EP1450862A1 - Replikationsfähige dual-ad-vektoren für vakzine und immunotherapeutische anwendungen - Google Patents

Replikationsfähige dual-ad-vektoren für vakzine und immunotherapeutische anwendungen

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Publication number
EP1450862A1
EP1450862A1 EP02770603A EP02770603A EP1450862A1 EP 1450862 A1 EP1450862 A1 EP 1450862A1 EP 02770603 A EP02770603 A EP 02770603A EP 02770603 A EP02770603 A EP 02770603A EP 1450862 A1 EP1450862 A1 EP 1450862A1
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Prior art keywords
cells
vector
vectors
cell
replication
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EP02770603A
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English (en)
French (fr)
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EP1450862A4 (de
Inventor
Sybille Sauter
Xiangming Fang
Amena Rahman
Nathan P. Englund
Scott Gallichan
Mark I. Fitchmun
Robert E. Sobol
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Magnum Therapeutics Inc
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Magnum Therapeutics Inc
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Publication of EP1450862A1 publication Critical patent/EP1450862A1/de
Publication of EP1450862A4 publication Critical patent/EP1450862A4/de
Withdrawn legal-status Critical Current

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    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
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    • C12N2830/00—Vector systems having a special element relevant for transcription
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    • 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.
  • 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.
  • 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. 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. 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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 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).
  • 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 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.
  • 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)
  • 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 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.
  • 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.
  • 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.
  • 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)
  • 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 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 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • Group A GTV8063 injected into a single site at 5 x 10 vp/tumor in 100 ⁇ l formulation buffer.
  • LNCaP "fast” cells a human prostate cancer cell line, passage 37; grown in RPMI1640 + 10% fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • 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.
  • Figure 1 Graph of tumor sizes. Arrow indicates time of treatment with viruses or control at week 3.
  • GTV8063 group B AdTK group
  • HIV Human immunodeficiency virus
  • 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.
  • 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.

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EP1450862A4 (de) 2005-03-02
WO2003061560A3 (en) 2007-10-18
WO2003061560A2 (en) 2003-07-31
WO2003033029A1 (en) 2003-04-24
JP2005511534A (ja) 2005-04-28
AU2002365260A1 (en) 2003-09-02
AU2002365260A8 (en) 2007-12-20
CA2463996A1 (en) 2003-04-24

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