and recombinant vaccines." Biotechnology, vol. 20, pp. 363-390; Morgan, A.J. (1992) "Epstein-Barr virus vaccines." Vaccine, vol. 10, no. 9, pp. 563-571 ; Osterhaus, A.D. & de Vries, P. (1992) "Vaccination against acute respiratory virus infections and measles in man." Immunobiology, vol. 184, nos. 2-3, pp. 180-192; Johnson, D.C. (1991) "Adenovirus vectors as potential vaccines against herpes simplex virus." Rev. Infect. Dis., vol. 13, suppl. 11 , pp. S912-S916; Graham, F.L. (1990) "Adenoviruses as expression vectors and recombinant vaccines." Trends Biotechnol, vol. 8, no. 4, pp. 85-87; Hung, P.P. et al. (1990) "Adenovirus vaccine strains genetically engineered to express HIV-1 or HBV antigens for use as live recombinant vaccines." Nat. Immun. Cell. Growth Regul, vol. 9, no. 3, pp. 160- 164; Chanda, P.K. (1990) "Helper independent recombinant adenovirus vectors: expression of HIV env or HBV surface antigen." Int. Rev. Immunol, vol. 7, no. 1 , pp. 67-77; Gurwith, M.J. et al. (1989) "Current use and future directions of adenovirus vaccine." Semin. Respir. Infect, vol. 4, no. 4, pp. 299-303; Tikchonenko, T.I. (1989) "Adenoviruses as vectors for the transfer of genetic information and for' the construction of new type vaccines." Adv. Exp. Med. Biol., vol. 257, pp. 193-204; as well as references in any of the foregoing)
U.S. Patents Nos. 5,643,579 and 4,920,209 describe oral vaccines using recombinant adenovirus coding for heterologous antigens of other infectious organisms. The vaccines disclosed therein are contained within enteric-coated formulations so that the adenovirus infects the intestinal tract and expresses the heterologous antigen as well as the adenovirus antigens.
The invention disclosed herein improves upon the adenovirus vaccines known in the art by providing a complementary dual-Ad system as the basis for vaccines. Such a system has a larger gene delivery capacity permitting the incorporation of multiple antigen and immune stimulatory DNA beyond other viral systems. Such a system allows the separation of functions into the component parts and yet remains fully functional upon introduction into a patient or subject in need of treatment.
SUMMARY OF THE INVENTION
The present invention provides novel vectors, compositions and methods for treating disease or other conditions. In one embodiment, the invention provides a composition. In one embodiment of the composition, first and second adenoviral vectors are provided having complementary function and being mutually dependent on each other for replication in a host organism or target cell. In one embodiment, one of said adenoviral vectors comprises a promoter that regulates expression of an early gene that controls propagation of the adenoviral vector in a host organism or target cell. In another embodiment, one of said adenoviral vectors comprises a host organism or target cell activated promoter and limits propagation of the adenoviral vectors to specific host organisms or target cells. In addition, another adenoviral vector of the composition comprises a partial Ad genome which can support the Ad replication cycle in the host organism or target cell. The replication of these vectors in the host organism or target cell directly or indirectly induces one or more immune response(s) that may alter the growth, function or other activity of the host organism, target cells, pathogens and/or infectious agents. The host organism, target cells, pathogens and/or infectious agents may be involved in a disease state, including, but not limited to, infection, cancer, autoimmune disease, cardiovascular disorder or other condition(s) known to those skilled in the art.
The invention is related to the invention described in the following published International Applications (and to any applications from which the
International Applications claim priority): Int. App. No. PCT/US97/10218, filed 30
May 1997 and published 4 December 1997 as WO 97/45550; Int. App. No.
PCT/US97/23685, filed 19 December 1997 and published 30 July 1998 as WO
98/32860; Int. App. No. PCT/US98/01301 , filed 23 January 1998 and published 13 August 1998 as WO 98/35028; Int. App. No. PCT/US98/03473, filed 23
February 1998 and published 11 September 1998 as WO 98/39411 ; and Int.
App. No. PCT/US98/10330, filed 19 May 1998 and published 3 December 1998 as WO 98/54345.
It will be understood by those skilled in the art that the present invention is not limited to the particular embodiments described herein. Other applications of the present invention are contemplated.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. Ad5 genome and transcription units. The length Ad5 genome is about 36 kb, divided into 100 map units (mu). The dotted arrows represent early (E) transcription and the solid arrows represent late (L) transcription. The directions of transcription are indicated by arrows. Gaps between arrows indicate intervening sequences.
The box represents location of the major later promoter and tripartite leader sequences (MLP). The solid triangle at 1 mu represents the location of the packaging signal. Figure 2. Principle of a complementary-Ad vector system. Shown are two major components of the system: the supplemental-Ad, and the controlled-Ad vector. With the E1-transactivation from the helper cell by activation of the specific promoter/enhancer carried by the controlled-Ad, the supplemental-Ad replicates itself and produces the late proteins to form capsids. However, in the embodiment shown in this figure, packaging of the supplemental-Ad genome into the capsid is inefficient due to the packaging attenuation associated with the supplemental-Ad. In the presence of the controlled-Ad vector genome, the supplemental-Ad also supports the DNA replication of the controlled-Ad vector genome, which is preferentially packaged due to its wild-type packaging signal that has high affinity to the limiting amount of the packaging proteins. Further purification of the Ad vectors can be achieved by a biochemical or physical method, such as ultracentrifugation.
Figure 3. Prototype supplemental-Ad and controlled-Ad vectors. Shown is the general structure of the helper virus and the controlled-Ad vectors. The latter have up to 36-kb gene delivery capacity that can be quipped with single or multiple gene expression cassettes.
Figure 4. Basic composition of an complementary dual-Ad vector system. The basic elements of the system are the supplemental-Ad vector and the controlled-Ad vector. (A) The supplemental-Ad is similar to the E1 -substituted first generation Ad and, in this embodiment, has a partial deletion of its packaging signal. The E1 region is replaced by a reporter gene and, in this embodiment, lacZ gene for β-gal protein. (B) The controlled-Ad, containing only the minimal cis- element of Ad genome (two ITRs with the packaging signal), carries a promoter/enhancer to drive the Ad E1 gene (Ad5E1 ). The promoter/enhancer is activated specifically in target cell, such as a cancerous or transformed cell. The AdE1 gene under the control of this element is specifically transcribed in tumor cells. The E1 protein then trans-activates the genome of the supplemental virus that has the E1 region substituted by a reporter gene and also has a manipulated packaging signal such that packaging of the controlled-Ad is preferred in the cell. The supplemental virus, activated by the E1 proteins, is able to replicate in the tumor cells, resulting in lysis of those cells.
Figure 5. Variation possibilities of complementary-Ad vectors. Shown are the variable structures of the supplemental-Ad (A) and controlled-Ad
(B) that can be designed in accordance with the need of application. The critical aspect of the variation is that the two vectors complement each other in Ad genome function such as E1 genes and/or other early genes. The complementation elements of the two vectors are listed in the Table (C). The transgene and supporting elements were mainly included in the controlled-Ad vectors, but this does not exclude the requirement for the supplemental-Ad for those elements in certain circumstances. Deletion of an early Ad gene that encodes the cell cycling inducer will abrogate the replicative capability of adenovirus in the normal resting cells, but allow the controlled-Ad specifically drive replication and propagation of the complementary-Ad vectors in tumor cells that have defect in the counterpart of the deleted viral gene product. Two examples are the viral proteins E1b-p55 and E1 b that are dispensible for virus propagation in p53-deleted (or mutated) or retinoblastoma-deleted (or mutated) tumor cells, respectively.
Figure 6. Methods for generation of the complementary-Ad vector system. To generate the complementary-viral vector system, two different complementation protocols may be used that give similar yields. In the first, the controlled-Ad plasmid is co-transfected with vDNA from AdHβ, and the helper cells are cultured until CPE is observed. In the second method, three days after an initial co-transfection of the controlled-Ad vector plasmid with pBHGIO, AdHβ is added as supplemental virus, and the cells are cultured until CPE is observed.
■ Figure 7. Clinical application approach of a complementary-Ad vector system. Controlled-Ad are generated by co-transfection of two plasmids in a helper cell line: The controlled-Ad plasmid may contain one or more Ad genes regulated by a cell-activated promoter/enhancer, the supplemental-Ad plasmid may contain the remainder of the Ad genes, and the helper cell line is a tumor- derived cell line in which the cell-activated promoter of the controlled-Ad is functional. The controlled-Ad and the supplemental-Ad complement each other in the supplemental cell line and propagate as a virus mixture. The vector mixture can be purified through CsCI gradients and injected locally or systemically into the tumor mass or the tumor bed following surgical debulking. The cell-activated promoter/enhancer specifically transcribes the AdE1 genes in target cells to produce E1A and E1 B proteins that transactivate transcription and replication of the supplemental-Ad. The controlled-Ad vector also replicates with the propagation of the supplemental-Ad. The replication of the controlled-Ad produces high copy numbers of the controlled-Ad genome, which supports high level expression of the immunomodulatory genes to induce specific anti-cancer immunity. Propagation of the supplemental-Ad in the target cells results in lysis of the target cells. This system may generate a local target cell destructive effect and a systemic target cell destructive response that results in rejection of distant target cells.
Figure 8. A MaxAd vector comprising 4 (four) expression cassettes. Shown is a schematic view of GTV8053, an exemplary, non-limiting vector according to the present invention. Figure 9. Dual Ad replication in permissive cells. Shown is representative dual Ad replication in 13 cell types, including LNCaP cells, A549 cells, Chang cells, Saos-2 cells, H358 cells, Huh-7 cells, DU145 cells, SKHep-1 cells, 2237 cells, U2OS cells, 293 cells, Hep-2 cells, and HepG2 cells.
Figure 10. Interferon-gamma (IFNgamma or IFNγ) expression in PSA Dual Ad infected cells.
Figure 11. B7.1 (CD80 or b7.1 ) expression in PSA Dual Ad infected cells.
Figure 12. Serum IgG antibody levels to GFP following MaxAd/GFP immunization of mice. Mice were immunized twice by the intranasal (i.n.) or intraperitoneal (i.p.) route (5 x 109 vp) and the serum examined for IgG antibodies to GFP. Control animals were immunized intranasally with a MaxAd virus expressing FVIII protein.
Figure 13. Induction of IgG and IgA antibodies in the genital tract of MaxAd/GFP immunized mice. Mice were immunized twice by the intranasal (i.n.) or intraperitoneal (i.p.) route (5 x 109 vp) and genital tracts washes were taken 3 weeks later and examined for IgA and IgG antibodies to GFP. Control animals were immunized intranasally with a MaxAd virus expressing FVIII protein (5 x 109 vp).
Figure 14. Lysis of GFP expressing and control cells by splenocytes from individual mice immunized intranasally or intraperitoneally twice with MaxAd/GFP (5 x 109 vp).
Figure 15. Anti-HIV p24 (capsid) Western blot of 293 cells transfected with HIV-1 DEN(3) or the safety-modified HIV-1 immunogen in pcDNA3.1. The primary antibody is a 1 :2,000 dil. mouse anti-p24 antibody (Cell Sciences) followed by the 1 :10,000 dil. secondary anti-mouse HRP- labeled antibody (Jackson ImmunoResearch).
DETAILED DESCRIPTION OF THE INVENTION
It will be appreciated by those skilled in the art that the present invention provides reagents and methodologies that substantially improve current vaccine and immunotherapy technologies and applications useful for the treatment of infectious diseases and other conditions. The present invention provides mutually-dependent complementary, replicable adenoviral (Ad) vector system useful for the treatment of infectious diseases and other conditions. In one embodiment, the system comprises a "controlled Ad" vector and a "supplemental Ad" vector. The controlled-Ad has minimal Ad c/s-elements (inverted terminal repeats (ITR) and a packaging signal) and a transcriptional regulatory sequence that a target cell is capable of activating or is activated ubiquitously through a generalized, "house-keeping" gene promoter. In certain embodiments, the transcriptional regulatory region may be a cell- or tissue-specific transcriptional regulatory region driving expression of the Ad E1 genes. In other embodiments, the transcriptional regulatory region may be a ubiquitous, general "house- keeping" transcriptional regulatory region driving expression of the Ad E1 genes. The controlled-Ad vector further comprises an expression cassette providing for expression of a gene of interest such as an immunomodulatory gene or an Ad early-region gene. The supplemental Ad may comprise a packaging signal, a deletion of the E1 region, and the remainder of the Ad genome with or without substitution. The Ad vectors thus produced using this system comprise a pair of recombinant adenoviruses that are mutually dependent, and are termed "complementary Ad vectors." It is also possible that a composition comprising more than one type of complementary Ad with a single type of supplemental Ad vector may be utilized in practicing the present invention.
In an embodiment, the system is useful for the treatment of conditions in which particular infectious agents or target cells are known to be involved. For instance, upon local or systemic injection to treat an infectious agent or target cell population, the vectors will replicate in the host organism or target cell and express an effector protein, such as an antigen, immunogen, epitope or immunomodulatory protein, resulting in a local and/or systemic prophylactic and/or therapeutic effect and induction of a local and/or systemic prophylactic and/or therapeutic immune response. The complementary Ad system provides Ad vectors that replicate locally and/or systemically, such as within a host organism or in the direct vicinity of an infectious agent or target cell (e.g., including, but not limited to, dendritic cells) capable of utilizing the E1 expression cassette of the controlled Ad vector.
Those skilled in the relevant art will recognize techniques useful in practicing the present invention. These techniques are described in many generalized references, including, but not limited to: Molecular Cloning: A Laboratory Manual (Sambrook, J. et al, Eds. (1989) Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY); Gene Expression Technology (Methods in Enzymology, Vol. 185, Goeddel, D., Ed. (1991) Academic Press: San Diego, CA); PCR Protocols: A Guide to Methods and Applications (Innis, et al, Eds., (1990) Academic Press: San Diego, CA); Culture of Animal Cells: A Manual of Basic Techniques (R.I. Freshney (1987), 2nd edition, Liss, Inc.: New York, NY); Antibodies: A Laboratory Manual (Hariow & Lane (1988) Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY); Guide to Protein Purification. Methods in Enzymology, vol. 182 (M.P. Deutscher, Ed. Academic Press: San Diego, CA); Kuby Immunology (Goldsby, R.A. et al. (2000) 4th ed., W.H. Freeman & Co.); Current Protocols in Immunology (Coligan, J. et al. (1999) John Wiley and Sons); Immunobiology (Janeway, C. et al. (2001) 5th ed., Garland Publishing); Cellular and Molecular Immunology (Abbas, A.K. et al. (2000) 4th ed., W.B. Sanders Co.); Roitt's Essential Immunology (Roitt, I. & Delves, P.J. (2001) 10th ed., Blackwell Science Inc.); Vaccines (Plotkin, S.A. et al. (1999) 3rd ed., W.B. Sanders Co.); The Cytokine Handbook (Thomson, A. (1998) 3rd ed., Academic Press); and the like; as well as references in any of the foregoing. Other suitable references beyond those listed here would also be known to the skilled artisan and available. All cited references are hereby incorporated by reference. As used herein, a "nucleic acid molecule" is defined a plasmid, virus, autonomously replicating sequence, phage or linear segment of a single- or double-stranded DNA or RNA derived from any source. A "transcriptional regulatory region" may comprise a promoter, enhancer, silencer or repressor element and is functionally associated with a nucleic acid of the present invention. Unless otherwise stated, the transcriptional regulatory region may alternatively be referred to as a promoter or an enhancer. Preferably, the transcriptional regulatory region drives high level gene expression in the target cell. Exemplary transcriptional regulatory regions suitable for use in the present invention include, but are not limited to, the human cytomegalovirus (CMV) immediate-early enhancer/promoter, the SV40 early enhancer/promoter, the JC polyomavirus promoter and the chicken β-actin promoter coupled to the CMV enhancer (Doll, R.F. et al. (1996) "Comparison of promoter strengths on gene delivery into mammalian brain cells using AAV vectors." Gene Ther., vol. 3, no. 5, pp. 437-447), the Rous sarcoma virus (RSV) promoter, the tyrosine kinase (TK) promoter, the Rous Sarcoma Virus (RSV) promoter, the α-fetoprotein (AFP) promoter, the DF-3 mucin enhancer, the tyrosinase promoter, the carcinoembryonic (CEA) promoter, the tyrosinase promoter, the prostate specific antigen (PSA) promoter, the Hi parvovirus promoter or other suitable promoter. It should be noted that many other suitable promoters are available to the skilled artisan and would be useful within the vectors described herein. An "expression cassette" may be a DNA fragment comprising a coding sequence for one or more reporter gene(s) and/or one or more effector gene(s) operably linked to a transcriptional regulatory region or a transcriptional control region sufficient for expression of the encoded protein in an appropriate cell type.
A "reporter gene" or "reporter construct" may be a subchromosomal and purified DNA molecule comprising a gene encoding an assayable product. An "effector gene" refers to any gene that, upon expression of the polypeptide encoded by the gene, confers an effect on an organism, tissue or cell. A "transgene" refers to a gene that has been inserted into the genome of an organism other than that normally present in the genome of the organism. A gene expressed in a "tissue-specific" or "tumor-specific" manner is that which demonstrates a greater amount of expression in one tissue as opposed to one or more second tissues in an organism (i.e., preferential expression in one tissue as compared to another cell or tissue). Similarly, then, a tissue-, cell- or tumor- specific promoter demonstrates greater activity in one tissue, cell or tumor, respectively, as compared to another tissue, cell or tumor.
An "antigen," "epitope," or "immunogen" refers to any molecule capable of inducing an immune response. Such a molecule includes, but is not limited to, cell-mediated antigens, those that induce a humoral and/or cellular immune response, those that interact with T-cell and/or B-cell receptors; those that induce an innate immune response, and the like.
An "immunomodulatory gene" refers to any gene that, upon expression of its nucleic acid or protein product, serves to alter an immune reaction, including both activation and repression or suppression of an immune response.
A "tumor suppressor gene" refers to a gene that, upon expression of its protein product, serves to suppress the development of a tumor including but not limited to growth suppression or induction of cell death. A "growth suppressor gene" is defined as a gene that, upon expression of its protein product, serves to suppress the growth of a cell. An "oncogene" is defined as a cancer-causing gene.
"Adenoviral particle" refers to an infectious adenovirus, including both wild type or recombinant. The adenovirus includes but is not limited to a DNA molecule encapsidated by a protein coat encoded within an adenoviral genome.
"Heterologous DNA" refers to DNA introduced into an adenoviral construct that was isolated from a source other than an adenoviral genome or other DNA, either in its natural state or as a recombinant, that is foreign to a host cell. A
"recombinant adenoviral particle" is defined as an infectious adenovirus having at least one portion of its genome derived from at least one other source, including both adenoviral genetic material as well as genetic material other than adenoviral genetic material.
A "treatable condition" refers to a condition of an organism that may be altered by administration of a form of treatment including but not limited to those treatments commonly defined as being of medicinal origin. A "genetic condition" is defined in this application as a condition of an organism that is at least partially the result of expression or suppression of at least one specific gene including but not limited to the wild-type form of that gene and any mutant form of that gene.
Most adenoviral vectors currently in use by those skilled in the art are deleted in the E1 region and propagated in a permissive cell line that may provide the missing E1 functions (Graham, F.L. et al. (1977) "Characteristics of a human cell line transformed by DNA from human adenovirus type 5." J. Gen. Virol, vol. 36, pp. 59-72). Alternatively, the E1 function(s) may be provided in trans through some other component of the system (e.g., including, but not limited to, the helper-Ad). In other cases, helper viruses have been used to package vectors containing large deletions of the viral genome. However, a major disadvantage of such systems is that the majority of the packaged virus is helper virus (similar to, in the present invention, the supplemental-Ad). Applicants have previously demonstrated, (see e.g., Related Applications, supra) complementation of large deletions in the Ad vector genome by a helper vector having a partial deletion of the packaging signal.
In a complementary Ad system as contemplated herein, the supplemental vector may have the E1 region substituted by a β-gal expression cassette or other cassette and may be propagated in a cell line that expresses E1 , such as A549 cells, during preparation for use in the complementary Ad system. The supplemental vector complements the controlled-Ad vectors having those the adenoviral genes deleted.
In the present invention, the term "controlled-Ad vector" refers to a vector in which, with the exception of the E1 gene region, adenoviral coding sequences are deleted. Further, the controlled Ad vector retains the minimal genetic elements necessary for replication and packaging. The "supplemental-Ad vector" provides proteins required to maintain replication and packaging of both the controlled-Ad vector and the supplemental-Ad vector. In the present invention, a controlled-Ad vector comprising one or more early Ad genes (such as E1, E4 or E2) not present within the genome of the supplemental-Ad vector, is provided. Thus, the controlled-Ad may also support replication of the supplemental-Ad (acting as a helper of the helper). Therefore, the two vectors, the supplemental- Ad and the controlled-Ad, are mutually dependent upon one another for propagation.
Expression of E1 from the complementary Ad vector may be driven by a constitutively active or "general" promoter that is active in many cell types, such as the CMV promoter/enhancer, SV40 early enhancer/promoter, the JC polyomavirus promoter, the chicken β-actin promoter coupled to the CMV enhancer (Doll, R.F. et al. (1996) supra), the Rous sarcoma virus (RSV) promoter, and the tyrosine kinase (TK) promoter, for example. In this manner, the complementary Ad system may be used to treat conditions in which are particular population of target cells are either difficult to specifically identify or target. Conditions such as these would include autoimmune conditions including systemic lupus erythematosis (SLE or "lupus"), arthritis, or infectious disease. In the case of arthritis, for example, the constitutive promoter in the controlled Ad allows for expression of a beneficial immunosuppressive cytokine in the local environment of an arthritic "flare-up" (ie, the areas in which the patient is experiencing pain). In such a case, it is not necessary to limit expression to a particular cell type, but it is more important that the cytokine be expressed as efficiently as possible within the local mileu. However, the complementary Ad system may still provide for some replication in the local area, thus providing expression of the immunomodulatory proteins for a substantial period of time.
Alternatively, expression of E1 may be limited to certain types of cells or tissues. To confer target cell-specific propagation of both the supplemental-Ad and the controlled-Ad in specific tissues or cells, the promoter of one or more of the early genes necessary for adenoviral replication is operably linked to a cell or tissue-specific promoter. Any adenoviral gene necessary for replication may be operably linked to the target cell-specific promoter such as E1, E2, and E4. In particular, where the target cell is an infectious cell any promoter showing activity in the target cell is suitable while cell-specific activity such as that provided by promoters for α-fetoprotein, carcinoembryonic antigen (CEA), melanotransferrin, Erb-B2, tyrosinase, MUC1, prostate specific antigen (PSA, osteocalcin (sarcomas, prostate cancer), tyrosinase promoter, DF3 promoter, chromogranin A (neuroendocrine tumors, SMLC), prostate specific membrane antigen (PSMA) promoter, or the glial fibrillary protein (GFP) promoter, for example, are preferred. Other suitable promoters are known in the art, and are encompassed by the present invention. In one embodiment, the present invention may comprise a controlled-Ad vector containing the E1 region with the E1A promoter substituted by the α-fetoprotein (AFP) promoter. Only cells that are able to utilize the α- fetoprotein (AFP) promoter and that have been co-infected with both the controlled-Ad and the supplemental-Ad (i.e., at the site of injection) support propagation of the controlled-Ad and the supplemental-Ad. As both viruses are produced in similar amounts (due to the effect of the partial deletion or modification in the packaging signal of the supplemental-Ad), neighboring cells will be co-infected and, if those cells are able drive E1 expression from the AFP promoter, such as hepatocellular carcinoma cells, both viruses will continue to propagate. The further the distance from the injection site (tumor or tumor bed after surgery), the lower the chances of co-infection and therefore vector spread is halted. This is an important safety advantage over a single oncolytic vector scenario where, if a leaky tumor-specific promoter were utilized, the vector spread could be systemic and detrimental to the patient.
Another advantage of this invention over currently available vectors is that the combination of two defective vectors provides added capacity for therapeutic heterologous DNA. Using the typical Ad vector carrying a tumor-specific promoter substituting the Ad E1 promoter, less than 4-kb is available for additional effector genes into the vector. In the present invention, the capacity of the controlled-Ad vector for exogenous non-viral DNA is up to 36 Kb. Any gene with an antigenic potential may be incorporated into the controlled-Ad. In one embodiment, the immuno-stimulatory genes encoding B7.1 (a co-stimulatory molecule) and the interferon (IFN-γ) are incorporated into a single controlled-Ad as two separate expression cassettes. Many other expression cassettes would be suitable for use in this system.
1. Complementary-Ad Vector System
a. Composition of the system: The complementary-Ad vector system consists of two major parts: (1) a packaging-attenuated supplemental-Ad vector which is E1 deleted or otherwise substituted and (2) the controlled Ad vector including only minimal c/s-elements of the Ad viral genome such as E1 or other early stage genes such as E2 or E4. These vectors can be propagated in Ad helper cell lines that allows E1 fraπs-activation like A549 cells and/or regulation of packaging signal for the supplemental-Ad. Alternatively, E1 function may be provide by a component of the system other than the cell line. The supplemental-Ad has all of the viral genes and elements required to replicate and frans-complement the controlled-Ad vectors, except that the supplemental Ad vector has an E1 deletion or substitution. In addition, the modified supplemental- Ad vector may further comprise a mutated (less efficient) packaging signal that selects for preferential packaging of the controlled-Ad vector. The controlled-Ad vector, on the other hand, has the inverted terminal repeats (ITRs) and wild-type packaging signal (the c/s-elements for Ad DNA replication and packaging). The controlled-Ad may be controlled using at least two different methods: promoter replacement and/or functional deletion. Other suitable methods of control would be understood by the skilled artisan.
(1) Promoter replacement: In one embodiment, the natural viral promoter of AdE1A is substituted with a constitutive promoter that drives expression of AdE1 in the host cell, such as the cytomegalovirus (CMV) promoter/enhancer. Other suitable promoters include SV40 early enhancer/promoter, the JC polyomavirus promoter and the chicken β-actin promoter coupled to the CMV enhancer (Doll, R.F. et al. (1996) supra), the Rous sarcoma virus (RSV) promoter, and the tyrosine kinase (TK) promoter, for example. Using a controlled Ad having a constitutive promoter driving E1 in combination with supplemental Ad is useful for treating conditions where targeting of particular cell types is either not preferable or feasible, as described above.
In another embodiment, the natural viral promoter of the early gene for control of viral replication may be substituted by a heterologous promoter, which is active or inducible only in target cells or production cells. For example, the E1a promoter of adenovirus is the initiator of the viral replication cycle. The E1a promoter may be replaced by the α-fetoprotein promoter, which is active mainly in hepatocarcinoma cells. Other examples of promoters that are lineage-specific or may be specifically activated in tumor cells are described herein.
(2) Functional alterations: An adenoviral gene that is not essential for the viral replicative cycle in the target cells can be deleted, which can differentiate the viral specific propagation in the target cells from that of non-permissive in non-target cells. For example, tumor cells are more likely to be cycling than non- tumor cells. As such, the typical adenoviral functions that induce cycling of normal resting of cells are not required. Deletion of the adenoviral gene encoding an Ad cell protein that induces the cell cycle will abrogate the replicative capability of Ad in normal resting cells. Two examples are of such proteins are Ad E1 b-p55 and E1b, each being dispensible for virus propagation in p53-deleted (or mutated to decrease function) and retinoblastoma-deleted (or mutated to decrease function) tumor cells, respectively. Thus, a supplemental Ad vector may be designed by deleting E1b-p55 or E1b. Alternatively, Ad helper cell lines similar to A549 cells may also be utilized to trans-activate supplemental- Ad transcription and replication. These cells may be used to propagate the supplemental-Ad without the aid of the controlled-Ad. Cell lines may also contain control mechanisms for the packaging attenuation of the supplemental-Ad. Such mechanisms may include expression of proteins that bind the supplemental Ad packaging signal or interfere with other such functions. b. Mechanism of operation of the system: This system is designed to limit Ad replication locally to either a specific area of the body or a particular target cell or tissue, or both. For treatment of conditions for which targeting specific cells or tissues is either not desired or feasible, replication may still be limited using complementary Ad and a constitutive promoter by virtue of the complementary Ad system. The complementary Ad vector mixture is locally injected where particular target cells are available, replication can be limited through the use of target cell-specific promoters and either local or systemic administration. Systemic administration of transcriptionally targeted vectors provides for replication in only those cells capable of expressing E1 from the target cell specific promoter. Use of a complementary Ad system with a consitutive promoter driving E1 is more appropriate for a local injection, and not necessarily the optimum reagent for systemic administration. Thus, in one embodiment, this system comprises a tissue-specific or tumor-activated promoter activated in the helper cells or target tumor cells. It is preferred that the controlled Ad comprise a promoter driving transcription of the E1 gene. The E1 gene products may in turn drive the transcription and replication of the supplemental-Ad genome and controlled-Ad genomes simultaneously.
As described above, it may be beneficial to modify the packing signal of the supplemental Ad to preferentially package the controlled-Ad vector. The packaging protein of Ad is a frans-acting factor present in low amount in the infected cells and is the rate-limiting step for the packaging of Ad. As the wild- type packaging signal is recognized by the packaging protein with higher affinity than the engineered signal, packaging of the supplemental viral genomes with mutated packaging signal is partially or completely suppressed in the presence of the controlled-Ad viral genomes having a wild-type packaging signal. This provides for preferential packaging of the controlled-Ad vector. Thus, the two viral vectors are mutually dependent.
Further deletion or substitution of the supplemental-Ad in the other early regions (E2, E3, and/or E4) may also be performed. Incorporation of the corresponding deleted genes in the controlled-Ad increases the packaging and titer of the controlled-Ad and provides for dependence of the supplemental-Ad on the controlled-Ad. This is particularly useful for development of, for example, anti-tumor complementary-Ad vectors. In preparing complementary-Ad compositions from complementary-Ad/supplemental-Ad compositions, standard biochemical techniques may be utilized. For example, the complementary-Ad vectors may be further purified from supplemental Ad vectors through biological, biochemical, or physical methods such as ultracentrifugation through CsCI gradient, if purification of the Ad vectors is required for application.
c. Capability of the system: High gene-delivery capacity is one of the major features of this complementary-Ad vector system. Both the complementary and the supplemental Ad vectors may be utilized for delivery of heterologous DNA to cells or tissues, or to increase or decrease express of genes in cells or tissues. Nucleic acids encoding various effector or reporter gene sequences may be incorporated into either the controlled Ad or the supplemental Ad, thus providing for expression of the gene sequence in an infected cell. The maximal packaging capacity of Ad is about 105% of the genome, or approximately 38 kb (Ghosh-Choudhury, G. et al. (1987) "Protein IX, a minor component of the human adenovirus capsid, is essential for the packaging of full-length genomes." EMBO J., vol. 6, pp. 1733-1739). The size of the viral c/s-element in the controlled-Ad vector may comprise less than 1 kb. Deletion of E1 and E3 genes in the supplemental-Ad provides an additional 8 kb capacity. As such, the total capacity of the complementary-Ad vector system for heterologous DNA is up to 42 kb. The heterologous DNA can be either transgene expression cassettes or regulatory elements. The expression cassettes can be single or multiple, bicistronic or polycistronic. The regulatory elements can be DNA sequences for controlling transgene retention, transcription, and vector targeting.
Multiple genes may be incorporated into the complementary Ad vectors to provide one or more cytokines, chemokines, interleukins, and/or immuno- modulating agents to optimize the systemic anti-target cell immune responses resulting from infection with the complementary Ad vectors. It is also possible to construct a complementary Ad system that delivers one or more cytotoxic and/or immunostimulatory genes singly or in combination to enhance a cytotoxic effect.
With respect to immunomodulatory genes, the complementary Ad system may be utilized to shift the immune response toward a T helper 1 type may avoid the antibody blockage of viral spread, thereby assisting in rejection of metastases. A transgene such as the antigen costimulatory molecule B7 may serve to elicit optimal cytolytic immune responses. In certain embodiments, target cells transduced by one of the complementary vectors (i.e., either the controlled Ad or the supplemental Ad) may not be lysed until co-infected by the complementary vector, providing expression and presentation of immunomodulatory molecules. It is important to compare infection by single and complementary replication-competent vectors for safety and efficacy in immunocompetent models cells that to identify those complementary Ad vectors with the greatest potential as therapeutic reagents.
Furthermore, transfer of nucleic acids encoding immunomodulatory proteins into target cells has resulted in significant anti-target cell immune responses in several animal models. For example, the multiple immunoregulatory roles of IFN-γ are well documented and include enhancement of antigen presentation by upregulation of class I and class II MHC molecules (see e.g., Boehm, U. et al. (1997) "Cellular responses to interferon-γ" Ann. Rev. Immunol, vol. 15. pp. 749-795). Importantly, IFN-γ gene expression has been shown to sensitize human and other mammalian target cells to the effects of secondary treatments (see e.g., Gruninger, L. et al. (1999) "Sensitizing human cervical cancer cells in vitro to ionizing radiation with interferon β or γ." Radiat. Res., vol. 152, pp. 493-498; Janssens, M.Y et al. (1998) "Activation of inducible nitric oxide synthase in nitric oxide-mediated radiosensitization of hypoxic EMT-6 tumor cells." Cancer Res., vol. 58, pp. 5646-5648).
The skilled artisan is increasingly aware of the fact that antigen recognition alone is not sufficient for T cell activation to effector functions. "Second signals," such as co-ligation of auxiliary molecules, are also critical for generating T cell mediated immunity (Mondino, A. & Jenkins, M.K. (1994) "Surface proteins involved in T cell costimulation." J. Leukoc. Biol., vol. 55, no. 6, pp. 805-815; June, OH. et al. (1990) "Role of the CD28 receptor in T-cell activation." Immunol. Today, vol. 11 , no. 6, pp. 211-216). Antigen recognition in the absence of these second signals can lead to tolerance or "anergy." Two co-stimulatory molecules in particular, B7.1 (CD80) and B7.2 (CD86), the ligands for CD28 and CTLA-4, have recently received a great deal of attention as potent co-stimulators for T cell function. The B7.1 (CD80) co-stimulatory molecule has a dramatic role in T cell- antigen presenting cell interactions and in generating effective T cell responses (see e.g., Lenschow, D.J. et al. (1996) "CD28/B7 system of T cell co-stimulation." Annu. Rev. Immunol, vol. 14, pp. 233-258). In humans, CD80 is expressed on dendritic cells and is induced on activated B cells, T cells, NK cells and macrophages (Azuma, M. et al. (1993) "Functional expression of B7/BB1 on activated T lymphocytes." J. Exp. Med., vol. 177, pp. 845-850; Freeman, G.J. et al. (1989) "B7, a new member of the Ig superfamily with unique expression on activated and neoplastic B cells." J. Immunol, vol. 143, pp. 2714-2722). Northern analysis for mRNA expression of CD80 revealed that most carcinomas, leukemias of B cell origin (including non-T cell ALL), prolymphocytic leukemia, hairy cell leukemia and chronic lymphocytic leukemia were CD80 negative while some non- Hodgkin's lymphomas were positive (Freeman, G.J. et al. (1989) supra). These results suggest that lack of CD80 expression by many target cells may contribute to their poor immunogenicity.
It may be advantageous to include nucleic acids encoding immunomodulatory proteins, including, but not limited to, IFN^y and CD80, into the complementary Ad system (i.e., by insertion of an expression cassette into a complementary Ad) to induce anti-target cell immune response(s).
The complementary Ad system is useful for lysis or destruction of cells, where, for example, the controlled Ad comprises E1 under the transcriptional control of a cell-specific promoter, and one or more nucleotide sequences encoding an immunomodulatory protein, such as IFN^y and/or CD80.
Administration of the complementary Ad, in combination with a supplemental Ad having either a non-functional, substantially incapacitated, or wild type packaging signal, provides a system capable of causing lysis of the target cells directly and via immunostimulation. Importantly, the immune stimulation also provides for lysis of distant target cells. Thus, in one embodiment, the complementary Ad system may be complete as a system for lysis of target cells by incorporating into the controlled Ad vector a cell- or tissue-specific or non-specific E1 expression cassette, an immunomodulatory effector gene expression cassette, and a supplemental Ad vector for complementation of Ad proteins that are not encoded by the controlled Ad.
However, in another embodiment, it may be useful to incorporate nucleic acids encoding various genes into the complementary or supplemental Ad that may aid in cell lysis by, for instance, inhibiting the cell cycle or causing apoptosis. As such, the complementary Ad system may also be utilized to modify in vivo target cell function or regulate target cell growth. Ad vectors have distinct advantages over other viral vectors (i.e., retroviruses, HSV) in that high titers may be produced, which is useful in preparing vectors for in vivo gene therapy. The complementary-Ad vectors may be useful for transiently suppressing cell- or tissue-specific proliferation following local administration. Therefore, the complementary Ad system may be useful for modifying in vivo target cell function or regulating target cell growth.
The complementary Ad system may also be useful for selective delivery of transgenes to target cells or tissues in vivo by surface modification of the vectors. In one emobidment, the capsid proteins (i.e., hexons and fibers) encoded by the supplemental Ad vector may be modified to increase the affinity for target cell surface proteins, thereby targeting replication to a particular target cell or tissue. For example, a hexon or fiber may be engineered to bind certain epitopes or ligands (i.e., protein A for binding to an Fc portion of an IgG molecules). As such, modified hexon and fiber genes may be incorporated into the recombinant viral genome for generation of complementary Ad preparations having surface sites that interact with particular ligands as targeting agents. The viral particles thus produced will provide tissue or cell recognition capabilities (see e.g., Curiel, D.T. (1999) "Strategies to adapt adenoviral vectors for targeted delivery." Ann. NY. Acad. Sci.; vol. 886, pp. 158-171).
In another embodiment, the complementary Ad system may be used for Ad-mediated vaccination via direct in vivo approaches. For the purpose of vaccination, the immunogenicity of the E1 -substituted Ad vectors may be beneficial, and this has been taken advantage of in the development of Ad-based recombinant vaccines. The contribution of the controlled-Ad vectors for this type of application may be realized by using the E1 -substituted Ad vectors as helper virus and co-delivering genes encoding antigens and immuno-enhancing proteins. As described above, it is possible to design E1 expression cassettes to control E1 expression using special promoter/enhancer, such as an inducible promoter.
It is important to note that the complementary Ad system may be used in combination with other gene transfer and\or gene therapy methodologies. Conventional Ad vectors have been used in combination with polylysine, liposome, and other conjugation materials as gene delivery complexes. The complementary-Ad vectors may also be used in these types of combinations. Many such combinations are known by those of skill in the art and would be applicable to the present invention.
Thus, the complementary Ad system may be utilized to deliver nucleic acids encoding one or more immunomodulatory gene(s) (e.g., including, but not limited to, interieukin (e.g., IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 , IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, or IL-18, for example), interferon (e.g., IFN-α, -β, -γ), a co-stimulatory molecule (e.g., B7.1 (CD80), B7.2), chemokine (CCR5), suicide gene (e.g.., HSV-TK, CDA, among others); a cell cycle gene (e.g., a gene having an effect on progession of the cell cycle such as p16, p15, WAF1/CIP1/p21 , p27, ataxia telangiectesia (ATM), p53, GADD45, p57/Kip2, Rb, p107, p130, a TOR family member, ubiquitin, lkB); a cell differentiation gene (e.g., a gene that induces or inhibits cell differentiation); a DNA synthesis gene (e.g., a gene involved in the synthesis of DNA by a cell), a DNA repair gene (e.g., a gene related to a DNA repair pathway in a cell such as MSH2, MLH1 , PMS2, PCNA); an anti-proliferative gene; a tumor suppressor gene (e.g., p53, Rb, PTPase); an angiogenesis gene that inhibits angiogenesis (e.g., anti-angiogenesis gene such as endostatin, angiostatin, a protease (e.g., MMP-2), a tumor suppressor gene / apoptosis inducer-gene (e.g., p53)); a growth factor gene (e.g., G-CSF, GM-CSF, M-CSF, VEGF) an apoptosis gene (e.g., an ICE family member, ataxia telangiectesia (ATM), c-myc, fas, fas ligand, tumor necrosis factor (TNF), reaper, hid, p53, bax, bad, bak, nbk, bcl-Xg, ICErdlll, MCH- 2, CPP32, ICH-IL, Nedd2, TX, ICErelll, ICH-2, ICE, a lamin, PARP, JGIP, c-jun); and, tumor antigens (e.g., MAGE, MART, CD80, CD86). The nucleotide and amino acid sequences of each of the nucleic acids listed above, as well as the many other suitable sequences, are well known in the art and widely available (e.g., GenBank) to the skilled artisan. Methods for generating complementary Ad (e.g., controlled Ad or supplemental Ad) constructs using these sequences, using the reagents and methodologies provided herein, is well within the capabilities of the skilled artisan.
The complementary Ad system may also provide a nucleotide sequence providing a sequence-specific ribozyme, a sequence-specific RNase P, or an antisense construct. A suitable effector sequence would inhibit the expression or function of a target sequence encoding a protein or the protein itself, where expression or function of such a protein causes a detrimental effect on the generation of an immune response in a patient or subject (e.g., including, but not limited to, TNFbeta (TNFβ), other immuno-responsive proteins and the like). The nucleotide and amino acid sequences of each of the targets described herein, as well as the many other suitable target sequences, are well known in the art and widely available to the skilled artisan. Methods for generating ribozymes (Perlman, H. et al. (2000) "Adenovirus-encoded hammerhead ribozyme to Bcl-2 inhibits neointimal hyperplasia and induces vascular smooth muscle cell apoptosis." Cardiovasc Res., vol. 45, no. 3, pp. 570-578), Rnase P molecules (Cobaleda, C. & Sanchez-Garcia, I. (2000) "In vivo inhibition by a site-specific catalytic RNA subunit of RNase P designed against the BCR-ABL oncogenic products: a novel approach for cancer treatment." Blood, vol. 95, no. 3, pp. 731- 737), and antisense constructs (Matsushita, H. et al. (2000) "Transfection of antisense p53 tumor suppressor gene oligodeoxynucleotides into rat carotid artery results in abnormal growth of vascular smooth muscle cells.are also widely available and well within the capabilities of the skilled artisan." Circulation, vol. 101 , no. 12, pp. 1447-52).
2. The Supplemental-Ad Vector
a. The basic structure of the supplemental-Ad: The supplemental Ad vector, in its most basic form, provides the proteins required for replication of the controlled Ad vector, which is deleted for the DNA encoding Ad structural and other proteins required for replication. It is possible to utilize a supplemental Ad vector having either a wild-type or an engineered packaging signal. In one embodiment, the supplemental-Ad vector has two main structural features: a partial wild-type Ad genome and engineered packaging signal. In addition, the supplemental-Ad is preferably defective in replication, such as by deletion or substitution of E1. For the purpose of limiting packaging of the supplemental Ad in the presence of the controlled-Ad vector, the supplemental is preferably attenuated in packaging. In one embodiment, then, the general structure of the supplemental-Ad may be summarized as an Ad vector comprising a wild-type Ad genome with the exception of an altered E1 region and packaging signal. However, it is also possible to manipulate other essential regulatory genes of Ad such as E2 and E4 by altering or substituting these sequences. The viral genome may also be fragmented and incorporated into separate supplemental Ad vectors to further inhibit the replication competence of the supplemental-Ad or to reduce the size of the supplemental-Ad genome. It is important that these manipulations do not significantly alter the titer of the supplemental-Ad during preparation of stock reagents. With this proviso, modifications affecting both supplemental-Ad replication and packaging attenuation of the supplemental-Ad should be considered in designing the vector.
b. Function of the supplemental-Ad: The primary function of the supplemental-Ad is to supply capsids for packaging of the controlled-Ad vectors and lyse the target cells through viral propagation. In order to fulfill this function, the supplemental-Ad must be able to reproduce itself, although it is dependent on the activation of a promoter by a target cell driving expression of E1 , provided by the controlled-Ad. It is preferred that DNA replication and transcription of the supplemental Ad genome is sufficient to yield functional quantities of the late gene products (the capsid proteins) such that the titer of the controlled-Ad vectors remains high.
It is also possible to utilize a supplemental-Ad vector having a wild-type or only slightly attenuated packaging signal. As such, the supplemental Ad will be more useful for co-expression of effector genes, along with those express from the controlled-Ad. For instance, where the controlled Ad vector contains DNA encoding E1 along with several effector proteins such that it is close to maximum capacity, it may be useful to utilize a supplemental Ad having a packaging signal that is only moderately disabled. In this way, the combined titer of the complementary Ad and the supplemental Ad is sufficient, and the system has an evern higher capacity for expressing functional amounts of effector proteins.
c. The designs for packaging attenuation: One purpose for attenuating packaging the supplemental-Ad is to reduce the potential for overgrowth of the supplemental-Ad as compared to the controlled-Ad vectors. This is important when relatively high titer of the controlled-Ad vectors is required for particular applications. The packaging function of the supplemental-Ad is designed to be defective but not completely disabled, as the supplemental-Ad is preferably maintained in cunjunction with the controlled-Ad vector, thus forming the complementary-Ad vector system. There are several design possibilities that will result in packaging attenuation of the supplemental-Ad, as described below:
1. Packaging signal mutation: The Ad5 packaging signal is composed of a repeated element that is functionally redundant (Hearing, et al. (1987)
"Identification of a repeated sequence element required for efficient encapsidation of the adenovirus type 5 genome." J. Virol, vol. 671 , pp. 2555-
2558). Partial deletions of the packaging signal elements have been shown to reduce the yield of mutant Ad from several fold to approximately a hundred fold that of the Ad with wild-type packaging signal (Grable, M. & Hearing, P. (1992) "cis and trans requirements for the selective packaging of adenovirus type 5 DNA." J. Virol, vol. 6, pp. 723-731). In one embodiment, then, the packaging signal is partially deleted in one or more A-repeats of the wild-type Ad packaging signal.
2. Synthetic packaging signal: As described above, the Ad5 packaging signal has a consensus A (adenosine) enriched motif (e.g., A-repeat: TAAATTTG). Therefore, incorporation of an array of tandem repeats of a selected A-repeat or any other synthetic DNA motif may alter the affinity of the artificial packaging signal to the packaging proteins and thus packaging of the supplemental-Ad.
3. Packaging signal interference: It is also possible to design mechanisms for interfering with the packaging signal. In order to interfere with the effective binding of the packaging proteins to the signal, DNA binding sequences can be incorporated into or adjacent to the A-repeat array of the packaging signal of the supplemental-Ad. The inserted binding sites provide for high-affinity binding by the respective DNA binding proteins resulting in positional competition for the Ad packaging proteins to the Ad packaging signal. In certain embodiments, binding sites may be incorporated into packaging signal structures that have been modified to reduce functionality.
4. Packaging signal relocation: In its native orientation, the Ad packaging signal is positioned at the left end of the wild-type Ad genome. It has been reported that the packaging signal may be positioned at the right end and retain functionality. As such, the packaging signal is relocatable. In one embodiment, then, an engineered packaging signal may be inserted at a non-wild-type location, further attenuating the packaging efficiency of the supplemental-Ad. This may assist in minimizing reversion of the supplemental-Ad to wild-type Ad by homologous recombination between the engineered packaging signal of the supplemental-Ad and the wild-type packaging signal of the controlled-Ad vectors. Thus, in attenuating the packaging of the supplemental-Ad, at least two basic aspects may be explored: c/s-elements and frans-acting factors. As such, useful designs may wither of these two basic aspects or some combination of these two configurations. The c/s-elements of the packaging signal are considered to be the A-repeats and the fraπs-acting factors are the packaging proteins. The skilled artisan would understand that many other methods are suitable for disabling the supplemental Ad packaging signal, and such methods are contemplated as part of the present invention.
d. Supplemental-Ad vectors with the wild-type packaging signal: As described above, it may be preferable to incorporate a packaging signal in the supplemental Ad that is only mildly disabled. In antoher embodiment, the supplemental-Ad vector may comprise a wild-type packaging signal. The final outcome of the process depends upon the relative propagation speed of the complementary vectors versus the tumor cells. The rate of propagation of the supplemental Ad vector limited by the number of viruses produced per infected cell and may be increased using a wild-type packaging signal in the supplemental vector. In contrast to the non-replicating mini-Ad vector system, the use of a wild-type packaging signal for both the controlled and supplemental vectors in this system is advantageous, because supplemental vector propagation depends upon the controlled-Ad vector and wee versa. The use of wild-type packaging signal should increase the propagation efficiency of the complementary vectors and therefore the potency of treatments using the complementary Ad system.
3. The Controlled-Ad Vector
In its most basic embodiment, the controlled Ad vector is utilized to deliver effector genes (e.g., including, but not limited to, E1 ) that limit expression of the complementary Ad vectors to cells that are capable of expressing an effector gene coding sequence carried by the controlled Ad. For instance, the controlled- Ad may provide a construct comprising a tissue-specific promoter such as a ubiquitous, generalized "house-keeping" promoter or a cell- or tissue-specific promoter operably linked to an E1 coding sequence. E1, then, will only be expressed in cells capable of utilizing the ubiquitous, generalized "housekeeping" promoter or the cell- or tissue-specific promoter. The supplemental Ad depends upon E1 for replication, and therefore upon the complementary Ad to provide E1. The supplemental Ad will not replicate in the absence of E1 and the complementary Ad will not replicate in the absence of proteins expressed only by the supplemental Ad vector. Therefore, replication will only occur in cells capable of utilizing the AFP promoter. In addition, the controlled Ad comprises DNA encoding effector proteins.
a. The basic structure of the controlled-Ad vector: Ad vectors are typically utilized as a circularized plasmid form through the fusion of ITRs (Graham, F. L. (1984) "Covalently closed circles of human adenovirus DNA." EMBO J., vol. 3, pp. 2917-2922.). Ad vectors may also be employed as linear DNA. A basic form of a controlled-Ad vector is a circular DNA containing an ITR fusion, a plasmid DNA replication origin, the Ad E1 gene, and one or more polycloning sites. The ITR fusion preferably contains the left end of the wild-type Ad from map unit 0 to 1 and the right end from map unit 99 to 100, in which the DNA replication origins of Ad are located in the both ITRs and the wild-type packaging signal located adjacent to the left ITR. However, many ITR structures may be suitable for practicing the present invention and are contemplated herein. The controlled-Ad vector may be controlled by at least two basic mechanisms: promoter replacement and functional deletion.
The Ad E1 -region genes, either naturally occurring or modified versions, are incorporated into a controlled-Ad within an expression cassette comprising a promoter sequence. In one embodiment, the promoter may be cell-specific, tissue-specific, tumor-activated or otherwise active in a particular cell to control expression of the E1 genes. Alternatively, the promoter may be a ubiquitous, generalized "house-keeping" gene promoter active in many cell and/or tissue types. Also, the promoter may be an adenoviral promoter. b. The structural and functional possibilities of the controlled-Ad vectors: It is also possible to modify the structure of the controlled Ad vector. Additional DNA sequences and elements may be modified or included as described below:
1. Expression cassettes of transgenes: As understood in the art, an expression cassette is a basic transcription unit. A basic expression cassette is
" typically a linear DNA structure comprising a promoter, the gene of interest, and a polyadenylation (polyA) signal. The expression cassette may comprise two or more genes of interest arranged as a bi- or poly-cistronic structure, provided additional elements for translation or splicing of RNA are provided between the genes. For example, multiple expression cassettes may be joined using an internal ribosome entry site (IRES) construct (Martinez-Salas, E. (1999) "Internal ribosome entry site biology and its use in expression vectors." Curr. Opin. Biotechnol, vol. 10, no. 5, pp. 458-464). Generally, controlled-Ad vectors may comprise one or multiple transgene expression cassettes. As described above, a broad range of gene sequences may be useful for practicing the present invention. Such gene sequences may be incorporated into the complementary Ad vector as part of expression cassettes, thus providing for expression in cells following infection by the vectors of the complementary Ad vector system.
2. Functional elements for vector DNA retention: It is also possible to include elements that assist in integrating the expression cassette into the target cell genome such as, for example, an adeno-associate virus inverted terminal repeat (AAV-ITR), homologous regions of DNA that stimulate recombinatinon of the expression cassette into the host cell genome. It is a further possibility to include elements that retain the controlled-Ad vectors as an episomal form in target cells such as origin of replication.
3. Regulatory elements for control of DNA transcription: DNA elements having transcriptional regulation functions such as enhancers, repressors or activator-binding sites, introns, 5' or 3'-untranslated regions (e.g., constitutive promoter) may also be incorporated into the complementary Ad vector. The regulatory elements may drive expression in multiple cell types or may restrict expression to particular cell types (i.e., cell-or tissue-specific promoter) . Various regulatory elements, such as weak promoters, silencers and the like may also be utilized to regulate expression in a particular cell type.
The regulatory elements may also restrict expression of, for example, E1 from the controlled-Ad vector due to the presence of DNA sequences capable of binding a ligand that interferes with transcription. Expression of large amounts of E1 can be toxic to certain host cells; a method for regulating the amount of E1 expressed in a cell is desireable. In addition, such a system would be useful for limiting the spread of Ad particles in vivo.
For instance, the CMV promoter may be engineered to comprise tetO sequences to which may bind the tetR/KRAB protein expressed in a producer cell line. Tetracycline binds to tetR KRAB. Therefore, addition of tetracycline to the cell culture would result in an inactive tetR/KRAB protein, and transcription from the CMV promoter would occur. Removal of tetracylcine from the cell culture results in free tetR/KRAB, which binds to the tetO sequences in the promoter and inhibits transcription. Thus, expression of coding sequences of the controlled-Ad may be regulated using the "tetracycline-reversed repression system".
An exemplary controlled-Ad construct for use in a tetracycline-reversed repression system may comprise the Ad 5'ITR/packaging signal, stuffer DNA, a tefO/CMV promoter containing 1-10, preferably 7, repeats of the tetO sequence operably linked to E1a/E1b coding sequences, an antibiotic selection marker for propagation of the plasmid in prokaryote host cells, and an expression cassette. One such expression cassette comprises human IL-3 under the transcriptional control of the Rous sarcoma virus (RSV) promoter. Propogation of this vector would be accomplished using a producer cell line that constituitively expresses tetR/KRAB, which binds to the tefO/CMV promoter and represses transcription of the E1a/E1b transcripts. The addition of tetracycline prevents the binding of TetR/KRAB to the tetOCMV promoter, and the sequences under the transcriptional control of the tefO/CMV promoter are expressed. 4. Elements for vector and transgene targeting: Targeting can be achieved at least at two levels: vector surface modification and tissue-specific expression. Tissue specific promoters can be utilized to avoid expression in any cell type but that targeted for delivery in vivo.
5. Other elements: Additional elements may comprise DNA replication origins of prokaryotic or eukaryotic cells, plasmid or vector selection markers, and backbones of the vectors, and the like. Also contemplated herein is the incorporation of of non-mammalian nucleic acid sequences that influence one or more immunes (e.g., including, but not limited to, CpG islands and the like)
c. The designs for high titer production of the controlled-Ad vectors: High- titer production of the controlled-Ad vectors is contemplated by the present invention. Advantages of the complementary Ad vector system over conventional vectors include the ability to obtain high-titer preparation This is mainly due to the large quantity of viral capsid protein provided by the supplemental Ad and the high copy number of the controlled Ad viral genome. The following provide examples of methods for generating high-titer controlled- Ad vectors:
1. Enhanced DNA replication: Ad has a unique enzymatic system for DNA replication. The E2 region proteins are the major frans-acting elements responsible for viral DNA replication. The replication origins are the cis-elements located at both ends of the viral genome. To enhance controlled-Ad genome replication, a sufficient amount of E2 protein expressed from the supplemental virus is preferably provided. High-level expression of E2 region proteins may also be achieved by including the E2 gene within the controlled-Ad genome in addition to that in the supplemental virus. Other mechanisms for increase in copy numbers of the controlled-Ad genome would be understood by the skilled artisan, and may include the following: 2. Enhanced packaging signal: Additional or enhanced packaging sequences may be utilized to enhance packaging by either adding more tandem repeats at one end or both, or generating synthetic packaging signals.
3. Enhanced packaging process: The packaging processes of Ad are not yet completely understood. Whether proteins binding to DNA at sites other than the packaging signal of Ad play synergistic roles for packaging is not yet certain. If so, the sequences for DNA-binding proteins, refereed to anchorage points for packaging, naturally existing in the Ad genome may need to put back to the controlled-Ad genome.
In addition, the controlled Ad and the supplemental Ad may also comprise DNA encoding effector proteins that are expressed in cells where viral replication results from co-infection of cells by the controlled and the supplemental Ad vectors. Infection of cells separately by the controlled Ad or the supplemental Ad still results in cellular expression of encoded antigens and contributes to the. generation of immunity against the expressed antigenic proteins. In summary, a dual vector vaccine system has several advantageous features for generating immune responses to encoded antigens.
First, such a system has a very large capacity for expressing multiple antigens and immunomodulatory proteins that exceeds the capabilities of standard adenoviral vector systems. Expression of multiple antigens and immunomodulatory genes is desirable to increase immune responses in a variety of clinical circumstances where immune responses to multiple antigens is desirable to better protect against or treat an infectious pathogen or to destroy tumor cells that comprise multiple antigenic targets.
Second, the vector system is capable of replication and it is generally appreciated that "live" replicating vaccines are typically more efficacious than those that do not replicate. This beneficial effect is likely due in part to the amplification of antigen expression provided by replicating vectors. The replication capability may be adjusted to provide desirable degrees of attenuated replication.
Third, each of the vectors may function as separate adenoviral vectors to express antigens providing an additional source of antigens to generate immune responses. Cells infected by just one of the vectors provides for several days to several weeks of antigen expression to further the development of immunity.
This combination of features 1) large gene delivery and expression capacity; 2) antigen/immunomodulatory gene expression by replication competent vectors and 3) antigen/immunomodulatory gene expression by replication incompetent vectors, all in one vaccination system goes beyond the capabilities of single replication-competent and single replication-incompetent systems. In addition, as the dual Ad vectors contain all of the genes required for their replication, it is not necessary to genetically engineer producer cell lines containing adenoviral genes for manufacturing the vectors and they may be readily adapted for production in cell lines accepted for vaccine production including, but not limited to, those that have been well characterized with safety histories for vaccine production purposes (e.g., including, but not limited to, WI38 cells, MRC5 cells, VERO cells, embryonic cells and cell lines, and the like). Yet, these dual ad vector systems still retain the advantageous features of standard adenoviral vectors including, but not limited to, highly efficient gene transfer and expression, broad host range of infectable human cell types, readily formulated for oral, nasal, mucosal, intradermal, intramuscular, intraperitoneal and systemic administration.
It should also be noted that other embodiments involving similar constructions of other multiple viral and non-viral vector systems are useful. In general, the vectors will be complementary and will together contain the complete complement of genes required for the replication of one or more of the component vectors. The complementing vectors may be of the same or different types as long as they together provide the complete gene functions required to propagate one or more of the vectors. The deleted regions of the vectors that are complemented by the supplemental vectors may be substituted to deliver expression cassettes for a variety of medicinal purposes.
4. The Ad helper cell lines
The controlled Ad and supplemental vectors may be propagated in any suitable cell line. Ad helper cell lines are used to efficiently produce complementary Ad vectors. The supplemental vector are propagated standard cell lines for adenovirus production, including, but not limited to, A549 cells, VIRO cells and the like. Alternatively, the cells may be of the type that do not express E1. The complementary Ad vectors may be produced in a helper cell line. Suitable cell lines are also described in the published applications referred to in the Related Applications section, supra.
The helper cell lines may trans-activate transcription of the AdE1 genes of the controlled-Ad, resulting in activation of transcription and replication of the supplemental-Ad genome. In one embodiment, the helper cells are unique from 293 cells in that the E1 fragment is carried within the controlled-Ad genome which has no overlapping sequence with the supplemental-Ad genome. The helper cell will preferably have the ability to activate the promoter controlling the Ad E1 genes in the controlled-Ad for the complementary-Ad system to function properly. For example, if E1 is under the transcriptional control of the PSA promoter, the cell line utilized for production may be LNCaP, a cell line that produces PSA and supports oncolytic adenovirus production. The helper cells may also be modified by inclusion of genes that support high copy-number production of the controlled-Ad vector, enhancing packaging of the controlled-Ad vector, and attenuation of packaging of the supplemental-Ad. For example, as described above, a cell line may be engineered to express tetR/KRAB or other protein capable of interfering with transcription from a promoter construct. It would be understood by the skilled artisan that other suitable helper cell lines may be constructed using standard techniques in the art.
The cell lines are preferably characterized to rule out the presence of adventitious agents, mycoplasma and other viral or infectious contaminants. Cell banking is preferably done using serum free and antibiotic free medium. The cells are grown to sufficient quantities in either suspension culture (spinner flask or suspension bioreactor) or as adherent cells in large capacity bioreactors (e.g., the CellCube system in a GMP manufacturing environment). A master cell bank may be made and a working cell bank derived therefrom. A master virus bank may then be made from viruses propagated in cells from the working bank.
The method of virus production depends on whether adherent or non- adherent cell cultures are used. The type of culture will depend on the amount of virus production that can be obtained (burst size). In a preferred embodiment, conventional suspension bioreactors are utilized for scalable production.
The virus preparations should also be tested for expression of IL-3 and specific activity determined by antigen and functional assays in vitro appropriate for detecting expression of a particular reporter or effector gene. Assays may also be performed to determined the amount of replication competent adenovirus (RCA) in a particular preparation. It is preferred that RCA is not detected at all in the preparations. The virus may also be tested for genome integrity. For clinical applications, efficacy and toxicity studies should be performed using animal model systems for each lot. Stability tests should also be performed on the specified formulation for a clinical trial, for example.
The following Examples are for illustrative purposes only and are not intended, nor should they be construed, as limiting the invention in any manner. Those skilled in the art will appreciate that variations and modifications can be made without violating the spirit or scope of the claimed invention.
EXAMPLES
Example 1 : Replication-competent adenoviral vectors with deletions other than
E1 for vaccine and immunotherapy applications Contemplated herein are adenoviral vectors, constructed using techniques known in the art, as well as derived adenoviral particles with one or more deletions in the adenoviral genome other than functional deletions in the E1 gene. Such deletions will not significantly affect the ability of the virus to replicate or to form viral progeny.
One example may be a partial or full deletion of the E3 gene. E3 represses an essential part of antigen presentation to the immune system, namely MHC class I expression (Burgert, H.G. & Blusch, J.H. (2000) "Immunomodulatory functions encoded by the E3 transcription unit of adenoviruses." Virus Genes, vol. 21, nos. 1-2, pp. 13-25; Sparer, T.E. & Gooding, L.R. (1998) "Suppression of MHC class I antigen presentation by human adenoviruses." Curr. Top. Microbiol. Immunol, vol. 232, pp. 135-147). The lack of functional E3 may allow better immune recognition of the immunogen(s) (i.e., antigen(s)) inserted into the adenoviral genome. Such insertions may be at the site of E3 deletion. Full deletion of the E3 gene would generate roughly 2 Kb of space in which to insert heterologous DNA sequence(s).
Another example may be full or partial deletion of the E4 gene. E4 deletion would not affect the ability of the virus to replicate or generate viral progeny and may disable all or part of the oncogenic potential reported for E4 (Nevels, M. et al. (2000) "Two distinct activities contribute to the oncogenic potential of the adenovirus type 5 E4orf6 protein." J. Virol, vol. 74, no. 11 , pp. 5168-5181). Full deletion of the E4 region would generate about 2 Kb of space in which to insert heterologous DNA sequence(s).
Other potential targets exist and may be similarly exploited.
Example 2: Wild-type Adenovirus Comprising Heterologous DNA Sequences
Another possibility for generating replication-competent adenoviral vectors would be to insert heterologous sequence(s) into a wild-type adenoviral genome (a so-called "add-Ad"). This is possible since adenoviral replication and packaging are known to accommodate up to about 105% of the size of the wild- type adenoviral genome (see above). Thus, about 2 Kb of heterologous sequence(s) may be added to the wild-type adenovirus genome.
Example 3: Testing of the Replication-competent Adenoviral Vectors
The transduction efficiency (i.e., infectivity) of the replication-competent adenoviral vectors may be tested in appropriate primary cells and cell lines, prior to in vivo animal tests. It is possible to test for transduction efficiency as well as viability of the transduced cell or cell line using techniques known in the art. Viability may be assessed using trypan blue exclusion, for example. Any of the cells and/or cell lines that adenoviruses are known to infect may be used for these analyses. Also, it is possible to use known techniques to test for expression of the heterologous DNA sequence(s) that are inserted into the adenoviral vector(s). For example, ELISA, immunoprecipitation and gel electrophoresis are techniques known in the art for analyzing gene expression and protein quantification. The effects of the expressed heterologous DNA sequences may be assessed by noting morphological changes of the transduced cell or cell line.
Once particular replication-competent adenovirus vectors have been generated and characterized, they will be tested in vivo using appropriate host organisms (e.g., mice, rabbits and/or non-human primates). The host organism may be exposed to the adenovirus by any route previously mentioned (e.g., orally, mucosally, intramuscularly, sub-cutaneously, etc.). The exposed host may then be analyzed for adenovirus infection and heterologous DNA sequence expression by any number of techniques known in the art. The ability of the heterologous DNA sequence expression to protect the host organism from subsequent infection by the disease agent from which the heterologous DNA sequence was derived may be assessed by exposing the host organism to the intact disease agent and observing the course of infection (if any) of the disease agent in the host organism. Example 4: Vaccine and Immunotherapy Applications for Replication-competent
Adenoviral Vectors
Immunological compositions and formulations comprising the replication- competent adenoviral vectors may be prepared using techniques and methods well known in the art (see e.g., Remington's Pharmaceutical Sciences (Martin, E. W. (Ed.) latest edition, Mack Publishing Co., Easton, PA; and the like). Adenoviral vaccines may be prepared using techniques known in the art (see e.g., Hilleman, M.R. (1958) "Efficacy of and indications for use of adenovirus vaccine." Am. J. Public Health, vol. 48, pp. 153-158; Gaydos, C.A. & J.C. Gaydos (1995) "Adenovirus vaccines in the U.S. military." Mil. /Wed, vol. 160, , no. 6, pp. 300-304; Gray, G.C. et al. (1999) "Respiratory diseases among U.S. military personnel: countering emerging threats." Emerg. Infect. Dis., vol. 5, no. 3, pp. 379-387; references in any of the foregoing; and the like). Effectiveness of adenoviral vaccines according to the invention may also be assessed using techniques known in the art (see e.g., Simpson, S. & Marshall, E. (2001 ) "Immune control, memory, and vaccines." Science, vol. 293, p. 233; Enserink, M. (2001) "Driving a stake into resurgent TB." Science, vol. 293, pp. 234-235; Cohen, J. (2001 ) "'Breeding' antigens for new vaccines." Science, vol. 293, pp. 236-238; Germain, R.N. (2001 ) "The art of the probable: system control in the adaptive immune system." Science, vol. 293, pp. 240-245; Sprent, J. & Tough, D.F. (2001) "T cell death and memory." Science, vol. 293, pp. 245-248; Fearon, D.T. et al. (2001 ) "Arrested differentiation, the self-renewing memory lymphocyte, and vaccination." Science, vol. 293, pp. 248-250; Zinkernagel, R.M. & Hengartner, H. (2001) "Regulation of the immune response by antigen." Science, vol. 293, pp. 251-253; Pulendran, B. et al. (2001) "Sensing pathogens and tuning immune responses." Science, vol. 293, pp. 253-256; Dupuis, S. et al. (2001) "Impairment of mycobacterial but not viral immunity by a germline human STAT1 mutation." Science, vol. 293, pp. 300-303; Binder, G.K. & Griffin, D.E. (2001) "Interferon-γ-mediated site-specific clearance of alphavirus from CNS neurons." Science, vol. 293, pp. 303-306; Lu, Q. & Lemke, G. (2001 ) "Homeostatic regulation of the immune system by receptor tyrosine kinases of the Tyro 3 family." Science, vol. 293, pp. 306-311 ; references in any of the foregoing; and the like).
Example 5: Replication-Competent Dual Adenovirus Vectors for Vaccine and Immunotherapy Applications
The invention comprises a mixture of two complementary adenoviral vector constructs and derived adenoviruses dependent on each other for replication. These two complementing adenoviral vector particles are capable to deliver one or multiple immunogens and/or immunomodulatory genes to cells. The first vector is the helper vector in form of a wildtype adenovirus construct deleted for functional E1 expression. Additional modifications may be generated in the helper virus with some modifications described in US08/658.961. The second vector consists of the "gutless" adenoviral vector deleted in all functional1 adenoviral proteins except for E1 while retaining both ITRs and the packaging signal. The general description of such a second vector may follow the examples in set out in the published applications referred to in the Related Applications section, supra, except that the vector will code for functional E1 as well. E1 can be driven either by its own promoter, by any heterologous constitutive promoter, by any inducible/repressable or tissue-specific promoter. The immunogen(s) and/or immunomodulatory gene(s) may be coded for by the gutless adenovirus or by the helper virus in the E1 -deleted region or in any other region not necessary for replication once E1 is provided.
This dual-Ad system is replication-competent in those cells that harbor both adenoviral constructs or derived viruses and thus turn into dual-Ad producing cells leading to an in vivo amplification of these vectors. The dual-Ad vectors can become a powerful gene delivery system for immunogens and other immunomodulatory genes for vaccines and immunotherapy applications. Current adenoviral vectors for vaccines are usually single adenoviral systems. There are several conceivable advantages of the above-described replication-competent system. First, replicating vectors are generally much more powerful when it comes to the induction of strong immune responses. Second, a lower dose of replication-competent dual-Ad vectors will most likely achieve comparable results seen with higher doses of a replication-incompetent Ad vectors, which in turn helps reduce costs. Third, certain adenoviral proteins (e.g., capsid proteins) are immunogenic and are known to induce an innate immune response (Molec. Ther.3:708, 2001 ; Molec. Ther.3:697, 2001 ; Molec. Ther. 3:757, 2001). The de novo generation of these adenoviral proteins may induce a general nonspecific immune response, which may help in the immune recognition of the delivered immunogen(s). Fourth, this vaccine/immunotherapeutic can be generated in any mammalian cell line suitable for the propagation of adenoviruses and the production of vaccines/immunotherapeutics without the need to express E1 sequences. In case E1 is driven by an inducible/repressable promoter, producer lines modified to induce/repress E1 are necessary. In case E1 is driven by a tissue-specific promoter, the producer line must be able to provide the component that allows for expression of E1 driven by the tissue-specific promoter. Another advantage of such a system would be reduced toxicity due to the complementary nature of the system and the divided functions between the component parts. Still another advantage of such a system, would be a combined benefit of vector replication coupled with long-term antigen expression and/or immunomodulatory gene expression.
The immune responses to immunogens delivered by replication- incompetent adenoviral vectors may be increased by the use of Dual-Ad replication-competent adenoviral vectors.
Potential antigen targets contemplated by the present invention are a variety of heterologous genes including those coded by the HIV virus, namely HIVgag, HIVpol, HIVtat, HIVrev, HIVenv, HIVvif, HIVnef, HIVvpu and HIVvpr genes as well as the HIV RRE sequence and combinations thereof. Also modifications of these genes to disable specific functions, such as deleting the transactivating function of tat (see e.g., Caputo et al. (1999) J. Immunol, vol. 162, pp. :5631-, Singer, D. et al. (1998) J.Acquir. Immune Defic. Syndr. Hum. Retrovirol. vol. 17, pp. 9-) and enzymes coded for by HIVpol such as reverse transcriptase, integrase and polymerase and the like. Also contemplated are partial gene sequences (epitopes, peptides) as well as fusion proteins thereof and the like.
Other heterologous genes include genes from the various HPV strains (eg HPV-16 with E1. E2, E4, E5, E6, E7, L1, L2), HSV (eg glycoproteins gB, gC, gD, gE, gG, gH, gl, gK, gL, gM plus capsid proteins ), RSV (proteins: NS1 , NS2, N, P, M, SH, G, F, M2, L), HCV (protein/glycoproteins: C, E1 , E2, p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B) and chiamydia (MOMP, 60 Kda cystein-rich membrane protein, protein E, OMP, Npt2cp, IpdA, OMPB).
In general, the invention disclosed herein contemplates modifications of any of the aforementioned heterologous genes, as well as others known to those skilled in the art, such that the genes express increased amounts via optimization of the codon usage as described by Pavlakis et al. (see e.g., U.S. Patent No. 5,965,726; U.S. Patent No. 6,174,666; U.S. Patent No. 6,291 ,664; and the like) and Seed, B. et al. (see e.g., U.S. Patent No. 5,786,464; U.S. Patent No. 5,795,737; U.S. Patent No. 6,114,148; and the like). Also, any known oncogenic properties or activities of these genes not necessary for immune induction may be eliminated by mutations in the respective areas of the genes.
Example 6: Exemplary Dual Ad Systems for Vaccine and Immunotherapy Applications
Figure 8 depicts a dual Ad system where in the MaxAd controlled vector: 1) the PSA promoter is utilized to drive E1 expression, 2) the RSV promoter is utilized to drive expression of the immunomodulatory gene, CD80 (b7.1), 3) the EF2 promoter is utilized to drive the expression of the immunomodulatory gene, interferon-gamma (IFNgamma or IFNγ) and 4) the SV40 promoter is utilized to drive the expression of the green fluorescent protein (GFP) antigen (Fig. 8).
Replication of such a dual Ad system has been documented in permissive cells that permit the expression of E1 (Fig. 9: LNCaP cells are PSA positive and are permissive for E1 expression from a PSA promoter) and in 293 control cells that constitutively express E1. Higher levels of interferon-gamma are observed in PSA positive LNCaP cells (Fig. 10) where the viruses can replicate compared to DU145 cells that are PSA negative and do not support viral replication. However, interferon-gamma expression is still observed in the PSA negative DU145 cell line indicating the ability of the Max-Ad to express target genes in the absence of replication. Expression of the immunomodulatory gene CD80 is depicted in the LNCaP cell line (Fig. 11).
Subsequent studies have demonstrated the ability of the Max-Ad to generate both mucosal and systemic humoral and cellular immunity to the GFP antigen.
Example 7: In vivo Murine Studies Utilizing a MaxAd/GFP Vector
In preliminary in vivo studies, the ability of the MaxAd system to induce immune responses following systemic and mucosal routes of vaccination has been examined. Immunization of mice with a MaxAd/GFP encoding the green fluorescence protein (GFP) as a model antigen resulted in the induction of both humoral (Figs. 12 and 13) and cellular immune responses (Fig. 14) to GFP.
Mice were immunized and boosted 2 weeks later by the intranasal (i.n.) or intraperitoneal (i.p.) route with MaxAd/GFP. Control animals were immunized intranasally (i.n.) with a MaxAd virus expressing an FVIII protein. At 5 weeks post primary immunization the serum was examined for anti-GFP IgG antibodies by ELISA (Fig. 12). Both intranasal and intraperitoneal immunization resulted in the generation of high levels of serum IgG antibodies to GFP. To examine whether mucosal or systemic routes of immunization resulted in antibodies in the mucosal tissues, washes of the genital tract were taken daily and examined for antibodies to GFP. Both i.n. and i.p. routes of immunization resulted in the presence of GFP-specific IgG antibodies in the genital tract (Fig. 13) at similar levels. In contrast, i.n. immunization resulted in much higher levels of IgA antibodies to GFP in mucosal washes (Fig. 13), highlighting the ability of MaxAd to induce additional immune parameters with mucosal vaccination. These results are consistent with those observed using a first generation vector where i.n. immunization induced similar systemic and mucosal-specific humoral immune responses.
To examine cellular immune responses in i.n. and i.p. immunized mice, splenocytes were isolated 5 weeks post primary and 3 weeks post secondary immunization and expanded in vitro. Both routes of immunization were found to induce strong CTL responses as seen by the high level of lysis of GFP expressing but not control targets (Fig. 14). The ability to induce strong GFP- specific CTL responses following mucosal immunization is proof-of-principle for a dual Ad system vaccine platform (e.g., including, but not limited to, a system comprising MaxAd as the controlled Ad).
Hence, the above studies demonstrate the ability of a dual Ad system to replicate in permissive cells, to express multiple target antigens and immunomodulatory genes and to induce immune responses to the target antigen carried by the controlled Ad (e.g., Max-Ad) component of the vector system.
In in vitro studies, the ability of a MaxAd system to activate antigen- presenting cells (APC) has been examined (Table 1). Normal murine splenocytes were transduced with MaxAd/GFP for 24h in vitro and the expression of MHC Class II, CD40, ICAM and the costimulatory molecules such as B7.1 and B7.2 were monitored by flow cytometry. These data suggest that MaxAd/GFP has been taken up by the APC and during the process, cell surface molecules that aid in antigen presentation (MHC Class II), costimulation (B7.1 and 2) and signal transduction (CD40 and ICAM) have been upregulated.
Table 1 : % Cells Positive for Cell Surface Marker
ICAM CD40 B7J 72 Class II
Control APC 65 35 13 8 40 MaxAd/GFP
Transduced APC 98 57 57 50 58
Table 1 : Phenotype of antigen-presenting cells (APC). Splenocytes were transduced with MaxAd/GFP and double stained with PE-conjugated macrophage, B and dendritic cell surface molecules and allophycocyanin-labeled antibodies to the cell surface molecules as shown above. The expression of cell surface markers on APC (B cells, macrophages and dendritic cells) was determined by flow cytometry.
Example 8: Controlled Ad Vector (e.g., MaxAd) Construction for HIV Vaccines
An HIV-1 immunogen derived from strain NL4-3 (Genbank accession #M19921) is safe and elicits HIV-specific CTL responses in vitro. The HIV-1 derived immunogen HIVgag/pol/tatnf/rev is driven by an EF1a promoter, generates viral particles, mimicking attenuated virus without the risk of infection. Safety regarding the emergence of an infectious virus is assured by deleting the accessory genes vif, vpr, vpu and nef as well as the envelope gene env. Additionally, all cis acting elements such as both HIV LTRs and the packaging signal are completely deleted.
The HIV-1 immunogen codes for the regulatory protein HIV Rev, which ensures export of HIV mRNA into the cytoplasm and itself can elicit immune responses to help combat HIV infection. The second regulatory protein, the transactivator Tat, is known to be a powerful immunogen as well and, hence, is retained in MaxAd/HIV. However, some undesired effects of Tat's transactivating potential were avoided by expressing a nonfunctional form of Tat that still retains its immunogenic potential.
The cDNA for the HIV-1 gag/pol/tat/rev was isolated from the previously described HIV-1 based lentiviral vector HIV-1 DEN(3) (Blood, vol. 96, pp. 1327- 1333, (2000)) and cloned into a derivative of the eukaryotic expression vector pcDNA3.1 For safety purposes, the maximum amount of non-functional HIV sequence was deleted including areas in the env, vif, vpr and vpu sequence while retaining the full gag/pol/tat/rev sequence.
To verify that the expression level of this safety-modified HIV-1 immunogen in the pcDNA3.1 plasmid is comparable to that in the original HIV-
1 DEN(3) construct, both plasmids were transfected into 293 cells and HIV p24 intracellular levels determined via anti-HIV p24 Western blot from cell lysates (Figure 15).
Example 9 Testing GTV8063 in LNCaP xenograft tumors in nude mice
MATERIALS AND METHODS:
Experimental design: Using the LNCaP xenograft tumor model in nude mice, determine the oncolytic effect of GTV8063 Dual Ad virus replication. Establish LNCaP tumors
6 with 1.5 x 10 LNCaP cells per injection site in 100 μl Matngel.
Group A: GTV8063 injected into a single site at 5 x 10 vp/tumor in 100 μl PBS + 10% glycerol.
Group B: AdTK injected into a single site at 5 x 10 vp/tumor in 100 μl PBS + 10% glycerol. Group C: 100 μl PBS + 10% glycerol injected into a single site.
MATERIALS:
15 nude mice, male, 4 weeks old (Harlan) Matrigel brand basement membrane matrix, cat. # 354234, lot 005110 (Collaborative
Biomedical Products/BD Pharmingen) g
1.36 x 10 LNCaP "fast" cells, a human prostate cancer cell line, passage 38; grown in RPMI1640 + 10% fetal bovine serum (FBS). "Fast" cells are a subclone of LNCaP cells that had been selected by GenStar for more rapid growth than LNCaP cells obtained from ATCC.
5 x 10 vp GTV8063 Dual Ad virus, lot 2 (chemical lysis), prepared 3-30-01, ref.
12 11 #0091-100; concentration 1.1 x 10 vp/ml; diluted to 5 x 10 vp/ml by using 500 μl of stock and adding 600 μl PBS + 10% glycerol
5 x 10 vp AdTK helper virus, Ref. # 0057-005-003; concentration 5 x 10 vp/ml; no dilution necessary.
PBS +10% glycerol 18 gauge needles 25 gauge needles 1 ml syringes
METHODS:
15 nude mice received from Harlan on 3-27-01, DOB 2-26-01 prebleeds done on all mice on 4-3-01 LNCaP cells were split on 4-3-01, 1 :4 and grown in 225 cm flasks in RPMI1640 + 10% FBS.
On 4-9-01, 3 flasks (225 cm) of LNCaP cells were harvested for injection using 5 ml trypsin-EDTA/flask, stopped with 10 ml DME + 10% FBS. LNCaP cells were pooled into one 50 ml conical tube, spun at 1000 rpm for 10 minutes in the Megafuge tabletop centrifuge, room temperature. Media was aspirated off, then cells washed in 25 ml PBS 1 time by tituration. Aliquot taken for counting. Spun rest of cells again at 1000 rpm for 10 minutes. Cells were counted on hemacytometer after making a 1/10 dilution of cells.
4 5 6
Avg: 54 cells/square x lO x lO = 54 x 10 cells/ml = 5.4 x 10 cells/ml
5.4 x 10 cells/mi x 25 ml = 136 x 10 cells total
To get 1.5 x 10 cells/injection: 136 x 10 /1.5 x 10 = 90.6 injections. For 100 μl injection, use 9.1 ml Matrigel to resuspend cells.
PBS aspirated off, cells resuspended in 9.1 ml Matrigel (with phenol red) on ice. Kept on ice at all times until injection. Tumors were established in 15 nude mice by injecting subcutaneously (S.Q.) in both legs,
100 μl per leg, on 4-9-01 with 1.5 x 10 cells per leg LNCaP "fast" cells, psg 38. Cells were injected S.Q. near the anterior region of the rectus femoris muscle.
Tumors were grown in mice for 5 weeks prior to treatment with virus or control. Tumor sizes were measured weekly with calipers. Volumes were calculated by taking length x width x depth x 0.52. At week 5, the 15 mice were randomized into 3 groups of 5 mice each based upon their average tumor volumes. The randomization attempted to have the average tumor sizes of each group be the same. Tumors were treated with virus or control by making the appropriate dilution of virus in 100 μl vehicle control (PBS + 10% glycerol). Virus or control were injected intratumorally in one site per tumor (groups A, B, C).
Statistical analyses were performed subsequently by a two-way repeated measures Analysis of Variance (RM-ANOV A).
RESULTS: GTV8063 is a dual adenovirus (Dual Ad virus) that contains a therapeutic vector expressing human IL-3 plus the Adenovirus El gene under control of the prostate specific antigen (PSA) promoter. GTV8063 is also composed of a second vector, the helper virus AdTK deleted for the El gene.
In the LNCaP xenograft tumor model, palpable, treatable tumors developed within 5 weeks after tumor cell injection into the flanks of male nude mice. Twelve weeks from the time of tumor cell injection, which was 7 weeks after virus treatment, the mice treated intratumorally with PBS vehicle control or AdTK helper virus either had died or were terminated due to their tumor burden. In contrast, the mice that received the GTV8063 Dual Ad virus survived, with tumors that were significantly smaller than in the AdTK or PBS treated control groups. GTV8063-treated tumors decreased in size during the first 3 weeks post virus treatment, unlike the control groups which increased in size and also in growth rate. As seen on the graph in Figure 1, there was no difference between the average tumor sizes of the Ad TK or PBS treated groups throughout the course of the experiment. The GTV8063 Dual Ad treated mice survived for the duration of the experiment, 12 weeks following tumor cell injection, without the tumor burden experienced by the control groups. Figure 1 represents the volume of the tumors injected for groups A, B and C over the course of the experiment. The arrow indicates point of treatment with virus or control.
Single site injection
Figure imgf000056_0001
CM to ■ _■ _0 \D r- oo σ> o — CM ___: .__. _*_ __c __. .___ -___ __c ,,— '"- ,— > > > > > > > > > Figure 1: Expt. 4 graph of average tumor sizes. Arrow indicates point of treatment at week 5 following tumor establishment.
Statistical Analyses
A two-way repeated measures Analysis of Variance (RM-ANOVA) was performed with Group and Week as classification variables. The output from this analysis is can be found in Tablel . The interaction term was significant, pO.OOOl . In this table, the pairwise group comparisons are rows, weeks are columns and the table values are p-values. Table 1 shows no differences at all time points between the two control groups, B and C. The GTV8063 DualAd group A was statistically different from the two control groups from week 8 through the study end in the comparisons of Groups A and B and Groups A and C.
Table 1. Summary of p-values from the RM-ANOVA
A: GTV8063 group
B: AdTK group
C: PBS + 10% glycerol buffer control
Figure imgf000057_0001
week 8 on.
COMMENTS: In this LNCaP tumor model, we determined the effect of Dual Ad virus GTV8063 on tumor growth. Because of the biological specificity of the virus for human cells, we observed the effects of viral replication only upon the human LNCaP tumors. As human Adenovirus does not replicate in mouse cells, biodistribution of the virus was not considered a factor in the results. Expression of the therapeutic gene for human IL-3 would not exert an effect on the tumors in this mouse model due to the species specificity of the cytokine. Human IL-3 protein by itself would not be expected to have an effect upon the tumors.
Histological analysis from previous experiments determined that adenovirus hexon protein was still present in the tumors at the termination of the experiment. The kinetics of viral replication over time were not addressed in this experiment.
GTV8063 Dual Ad demonstrated its oncolytic efficacy by preventing unchecked tumor growth in the LNCaP xenograft model in nude mice. Neither the helper virus AdTK alone nor the buffer was effective at reducing tumor size. There was a difference in tumor sizes between the GTV8063 and PBS treated group as early as 1 week following virus treatment. A significant difference became evident between the GTV8063-treated group and both of the control groups from the third week following virus treatment until the termination of the experiment. Tumors in the groups treated with AdTK or PBS continued to grow during the course of the experiment, increasing in size rapidly up to the termination of the experiment at week 12. However, GTV8063 treated tumors diminished in size for 3 weeks following virus treatment and remained small through week 12. Because AdTK helper virus could not replicate on its own due to a lack of El, treating tumors with AdTK helper virus had no effect upon their growth, similar to the PBS treated control group. Therefore it was not the presence of a non-replicating adenovirus that exerted an oncolytic effect, but rather the Dual Ad virus that curtailed tumor growth in the GTV8063 treated group. This is attributed to viral replication within the tumors.
Example 10 Testing GTV8063 in LNCaP xenograft tumors in nude mice
MATERIALS AND METHODS:
Experimental design: Using the LNCaP xenograft tumor model in nude mice, determine the oncolytic effect of GTV8063 Dual Ad virus replication. Establish LNCaP tumors with 1.5 x 10 LNCaP cells per injection site in 100 μl Matrigel.
Group A: GTV8063 injected into a single site at 5 x 10 vp/tumor in 100 μl formulation buffer.
10 Group B: AdTK injected into a single site at 2.5 x 10 vp/tumor in 100 μl formulation buffer. Group C: 100 μl formulation buffer injected into a single site.
MATERIALS:
15 nude mice, male, 4 weeks old (Harlan). Matrigel brand basement membrane matrix, cat. # 354234, lot 0051 10 (Collaborative Biomedical Products/BD Pharmingen)
3 x 10 LNCaP "fast" cells, a human prostate cancer cell line, passage 37; grown in RPMI1640 + 10% fetal bovine serum (FBS). "Fast" cells are a subclone of LNCaP cells that had been selected by GenStar for more rapid growth than LNCaP cells obtained from ATCC.
5 10 vp GTV8063 Dual Ad virus, ref. # 0054-111 ; concentration 5 x 10 vp/ml; no dilution necessary.
2.5 x 10 vp AdTK helper virus, Ref. #0054-115; concentration 2.4 x 10 vp/ml
Formulation buffer: 40mg/ml Mannitol, lOmg/ml Sucrose, lOmM Tris, 0.001% Lutrol F-68, pH 7.2. Tracker number 111-184-001. 18 gauge needles 25 gauge needles 1 ml syringes METHODS:
15 nude male mice received from Harlan on 12-11-01, DOB 11-16-01. prebleeds done on all mice on 12-12-01 LNCaP cells were split on 1-16-02, 1 :5 and grown in 225 cm flasks in RPMI1640 + 10% FBS.
On 1-22-02, 7 flasks of 225 cm were harvested for injection using 4 ml trypsin- EDTA/flask, stopped with 10 ml DME + 10% FBS. LNCaP cells were pooled into one 50 ml conical tube, spun at 1200 for 7 minutes in the Megafuge tabletop centrifuge, room temperature. Media was aspirated off, then cells washed in 25 ml PBS 1 time by tituration. Aliquot taken for counting.
To get 1.5 x 10 cells/injection: g Removed 3 x 10 cells, enough for 100 mice (200 injections) into 50 ml tube. Spin in
Megafuge, 1200 φm, 7 minutes. PBS aspirated off. For 100 μl injection, use 20 ml Matrigel to resuspend cells. Kept on ice at all times until injection.
Tumors were established in 15 nude mice by injecting subcutaneously (S.Q.) in both legs, 100 μl per leg, on 1-22-02 with 1.5 x 10 cells per leg LNCaP "fast" cells, psg 37. Cells were injected S.Q. near the anterior region of the rectus femoris muscle.
Tumors were grown in mice for 3 weeks prior to treatment with virus or control. Tumor sizes were measured weekly with calipers. Volumes were calculated by taking length x width x depth x 0.52. At week 3, the 15 mice were randomized into 3 groups of 5 mice each based upon their average tumor volumes.
Tumors were treated with virus or control by making the appropriate dilution of virus in 100 μl formulation buffer vehicle control. Virus or control were injected intratumorally in one site per tumor (groups A, B, C).
Statistical analyses were performed subsequently by a two-way repeated measures Analysis of Variance (RM-ANOVA).
RESULTS: GTV8063 is a dual adenovirus (Dual Ad virus) that contains a therapeutic vector expressing human IL-3 plus the Adenovirus El gene under control of the prostate specific antigen (PSA) promoter. GTV8063 is also composed of a second vector, the helper virus AdTK deleted for the El gene. In the LNCaP xenograft tumor model, palpable, treatable tumors developed within 3 weeks after tumor cell injection into the flanks of male nude mice. Twelve weeks from the time of tumor cell injection, which was nine weeks after virus treatment, the mice treated intratumorally with formulation buffer vehicle control or AdTK helper virus either had died or were terminated due to their tumor burden. In contrast, the mice that received the GTV8063 Dual Ad virus survived, with tumors that were significantly smaller than in the AdTK or vehicle treated control groups. GTV8063 -treated tumors decreased in size during the first 3 weeks post virus treatment, unlike the control groups which increased in size and also in growth rate. As seen on the graph in Figure 1, there was no difference between the average tumor sizes of the AdTK or vehicle treated groups. The GTV8063 Dual Ad treated mice survived for the duration of the experiment, 12 weeks following tumor cell injection, without the tumor burden experienced by the control groups. Figure 1 represents the volume of the tumors for the groups A, B and C over the course of the experiment.
Figure imgf000060_0001
week 2 week4 week 6 week 8 week week week week
10 12 14 16
Time post LNCap cell injection 1.5x10Λ6 cells/tumor
Figure 1: Graph of tumor sizes. Arrow indicates time of treatment with viruses or control at week 3.
Statistical Analyses Methods
A two-way repeated measures Analysis of Variance (RM-ANOVA) was performed with Group and Week as classification variables. The output from this analysis can be found in Table 1. The interaction term was significant, pO.OOOl . In this table, the pairwise group comparisons are rows, weeks are columns and the table values are p-values. Table 1 shows no significant differences at all time points between the two control groups, B and C. The GTV8063 DualAd group A was statistically different from the two control groups from week 8 through the study end in the comparisons of Groups A and B and Groups A and C. Table 1. Summary of p-values from the RM-ANOVA
A: GTV8063 group B: AdTK group
C: formulation buffer
Figure imgf000061_0001
GTV8063 (group A) was statistically different from AdTK (group B) and PBS (group C) from week 8 on.
COMMENTS: In this LNCaP tumor model, we determined the effect of Dual Ad virus GTV8063 on tumor growth. Because of the biological specificity of the virus for human cells, the lytic effects of viral replication are only expected in the human LNCaP tumor in this xenograft model. Since human adenovirus does not replicate in mouse cells, biodistribution of the virus was not considered a factor in the results. Expression of the therapeutic gene for human IL-3 would not exert an effect on the tumors in this mouse model due to the species specificity of the cytokine. Human IL-3 protein by itself would not be expected to have an effect upon the tumors.
Histological analysis from previous experiments determined that adenovirus hexon protein was still present in the tumors at the termination of the experiment. Following the replication of the virus over time was not addressed in this experiment.
GTV8063 Dual Ad demonstrated its oncolytic efficacy by preventing unchecked tumor growth in the LNCaP xenograft model in nude mice. Neither the helper virus AdTK alone nor the formulation buffer was effective at reducing tumor size. There was a difference in tumor sizes between the GTV8063 and vehicle treated group as early as the second week following virus treatment until the termination of the experiment. This difference became statistically significant five weeks after injection of the tumors. Tumors in the groups treated with AdTK or vehicle continued to grow during the course of the experiment, increasing in size rapidly up to the termination of the animals at week 12 or 13 due to morbidity from their tumor burden. However, the mice with GTV8063- treated tumors were alive at the study end with much smaller tumor burden.
Because AdTK helper virus could not replicate on its own due to a lack of El, treating tumors with AdTK helper virus had no effect upon their growth, similar to the vehicle treated control group. Therefore, it was not the presence of a non-replicating adenovirus that exerted an oncolytic effect, but the combination Dual Ad virus replication that curtailed tumor growth in the GTV8063 group.
REFERENCES
Kaul, R., D. Trabattoni, J. J. Bwayo, D. Arienti, A. Zagliani, F. M. Mwangi, C. Kariuki, E. N. Ngugi, K. S. MacDonald, T. B. Ball, M. Clerici, and F. A. Plummer. 1999. HIV-1 -specific mucosal IgA in a cohort of HIV-1 -resistant Kenyan sex workers. Aids 13:23.
Mazzoli, S., L. Lopalco, A. Salvi, D. Trabattoni, S. Lo Caputo, F. Semplici, M. Biasin, C. Bl, A. Cosma, C. Pastori, F. Meacci, F. Mazzotta, M. L. Villa, A.
G. Siccardi, and M. Clerici. 1999. Human immunodeficiency virus (HIV)- specific IgA and HIV neutralizing activity in the serum of exposed seronegative partners of HIV- seropositive persons. J Infect Dis 180:871.
Mazzoli, S., D. Trabattoni, S. Lo Caputo, S. Piconi, C. Ble, F. Meacci, S. Ruzzante, A. Salvi, F. Semplici, R. Longhi, M. L. Fusi, N. Tofani, M. Biasin, M. L. Villa, F. Mazzotta, and M. Clerici. 1997. HIV-specific mucosal and cellular immunity in HIV-seronegative partners of HIV-seropositive individuals [see comments]. Nat Med 3:1250.
Kaul, R., F. A. Plummer, J. Kimani, T. Dong, P. Kiama, T. Rostron, E. Njagi, K. S. MacDonald, J. J. Bwayo, A. J. McMichael, and S. L. Rowland-Jones. 2000. HIV-1 -specific mucosal CD8+ lymphocyte responses in the cervix of HIV-1- resistant prostitutes in Nairobi. J Immunol 164:1602.
Gallichan, W. S., D. C. Johnson, F. L. Graham, and K. L. Rosenthal. 1993. Mucosal immunity and protection after intranasal immunization with recombinant adenovirus expressing herpes simplex virus glycoprotein B. J Infect Dis 168:622.
Gallichan, W. S., and K. L. Rosenthal. 1995. Specific secretory immune responses in the female genital tract following intranasal immunization with a recombinant adenovirus expressing glycoprotein B of herpes simplex virus. Vaccine 13:1589.
Gallichan, W. S., and K. L. Rosenthal. 1996. Long-lived cytotoxic T lymphocyte memory in mucosal tissues after mucosal but not systemic immunization. J Exp Med 184:1879. Gallichan, W. S., and K. L. Rosenthal. 1998. Long-term immunity and protection against herpes simplex virus type 2 in the murine female genital tract after mucosal but not systemic immunization. J Infect Dis 177:1155.
J lnf Dis 168:622-629
Virology 224:487-497
Bruce, C. B., A. Akrigg, S. A. Sharpe, T. Hanke, G. W. Wilkinson, and M. P. Cranage. 1999. Replication-deficient recombinant adenoviruses expressing the human immunodeficiency virus Env antigen can induce both humoral and CTL immune responses in mice. J Gen Virol 80:2621.
Lubeck, M. D., R. J. Natuk, M. Chengalvala, P. K. Chanda, K. K. Murthy, S. Murthy, S. Mizutani, S. G. Lee, M. S. Wade, B. M. Bhat, and et al. 1994. Immunogenicity of recombinant adenovirus-human immunodeficiency virus vaccines in chimpanzees following intranasal administration [published erratum appears in AIDS Res Hum Retroviruses 1995 Jan;1 1 (1 ):189]. AIDS Res Hum Retroviruses 10:1443.
Natuk, R. J., M. D. Lubeck, P. K. Chanda, M. Chengalvala, M. S. Wade, S. C.
Murthy, J. Wilhelm, S. K. Vernon, S. K. Dheer, S. Mizutani, and et al.
1993. Immunogenicity of recombinant human adenovirus-human immunodeficiency virus vaccines in chimpanzees. AIDS Res Hum
Retroviruses 9:395.
Buge, S. L., L. Murty, K. Arora, V. S. Kalyanaraman, P. D. Markham, E. S. Richardson, K. Aldrich, L. J. Patterson, C. J. Miller, S. M. Cheng, and M. Robert-Guroff. 1999. Factors associated with slow disease progression in macaques immunized with an adenovirus-simian immunodeficiency virus (SIV) envelope priming- gp120 boosting regimen and challenged vaginally with SIVmac251 . J Virol 73:7430. Zhang, W. W., S. F. Josephs, J. Zhou, X. Fang, R. Alemany, C. Balague, Y. Dai,
D. Ayares, E. Prokopenko, Y. C. Lou, E. Sethi, D. Hubert-Leslie, M.
Kennedy, L. Ruiz, and S. Rockow-Magnone. 1999. Development and application of a minimal-adenoviral vector system for gene therapy of hemophilia A. Thromb Haemost 82:562.
Zhang, W. 1997. Adenoviral vectors: development and application. Exp Opin Invest Drugs 6:1420.
Balague, C, J. Zhou, Y. Dai, R. Alemany, S. F. Josephs, G. Andreason, M. Hariharan, E. Sethi, E. Prokopenko, H. Y. Jan, Y. C. Lou, D. Hubert- Leslie, L. Ruiz, and W. W. Zhang. 2000. Sustained high-level expression of full-length human factor VIII and restoration of clotting activity in hemophilic mice using a minimal adenovirus vector. Blood 95:820.
Borgland, S. L., G. P. Bowen, N. C. Wong, T. A. Libermann, and D. A. Muruve. 2000. Adenovirus vector-induced expression of the C-X-C chemokine IP- 10 is mediated through capsid-dependent activation of NF-kappaB. J Virol 74:3941.
Hum Gene Ther 70:965-976
Am J Repir Cell Mol Biol 21 : 388-394
J Virol 74:11 173-11180
Science 290:486-492
Haddad, K., J. Ramprakash, M. Sedegah, Y. Charoenvit, R. Baumgartner, S. Kumar, S. L. Hoffman, and W. R. Weiss. 2000. Plasmid vaccine expressing GM-CSF attracts infiltrates including immature dendritic cells into injected muscles. Journal Immunology 165:3772. Tarr, P. E. 1996. Granulocyte-macrophage colony-stimulating factor and the immune system. Med Oncol 13:133.
Kurzrock, R., M. Talpaz, and J. U. Gutterman. 1992. Very low doses of GM-CSF administered alone or with erythropoietin in aplastic anemia. Am J Med 93:41.
McNeel, D. G., K. Schiffman, and M. L. Disis. 1999. Immunization with recombinant human granulocyte-macrophage colony- stimulating factor as a vaccine adjuvant elicits both a cellular and humoral response to recombinant human granulocyte-macrophage colony- stimulating factor.
Blood 93:2653.
Trillo-Pazos, G., E. McFariane-Abdulla, I. C. Campbell, G. J. Pilkington, and I. P. Everall. 2000. Recombinant nef HIV-IIIB protein is toxic to human neurons in culture. Brain Res 864:315.
Filler, S. O, P. Jans, P. W. Gage, and D. A. Jans. 1998. Extracellular HIV-1 virus protein R causes a large inward current and cell death in cultured hippocampal neurons: implications for AIDS pathology. Proc Natl Acad Sci U S A 95:4595.
Chan, S. Y., M. C. Louie, J. R. Piccotti, G. Iyer, X. Ling, Z. Y. Yang, G. J. Nabel, and D. K. Bishop. 1998. Genetic vaccination-induced immune responses to the human immunodeficiency virus protein Rev: emergence of the interieukin 2- producing helper T lymphocyte. Hum Gene Ther 9:2187.
Blazevic, V., M. Heino, A. Lagerstedt, A. Ranki, and K. J. Krohn. 1996. lnterleukin-10 gene expression induced by HIV-1 Tat and Rev in the cells of HIV-1 infected individuals. J Acquir Immune Defic Syndr Hum Retrovirol 13:208. Cafaro, A., A. Caputo, C. Fracasso, M. T. Maggiorella, D. Goletti, S. Baroncelli, M. Pace, L. Sernicola, M. L. Koanga-Mogtomo, M. Betti, A. Borsetti, R. Belli, L. Akerblom, F. Corrias, S. Butto, J. Heeney, P. Verani, F. Titti, and
B. Ensoli. 1999. Control of SHIV-89.6P-infection of cynomolgus monkeys by HIV-1 Tat protein vaccine [see comments]. Nat Med 5:643.
Barillari, G., C. Sgadari, C. Palladino, R. Gendelman, A. Caputo, C. B. Morris, B.
C. Nair, P. Markham, A. Nel, M. Sturzl, and B. Ensoli. 1999. Inflammatory cytokines synergize with the HIV-1 Tat protein to promote angiogenesis and Kaposi's sarcoma via induction of basic fibroblast growth factor and the alpha v beta 3 integrin. J Immunol 163:1929.
Barillari, G., C. Sgadari, V. Fiorelli, F. Samaniego, S. Colombini, V. Manzari, A. Modesti, B. C. Nair, A. Cafaro, M. Sturzl, and B. Ensoli. 1999. The Tat protein of human immunodeficiency virus type-1 promotes vascular cell growth and locomotion by engaging the alphaδbetal and alphabeta3 integrins and by mobilizing sequestered basic fibroblast growth factor. Blood 94:663.
Brother, M. B., H. K. Chang, J. Lisziewicz, D. Su, L. C. Murty, and B. Ensoli. 1996. Block of Tat-mediated transactivation of tumor necrosis factor beta gene expression by polymeric-TAR decoys. Virology 222:252.
Ensoli, B., L. Buonaguro, G. Barillari, V. Fiorelli, R. Gendelman, R. A. Morgan, P. Wingfield, and R. C. Gallo. 1993. Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat protein on cell growth and viral transactivation. J Virol 67:277.
Chang, H. C, F. Samaniego, B. C. Nair, L. Buonaguro, and B. Ensoli. 1997. HIV- 1 Tat protein exits from cells via a leaderless secretory pathway and binds to extracellular matrix-associated heparan sulfate proteoglycans through its basic region. Aids 11:1421. Helland, D. E., J. L. Welles, A. Caputo, and W. A. Haseltine. 1991 . Transcellular transactivation by the human immunodeficiency virus type 1 tat protein. J Virol 65:4547.
Gibellini, D., A. Bassini, S. Pierpaoli, L. Bertolaso, D. Milani, S. Capitani, M. La Placa, and G. Zauli. 1998. Extracellular HIV-1 Tat protein induces the rapid Ser133 phosphorylation and activation of CREB transcription factor in both Jurkat lymphoblastoid T cells and primary peripheral blood mononuclear cells. J Immunol 160:3891.
Huang, L., I. Bosch, W. Hofmann, J. Sodroski, and A. B. Pardee. 1998. Tat protein induces human immunodeficiency virus type 1 (HIV-1 ) coreceptors and promotes infection with both macrophage-tropic and T- lymphotropic HIV-1 strains. J Virol 72:8952.
Caselli, E., M. Betti, M. P. Grossi, P. G. Balboni, C. Rossi, C. Boarini, A. Cafaro, G. Barbanti-Brodano, B. Ensoli, and A. Caputo. 1999. DNA immunization with HIV-1 tat mutated in the trans activation domain induces humoral and cellular immune responses against wild-type Tat. J Immunol 162:5631.
Gruber, A., J. Kan-Mitchell, K. L. Kuhen, T. Mukai, and F. Wong-Staal. 2000. Dendritic cells transduced by multiply deleted HIV-1 vectors exhibit normal phenotypes and functions and elicit an HIV-specific cytotoxic T- lymphocyte response in vitro. Blood 96:1327.
All of the patents, published and unpublished patent applications as well as any other scientific or technical writings referred to herein are expressly incorporated by reference to the extent that they are not contradictory.
While preferred embodiments of the invention have been described and shown herein, since variations in the preferred form will be apparent to those skilled in the art, the invention should not be construed as limited to the specific embodiments shown and described, but rather as set forth in the claims.

Claims

CLAIMSWe claim
1. A biological composition comprising a first and second adenoviral vector having complementary function and being dependent upon one another for replication in one or more host organisms and/or target cells.
2. The composition of claim 1 , wherein the first adenoviral vector comprises a promoter that regulates expression of an early gene that controls propagation of the adenoviral vectors in the host organism and/or target cell.
3. The composition of claim 2, wherein the promoter is only active in one or more host organisms or target cells and propagation is limited to the one or more host organisms and/or target cells.
4. The composition of claim 1 , wherein the second adenoviral vector comprises a partial adenoviral genome that can support adenoviral replication in the one or more host organisms and/or target cells.
5. The composition of claim 4, wherein the replication of the first and second adenoviral vectors in the one or more host organisms and/or target cells directly and/or indirectly induces one or more immune responses that can alter the growth, function and/or other activity of the one or more host organisms and/or target cells.
6. The composition of claim 4, wherein the replication of the first and second adenoviral vectors in the one or more host organisms and/or target cells directly and/or indirectly induces one or more immune responses that can alter the growth, function and/or other activity of the one or more host organisms and/or target cells with respect to one or more pathogens and/or infectious agents that affect the one or more host organisms and/or target cells.
7. The composition of claim 6, wherein the host organisms, target cells, pathogens and/or infectious agents can be involved in one or more disease states selected from the group consisting of infections, cancers, autoimmune diseases, and cardiovascular disorders.
8. A biological composition for targeting one or more infectious agent(s) and/or target cell(s), the composition comprising a first adenoviral vector and a second adenoviral vector, wherein each vector is dependent upon the other for replication.
9. The composition of claim 8, wherein the first adenoviral vector can comprise an E1 expression cassette from which E1 is expressed within one or more infectious agent(s) and/or target cell(s) from a promoter and wherein the first adenoviral vector does not express other adenoviral proteins.
10. The composition of claim 8 or 9, wherein the second adenoviral vector provides adenoviral proteins required for packaging and replication.
1 1. The composition of any one of the preceding claims, wherein the composition is used for the delivery and expression of one or more prophylactic genes and/or therapeutic genes, wherein some of the one or more prophylactic genes and/or therapeutic genes demonstrate direct cytotoxic effects, indirect cytotoxic effects, and or sensitize target cells to the effects of other treatments.
12. The composition of any one of claims 1-10, wherein the first or second adenoviral vectors encode one or more antigen(s), with or without immunomodulatory genes, to induce one or more immune response(s) to the encoded antigen or antigens.
PCT/US2002/033162 2001-10-16 2002-10-16 Replication competent dual-ad vectors for vaccine and immunotherapy applications WO2003033029A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP2003535831A JP2005511534A (en) 2001-10-16 2002-10-16 Self-replicating dual Ad vectors for vaccine and immunotherapy applications
CA 2463996 CA2463996A1 (en) 2001-10-16 2002-10-16 Replication competent dual-ad vectors for vaccine and immunotherapy applications
EP02770603A EP1450862A4 (en) 2001-10-16 2002-10-16 Replication competent dual-ad vectors for vaccine and immunotherapy applications

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US32979801P 2001-10-16 2001-10-16
US60/329,798 2001-10-16

Publications (1)

Publication Number Publication Date
WO2003033029A1 true WO2003033029A1 (en) 2003-04-24

Family

ID=23287062

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/US2002/033079 WO2003061560A2 (en) 2001-10-16 2002-10-16 Replication competent dual-ad vectors for vaccine and immunotherapy applications
PCT/US2002/033162 WO2003033029A1 (en) 2001-10-16 2002-10-16 Replication competent dual-ad vectors for vaccine and immunotherapy applications

Family Applications Before (1)

Application Number Title Priority Date Filing Date
PCT/US2002/033079 WO2003061560A2 (en) 2001-10-16 2002-10-16 Replication competent dual-ad vectors for vaccine and immunotherapy applications

Country Status (5)

Country Link
EP (1) EP1450862A4 (en)
JP (1) JP2005511534A (en)
AU (1) AU2002365260A1 (en)
CA (1) CA2463996A1 (en)
WO (2) WO2003061560A2 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005025671A1 (en) 2003-09-17 2005-03-24 Djalma Luiz Rodrigues A phototherapy equipment, particularly for the treatment of hyperbilirubinemia
WO2009094647A2 (en) 2008-01-25 2009-07-30 Introgen Therapeutics, Inc. P53 biomarkers

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998035028A2 (en) * 1997-02-10 1998-08-13 Baxter International Inc. An oncolytic/immunogenic complementary-adenoviral vector system
WO2001092550A2 (en) * 2000-05-31 2001-12-06 Human Gene Therapy Research Institute Methods and compositions for efficient gene transfer using transcomplementary vectors

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998035028A2 (en) * 1997-02-10 1998-08-13 Baxter International Inc. An oncolytic/immunogenic complementary-adenoviral vector system
US6403370B1 (en) * 1997-02-10 2002-06-11 Genstar Therapeutics Corporation Oncolytic/immunogenic complementary-adenoviral vector system
WO2001092550A2 (en) * 2000-05-31 2001-12-06 Human Gene Therapy Research Institute Methods and compositions for efficient gene transfer using transcomplementary vectors
US20020042139A1 (en) * 2000-05-31 2002-04-11 Higginbotham James N. Methods and compositions for efficient gene transfer using transcomplementary vectors

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ALEMANY R.: "Complementary adenoviral vectors for oncolysis", CANCER GENE THERAPY, vol. 6, no. 1, January 1999 (1999-01-01) - February 1999 (1999-02-01), pages 21 - 25, XP001037527 *
See also references of EP1450862A4 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005025671A1 (en) 2003-09-17 2005-03-24 Djalma Luiz Rodrigues A phototherapy equipment, particularly for the treatment of hyperbilirubinemia
WO2009094647A2 (en) 2008-01-25 2009-07-30 Introgen Therapeutics, Inc. P53 biomarkers

Also Published As

Publication number Publication date
JP2005511534A (en) 2005-04-28
EP1450862A1 (en) 2004-09-01
AU2002365260A1 (en) 2003-09-02
AU2002365260A8 (en) 2007-12-20
WO2003061560A3 (en) 2007-10-18
WO2003061560A2 (en) 2003-07-31
EP1450862A4 (en) 2005-03-02
CA2463996A1 (en) 2003-04-24

Similar Documents

Publication Publication Date Title
EP1032696B1 (en) Vector for tissue-specific replication and gene expression
EP0870049B1 (en) Complementary adenoviral vector systems and cell lines
EP0791050B1 (en) Vectors for tissue-specific replication
JP3816952B2 (en) Defective adenovirus containing therapeutic and immunoprotective genes
US6403370B1 (en) Oncolytic/immunogenic complementary-adenoviral vector system
RU2361611C2 (en) Designing of carcinolytic adenovirus recombinant, specifically expressing immunomodulatory factor gm-csf in tumoral cells and its application
US20240018546A1 (en) Single cycle replicating adenovirus vectors
WO1998035028A9 (en) An oncolytic/immunogenic complementary-adenoviral vector system
JP2000106875A (en) Use of adenovirus e4 leading frme for improving expression of target gene
CA2973109A1 (en) Methods and compositions for ebola virus vaccination
US20080292592A1 (en) Oncolytic Adenovirus Armed with Therapeutic Genes
KR100368292B1 (en) Recombinant Adenovirus for Gene Therapy of Cancer
EP0181117A2 (en) Oral vaccines
CN100471957C (en) Adenoviral vectors for immunotherapy
Melcher et al. Heat shock protein expression in target cells infected with low levels of replication-competent virus contributes to the immunogenicity of adenoviral vectors
JP2004519201A (en) Chimeric adenovirus vector
EP1450862A1 (en) Replication competent dual-ad vectors for vaccine and immunotherapy applications
EP1127151A2 (en) Bovine cells expressing adenovirus essential functions for propagation of recombinant adenoviral vectors
Perkus et al. 3.6 Recombinant virus as vaccination carrier of heterologous antigens
JP2005519959A (en) Methods for inducing an enhanced immune response against HIV
MXPA98004716A (en) Complementary systems of adenoviral vectors and cell lines

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BY BZ CA CH CN CO CR CU CZ DE DM DZ EC EE ES FI GB GD GE GH HR HU ID IL IN IS JP KE KG KP KR LC LK LR LS LT LU LV MA MD MG MN MW MX MZ NO NZ OM PH PL PT RU SD SE SG SI SK SL TJ TM TN TR TZ UA UG US UZ VN YU ZA ZM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ UG ZM ZW AM AZ BY KG KZ RU TJ TM AT BE BG CH CY CZ DK EE ES FI FR GB GR IE IT LU MC PT SE SK TR BF BJ CF CG CI GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2003535831

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 2463996

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 2002770603

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2002770603

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 2002770603

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载