[go: up one dir, main page]

WO2005117993A2 - Methode permettant une utilisation plus sure et plus efficace de vecteurs adenoviraux pour le traitement des cancers - Google Patents

Methode permettant une utilisation plus sure et plus efficace de vecteurs adenoviraux pour le traitement des cancers Download PDF

Info

Publication number
WO2005117993A2
WO2005117993A2 PCT/US2005/019630 US2005019630W WO2005117993A2 WO 2005117993 A2 WO2005117993 A2 WO 2005117993A2 US 2005019630 W US2005019630 W US 2005019630W WO 2005117993 A2 WO2005117993 A2 WO 2005117993A2
Authority
WO
WIPO (PCT)
Prior art keywords
replication
adenoviral vector
adenoviral
deficient
acid sequence
Prior art date
Application number
PCT/US2005/019630
Other languages
English (en)
Other versions
WO2005117993A3 (fr
Inventor
Thomas J. Wickham
Masaki Akiyama
Selva Murgesan
David Einfeld
Tomoyuki Abe
Hiroshi Yoshida
Osamu Yamada
Hiromasa Araki
Original Assignee
Genvec, Inc.
Fuso Pharmaceutical Industries, Ltd.
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 Genvec, Inc., Fuso Pharmaceutical Industries, Ltd. filed Critical Genvec, Inc.
Publication of WO2005117993A2 publication Critical patent/WO2005117993A2/fr
Publication of WO2005117993A3 publication Critical patent/WO2005117993A3/fr

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
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10332—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
    • 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/40—Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source
    • C12N2810/405—Vectors comprising RGD peptide
    • 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

Definitions

  • This invention pertains to methods of treating rumors located within the peritoneal cavity of a mammal.
  • Gene therapy is gaining acceptance in the scientific community as a promising treatment for a variety of ailments.
  • Gene transfer vectors derived from adenovirus have proven to have many attractive characteristics in the context of gene therapy including substantial and transient gene expression, the ability to be propagated in high titers, and the ability to transduce a wide variety of cell types.
  • adenoviral vectors suffer from limitations similar to those of other gene transfer vectors with respect to achieving delivery to tissues affected by a specific disease (e.g., cancer), while sparing normal tissue.
  • Viral vectors inherently encode and/or display antigenic epitopes that are recognized by a host immune system.
  • the immunogenicity of viral vectors, including adenoviral vectors is a major impediment in the use of these gene transfer vehicles in vivo.
  • a majority of the human population has been exposed to adenovirus and, therefore, has pre-existing immunity to adenoviral vectors based on human adenovirus serotypes, which limits the effectiveness of the virus as a gene transfer vector.
  • adenoviral vector administration induces inflammation and activates both innate and acquired immune mechanisms.
  • Adenoviral vectors activate antigen-specific (e.g., T-cell dependent) immune responses, which limit the duration of transgene expression following an initial administration of the vector.
  • exposure to adenoviral vectors stimulates production of neutralizing antibodies by B cells, which precludes gene expression from subsequent doses of adenoviral vector (Wilson & Kay, Nat. Med., 3(9), 887-889 (1995)). Indeed, the effectiveness of repeated administration of the vector can be severely limited by host immunity.
  • adenoviral serotype 2 or 5 vector can result in the production of neutralizing antibodies directed against the vector which prevent expression from the same serotype vector administered 1 to 2 weeks later (see, for example, Kass- Eisler et al, Gene Therapy, 1, 395-402 (1994), and Kass-Eisler et al., Gene Therapy, 3, 154- 162 (1996)).
  • Adenoviral vectors are typically cleared from circulation within minutes and are cleared from the body within about 7-10 days. Within the first two days of infection, approximately 90% of adenoviral vector DNA is eliminated (Elkon et al., PNAS, 94, 9814-9819 (1997)). The rapid clearance of adenoviral vectors decreases circulation time and prevents efficient delivery to target cells via systemic circulation, which may be required to treat diseases such as disseminated cancers.
  • Adenoviral fiber, penton, and hexon proteins have received the most attention as these represent the first exposure of the virus to the host's immune and clearance systems, and are involved in adenovirus binding to cell surfaces. For example, U.S.
  • Patent 6,153,435 describes adenoviral vectors having a chimeric adenovirus coat protein with a decreased ability or inability to be recognized by a neutralizing antibody directed against the corresponding wild-type adenovirus coat protein.
  • U.S. Patent 6,576,456 describes adenoviral vectors having a chimeric adenovirus fiber protein that binds to a cell surface receptor other than a native cell surface receptor. Genetic manipulation of adenoviral coat proteins has resulted in success, although somewhat limited, in selectively targeting diseased tissue and avoiding host immunity.
  • the invention provides a method of destroying tumor cells in a mammal.
  • the method comprises administering to the mammal intraperitoneally a dose of replication- deficient adenoviral vector comprising (a) an exogenous nucleic acid sequence encoding a human TNF- ⁇ which is operably linked to a tumor cell-selective promoter, (b) a fiber protein wherein a native CAR-binding site is disrupted, and (c) and a penton base protein wherein a native integrin-binding site is disrupted, wherein the tumor is located in the peritoneal cavity of the mammal, such that the TNF- ⁇ is produced and tumor cells in the mammal are destroyed.
  • Figure 1 is a graph illustrating the change in percent (%) body weight of mice over time after intraperitoneal injection of the adenoviral vector Adc M v /EGR TNF (•), Adc M v /EGR TNF** (A), and Adc MV/EGR TNF**RGD ( ⁇ ) compared to naive control mice ( ⁇ ).
  • Figure 2a is a graph illustrating tumor weight loss in mice after intraperitoneal administration of the adenoviral vectors Adj NF EGR, Ad ⁇ NF CMV/EGR-1, Adi NF RSV, and Ad ⁇ NF DF3.
  • Figure 2b is a graph illustrating the change in (%) body weight in mice after intraperitoneal administration of the adenoviral vectors Ad NF EGR, Ad ⁇ NF CMV/EGR-1, Ad TOF RSF, and ADTMFDF3.
  • Figure 3 a is a graph illustrating the change in percent (%) body weight of mice over time after intraperitoneal injection of the adenoviral vector AdCMV/EGR-l ⁇ NF **RGD.
  • Figure 3b is a graph illustrating the change in percent (%) body weight of mice over time after intraperitoneal injection of the adenoviral vector AdE2F ⁇ F **RGD.
  • Figure 4 is a graph illustrating the change in percent (%) body weight of mice over time after intraperitoneal injection of the adenoviral vector AdE2F ⁇ NF **RGD in combination with intraperitoneal injection of cisplatin.
  • Figure 5a is a graph illustrating the percent (%) change in Meth-A tumor circumference over time after intraperitoneal injection of the adenoviral vector AdCMV/EGR-l ⁇ NF **RGD in combination with intraperitoneal injection of cisplatin.
  • Figure 5b is a graph illustrating the percent (%) change in Meth-A tumor circumference over time after intraperitoneal injection of the adenoviral vector AdE2F ⁇ NF **RGD in combination with intraperitoneal injection of cisplatin.
  • Figure 6a is a graph illustrating percent (%) survival and change in percent (%) body weight of Meth-A tumor-bearing mice after intraperitoneal injection of the adenoviral vector AdCMV/EGR-l ⁇ NF **RGD in combination with intraperitoneal injection of cisplatin.
  • Figure 6b is a graph illustrating percent (%) survival and change in percent (%) body weight of Meth-A tumor-bearing mice after intraperitoneal injection of the adenoviral vector AdE2F ⁇ NF **RGD in combination with intraperitoneal injection of cisplatin.
  • the invention provides a method of destroying tumor cells in a mammal.
  • the method comprises administering to the mammal intraperitoneally a dose of replication- deficient adenoviral vector comprising an exogenous nucleic acid sequence encoding a human TNF- ⁇ which is operably linked to a tumor cell-selective promoter.
  • the replication- deficient adenoviral vector further comprises (a) a fiber protein wherein a native CAR- binding site is disrupted, and (b) and a penton base protein wherein a native integrin-binding site is disrupted.
  • the tumor is located in the peritoneal cavity of the mammal.
  • Adenovirus from any origin, any subtype, mixture of subtypes, or any chimeric adenovirus can be used as the source of the viral genome for the replication-deficient or conditionally-replicating adenoviral vector. While non-human adenovirus (e.g., simian, avian, canine, ovine, or bovine adenoviruses) can be used to generate the replication- deficient adenoviral vector, a human adenovirus preferably is used as the source of the viral genome for the replication-deficient or conditionally-replicating adenoviral vector of the inventive method.
  • the adenovirus can be of any subgroup or serotype.
  • an adenovirus can be of subgroup A (e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, 35, and 50), subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-48), subgroup E (e.g., serotype 4), subgroup F (e.g., serotypes 40 and 41), an unclassified serogroup (e.g., serotypes 49 and 51), or any other adenoviral serotype.
  • subgroup A e.g., serotypes 12, 18, and 31
  • subgroup B e.g., serotypes 3, 7, 11, 14, 16, 21, 34, 35, and 50
  • subgroup C e.g., serotypes 1, 2, 5, and 6
  • subgroup D e.g., serotypes
  • Adenoviral serotypes 1 through 51 are available from the American Type Culture Collection (ATCC, Manassas, VA).
  • the adenoviral vector is of human subgroup C, especially serotype 2 or even more desirably serotype 5.
  • Adenoviral vectors of serotype 35 or serotype 41 also are appropriate for use in the context of the invention.
  • An adenoviral vector containing an adenoviral genome comprising a mixture of adenoviral serotypes i.e., a chimeric adenoviral vector
  • a chimeric adenoviral vector also is within the scope of the invention.
  • a particularly preferred adenoviral vector contains an adenoviral genome comprising a portion of a serotype 2 genome and a portion of a serotype 5 genome.
  • nucleotides 1-456 of such an adenoviral vector are derived from a serotype 2 genome, while the remainder of the adenoviral genome is derived from a serotype 5 genome.
  • the adenoviral vector comprises an adenoviral genome that lacks at least one replication-essential gene function (i.e., such that the adenoviral vector does not replicate in typical host cells, especially those in a human patient that could be infected by the adenoviral vector in the course of treatment in accordance with the invention).
  • a deficiency in a gene, gene function, or gene or genomic region, as used herein, is defined as a deletion or mutation of sufficient genetic material of the viral genome to impair or obliterate the function of the gene whose nucleic acid sequence was deleted or mutated in whole or in part.
  • Replication-essential gene functions are those gene functions that are required for replication (e.g., propagation) and are encoded by, for example, the adenoviral early regions (e.g., the El, E2, and E4 regions), late regions (e.g., the L1-L5 regions), genes involved in viral packaging (e.g., the IVa2 gene), and virus- associated RNAs (e.g., VA-RNA1 and/or VA-RNA2). More preferably, the replication- deficient adenoviral vector comprises an adenoviral genome deficient in at least one replication-essential gene function of one or more regions of the adenoviral genome.
  • the adenoviral vector is deficient in at least one gene function of the El region of the adenoviral genome required for viral replication (denoted an El -deficient adenoviral vector).
  • the recombinant adenovirus also can have a mutation in the major late promoter (MLP), as discussed in International Patent Application WO 00/00628.
  • MLP major late promoter
  • the adenoviral vector is deficient in at least one replication-essential gene function (desirably all replication-essential gene functions) of the El region and at least part of the ' nonessential E3 region (e.g., an Xba I deletion of the E3 region) (denoted an E1/E3 -deficient adenoviral vector).
  • the adenoviral vector can be deficient in part or all of the El A region and part or all of the E1B region, e.g., in at least one replication-essential gene function of each of the El A and E1B regions.
  • the adenoviral vector When the adenoviral vector is deficient in at least one replication-essential gene function in one region of the adenoviral genome (e.g., an El- or E1/E3 -deficient adenoviral vector), the adenoviral vector is referred to as "singly replication-deficient.”
  • a particularly preferred singly replication-deficient adenoviral vector is that described in the Examples herein, i.e., a replication-deficient adenoviral vector requiring, at most, complementation of the El region of the adenoviral genome, so as to propagate the adenoviral vector (e.g., to form adenoviral vector particles).
  • the adenoviral vector can be "multiply replication-deficient,” meaning that the adenoviral vector is deficient in one or more replication-essential gene functions in each of two or more regions of the adenoviral genome.
  • the aforementioned El- deficient or E1/E3 -deficient adenoviral vector can be further deficient in at least one replication-essential gene function of the E4 region (denoted an E1/E4- or E1/E3/E4- deficient adenoviral vector), and/or the E2 region (denoted an E1/E2- or E1/E2/E3 -deficient adenoviral vector), preferably the E2A region (denoted an E1/E2A- or E1/E2A/E3 -deficient adenoviral vector).
  • the adenoviral vector lacks replication-essential gene functions of only those replication-essential gene functions encoded by the early regions of the adenoviral genome, i.e., a replication-deficient adenoviral vector requiring at most complementation of the El and E4 regions, El and E2A regions, or El, E2A, and E4 regions of the adenoviral genome so as to propagate the adenoviral vector (e.g., to form adenoviral vector particles), although this is not required in all contexts of the invention.
  • a preferred multiply-deficient adenoviral vector comprises an adenoviral genome having deletions of nucleotides 457-3332 of the El region, nucleotides 28593-30470 of the E3 region, nucleotides 32826-35561 of the E4 region, and, optionally, nucleotides 10594- 10595 of the region encoding VA-RNA1.
  • Nucleotides 356-3329 or 356-3510 can be removed to create a deficiency in replication- essential El gene functions.
  • Nucleotides 28594-30469 can be deleted from the E3 region of the adenoviral genome.
  • the adenoviral vector when multiply replication-deficient, especially in replication-essential gene functions of the El and E4 regions, preferably includes a spacer element to provide viral growth in a complementing cell line similar to that achieved by singly replication-deficient adenoviral vectors, particularly an El -deficient adenoviral vector.
  • the spacer element can contain any sequence or sequences which are of a desired length, such as sequences at least about 15 base pairs (e.g., between about 15 base pairs and about 12,000 base pairs), preferably about 100 base pairs to about 10,000 base pairs, more preferably about 500 base pairs to about 8,000 base pairs, even more preferably about 1,500 base pairs to about 6,000 base pairs, and most preferably about 2,000 to about 3,000 base pairs in length.
  • the spacer element sequence can be coding and/or non-coding and native and/or non-native with respect to the adenoviral genome, but does not restore the replication-essential function to the deficient region.
  • the replication-deficient or conditionally-replicating adenoviral vector is an El/E4-deficient adenoviral vector wherein the L5 fiber region is retained, and a spacer is located between the L5 fiber region and the right-side ITR.
  • the E4 polyadenylation sequence alone or, most preferably, in combination with another sequence exists between the L5 fiber region and the right-side ITR, so as to sufficiently separate the retained L5 fiber region from the right- side ITR, such that viral production of such a vector approaches that of a singly replication- deficient adenoviral vector, particularly an El -deficient adenoviral vector.
  • the E4 polyadenylation sequence and the E4 promoter are retained in the adenoviral genome.
  • the adenoviral vector can be deficient in replication-essential gene functions of only the early regions of the adenoviral genome, only the late regions of the adenoviral genome, and both the early and late regions of the adenoviral genome.
  • the adenoviral vector also can have essentially the entire adenoviral genome removed, in which case it is preferred that at least either the viral inverted terminal repeats (ITRs) and one or more promoters or the viral ITRs and a packaging signal are left intact (i.e., an adenoviral amplicon).
  • ITRs inverted terminal repeats
  • the 5' or 3' regions of the adenoviral genome comprising ITRs and packaging sequence need not originate from the same adenoviral serotype as the remainder of the viral genome.
  • an adenoviral serotype 5 genome i.e., the region of the genome 5' to the adenoviral El region
  • the corresponding region of an adenoviral serotype 2 genome e.g., the Ad5 genome region 5' to the El region of the adenoviral genome is replaced with nucleotides 1-456 of the Ad2 genome.
  • Suitable replication-deficient adenoviral vectors including multiply replication-deficient adenoviral vectors, are disclosed in U.S. Patents 5,837,511; 5,851,806; 5,994,106; and 6,127,175; U.S. Patent Application Publication Nos.
  • the replication-deficient or conditionally-replicating adenoviral vector is used in the context of the invention in the form of an adenoviral vector composition, especially a pharmaceutical composition, which is virtually free of replication-competent adenovirus (RCA) contamination (e.g., the composition comprises less than about 1% of RCA contamination).
  • RCA replication-competent adenovirus
  • the composition is RCA-free.
  • Adenoviral vector compositions and stocks that are RCA-free are described in U.S. Patents 5,944,106 and 6,482,616, U.S. Patent Application Publication No. 2002/0110545 Al, and International Patent Application WO 95/34671.
  • Replication-deficient adenoviral vectors are typically produced in complementing cell lines that provide gene functions not present in the replication-deficient adenoviral vectors, but required for viral propagation, at appropriate levels in order to generate high titers of viral vector stock.
  • a preferred cell line complements for at least one and preferably all replication-essential gene functions not present in a replication-deficient adenovirus.
  • the complementing cell line can complement for a deficiency in at least one replication-essential gene function encoded by the early regions, late regions, viral packaging regions, virus-associated RNA regions, or combinations thereof, including all adenoviral functions (e.g., to enable propagation of adenoviral amplicons).
  • the complementing cell line complements for a deficiency in at least one replication-essential gene function (e.g., two or more replication-essential gene functions) of the El region of the adenoviral genome, particularly a deficiency in a replication-essential gene function of each of the El A and EIB regions.
  • the complementing cell line can complement for a deficiency in at least one replication-essential gene function of the E2 (particularly as concerns the adenoviral DNA polymerase and terminal protein) and/or E4 regions of the adenoviral genome.
  • a cell that complements for a deficiency in the E4 region comprises the E4-ORF6 gene sequence and produces the E4-ORF6 protein.
  • Such a cell desirably comprises at least ORF6 and no other ORF of the E4 region of the adenoviral genome.
  • the cell line preferably is further characterized in that it contains the complementing genes in a non-overlapping fashion with the adenoviral vector, which minimizes, and practically eliminates, the possibility of the vector genome recombining with the cellular DNA. Accordingly, the presence of replication competent adeno viruses (RCA) is minimized if not avoided in the vector stock, which, therefore, is suitable for certain therapeutic purposes, especially gene therapy purposes.
  • the lack of RCA in the vector stock avoids the replication of the adenoviral vector in non-complementing cells.
  • Complementing cell lines for producing the adenoviral vector include, but are not limited to, 293 cells (described in, e.g., Graham et al., J. Gen. Virol, 36, 59-72 (1977)), PER.C6 cells (described in, e.g., International Patent Application WO 97/00326, and U.S. Patents 5,994,128 and 6,033,908), and 293-ORF6 cells (described in, e.g., International Patent Application WO 95/34671 and Brough et al., J. Virol, 71, 9206-9213 (1997)). In some instances, the complementing cell will not complement for all required adenoviral gene functions.
  • Helper viruses can be employed to provide the gene functions in trans that are not encoded by the cellular or adenoviral genomes to enable replication of the adenoviral vector.
  • Adenoviral vectors can be constructed, propagated, and/or purified using the materials and methods set forth, for example, in U.S. Patents 5,965,358, 5,994,128, 6,033,908, 6,168,941, 6,329,200, 6,383,795, 6,440,728, 6,447,995, and 6,475,757, U.S. Patent Application Publication No.
  • Non-group C adenoviral vectors including adenoviral serotype 35 vectors, can be produced using the methods set forth in, for example, U.S. Patents 5,837,511 and 5,849,561, and International Patent Applications WO 97/12986 and WO 98/53087. Moreover, numerous adenoviral vectors are available commercially.
  • the adenoviral vector is not replication-deficient, ideally the adenoviral vector is manipulated to limit replication of the vector to within the target tissue.
  • the adenoviral vector can be a conditionally-replicating adenoviral vector, which is engineered to replicate under conditions pre-determined by the practitioner.
  • replication- essential gene functions e.g., gene functions encoded by the adenoviral early regions, can be operably linked to an inducible, repressible, or tissue-specific transcription control sequence, e.g., promoter.
  • replication requires the presence or absence of specific factors that interact with the transcription control sequence.
  • Conditionally- replicating adenoviral vectors are particularly useful in delivering exogenous nucleic acids with the purpose of destroying target cells. Replication of the adenoviral vector can be limited to a target tissue, thereby allowing greater distribution of the vector throughout the tissue while exploiting adenovirus' natural ability to lyse cells during the replication cycle. In cancer therapy, conditionally-replicating adenovirus provides a mode of destroying tumor cells in addition to delivery of lethal exogenous nucleic acids. Conditionally-replicating adenoviral vectors are described further in U.S. Patent 5,998,205.
  • the replication-deficient or conditionally-replicating adenoviral vector has a reduced ability to transduce mesothelial cells and hepatocytes compared to wild-type adenovirus of the same serotype of the replication-deficient or conditionally-replicating adenoviral vector.
  • the replication-deficient or conditionally-replicating adenoviral vector is a chimeric adenoviral vector (e.g., a serotype 2 and serotype 5 chimera)
  • adenoviral vector has a reduced ability to transduce mesothelial cells and hepatocytes compared to wild-type adenovirus of any one of the serotypes of the chimeric replication- deficient or conditionally-replicating adenoviral vector.
  • Adenoviruses that do not naturally transduce mesothelial cells and hepatocytes, such as some non-human adenoviruses, can be used in the context of the invention.
  • adenoviral vectors based on serotypes of human adenovirus that naturally infect cells of the mesothelium and liver are modified to reduce binding to these cells.
  • reduced transduction or binding is meant that transduction levels of a target cell, such as a mesothelial cell or hepatocyte, by the replication-deficient or conditionally-replicating adenoviral vector is at least approximately 3-fold less (e.g., at least approximately 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 35-fold, 45-fold, or 50-fold less) than transduction levels mediated by wild-type adenovirus of the same serotype of the replication-deficient or conditionally-replicating adenoviral vector.
  • the reduction in transduction efficiency is a substantial reduction (such as at least an order of magnitude, and preferably more).
  • the replication-deficient or conditionally-replicating adenoviral vector does not transduce mesothelial cells or hepatocytes.
  • the native binding sites located on adenoviral coat proteins which mediate cell entry are absent or disrupted. Two or more of the adenoviral coat proteins are believed to mediate attachment to cell surfaces (e.g., the fiber and penton base).
  • Any suitable technique for altering native binding to a host cell e.g., a mesothelial cell or hepatocyte
  • exploiting differing fiber lengths to ablate native binding to cells can be accomplished via the addition of a binding sequence to the penton base or fiber knob.
  • the adenoviral fiber protein can be modified to reduce the number of amino acids in the fiber shaft, thereby creating a "short-shafted" fiber (as described in, for example, U.S. Patent 5,962,311).
  • the fiber proteins of some adenoviral serotypes are naturally shorter than others, and these fiber proteins can be used in place of the native fiber protein to reduce native binding of the adenovirus to its native receptor.
  • the native fiber protein of an adenoviral vector derived from serotype 5 adenovirus can be switched with the fiber protein from adenovirus serotypes 40 or 41.
  • the nucleic acid residues associated with native substrate binding can be mutated (see, e.g., International Patent Application WO 00/15823; Einfeld et al., J Virol, 75(23), 11284-11291 (2001); and van Beusechem et al, J Virol, 76(6), 2753- 2762 (2002)) such that the adenoviral vector incorporating the mutated nucleic acid residues is less able to bind its native substrate.
  • adenovirus serotypes 2 and 5 transduce cells via binding of the adenoviral fiber protein to the coxsackievirus and adenovirus receptor (CAR) and binding of penton proteins to integrins located on the cell surface.
  • the replication-deficient or conditionally-replicating adenoviral vector of the inventive method can lack native binding to CAR and/or exhibit reduced native binding to integrins.
  • the native CAR and/or integrin binding sites e.g., the RGD sequence located in the adenoviral penton base
  • the replication-deficient or conditionally-replicating adenoviral vector also can comprise a chimeric coat protein comprising a non-native amino acid sequence that binds a substrate (i.e., a ligand).
  • a substrate i.e., a ligand
  • the inventive method allows an adenoviral vector to remain in circulation for extended periods of time, the inventive method is particularly suited for use of "targeted" adenoviral vectors, which comprise a non-native amino acid sequence that preferentially binds a target cell.
  • the non-native amino acid sequence of the chimeric adenoviral coat protein allows an adenoviral vector comprising the chimeric coat protein to bind and, desirably, infect host cells not naturally infected by the corresponding adenovirus without the non-native amino acid sequence (i.e., host cells not infected by the corresponding wild-type adenovirus), to bind to host cells naturally infected by the corresponding adenovirus with greater affinity than the corresponding adenovirus without the non-native amino acid sequence, or to bind to particular target cells with greater affinity than non-target cells.
  • non-native amino acid sequence can comprise an amino acid sequence not naturally present in the adenoviral coat protein or an amino acid sequence found in the adenoviral coat but located in a non-native position within the capsid.
  • preferentially binds is meant that the non-native amino acid sequence binds a receptor, such as, for instance, ⁇ v ⁇ 3 integrin, with at least about 3-fold greater affinity (e.g., at least about 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 35-fold, 45-fold, or 50-fold greater affinity) than the non-native ligand binds a different receptor, such as, for instance, ⁇ v ⁇ l integrin.
  • the non-native amino acid sequence can be conjugated to any of the adenoviral coat proteins to form a chimeric coat protein. Therefore, for example, the non-native amino acid sequence of the invention can be conjugated to, inserted into, or attached to a fiber protein, a penton base protein, a hexon protein, proteins IX, VI, or Ilia, etc.
  • the sequences of such proteins, and methods for employing them in recombinant proteins, are well known in the art (see, e.g., U.S.
  • the coat protein portion of the chimeric coat protein can be a full-length adenoviral coat protein to which the ligand domain is appended, or it can be truncated, e.g., internally or at the C- and/or N- terminus.
  • the coat protein portion need not, itself, be native to the adenoviral vector.
  • the coat protein can be an adenoviral serotype 4 (Ad4) fiber protein incorporated into an adenoviral serotype 5 vector, wherein the native CAR binding motif of the Ad4 fiber is preferably ablated.
  • Ad4 adenoviral serotype 4
  • SAV-25 simian adenovirus type 25
  • Native binding of the SAV-25 fiber can be ablated by mutating the AB loop and ⁇ sheet of the fiber protein, and, optionally, a non-native amino acid sequence can be inserted into the HI loop or attached to the C-terminus of the fiber protein.
  • the chimeric coat protein preferably is able to incorporate into an adenoviral capsid as its native counterpart coat protein.
  • a given non-native amino acid sequence can be incorporated into any location of the virus capable of interacting with a substrate (i.e., the viral surface).
  • the ligand can be incorporated into the fiber, the penton base, the hexon, protein IX, VI, or Ilia, or other suitable location. Where the ligand is attached to the fiber protein, preferably it does not disturb the interaction between viral proteins or fiber monomers.
  • the non-native amino acid sequence preferably is not itself an oligomerization domain, as such can adversely interact with the trimerization domain of the adenovirus fiber.
  • the ligand is added to the virion protein, and is incorporated in such a manner as to be readily exposed to the substrate (e.g., at the N- or C- terminus of the protein, attached to a residue facing the substrate, positioned on a peptide spacer to contact the substrate, etc.) to maximally present the non-native amino acid sequence to the substrate.
  • the non-native amino acid sequence is incorporated into an adenoviral fiber protein at the C-terminus of the fiber protein (and attached via a spacer) or incorporated into an exposed loop (e.g., the HI loop) of the fiber to create a chimeric coat protein.
  • the non-native amino acid sequence is attached to or replaces a portion of the penton base, preferably it is within the hypervariable regions to ensure that it contacts the substrate.
  • the non-native amino acid sequence is attached to the hexon, preferably it is within a hypervariable region (Miksza et al, J Virol, 70(3), 1836-44 (1996)).
  • Binding affinity of a non-native amino acid sequence to a cellular receptor can be determined by any suitable assay, a variety of which assays are known, and is useful in selecting a non-native amino acid sequence for incorporating into an adenoviral coat protein. Desirably, the transduction levels of host cells are utilized in determining relative binding efficiency.
  • host cells displaying ⁇ v ⁇ 3 integrin on the cell surface can be exposed to a replication-deficient or conditionally- replicating adenoviral vector comprising the chimeric coat protein and the corresponding adenovirus without the non-native amino acid sequence, and then transduction efficiencies can be compared to determine relative binding affinity.
  • both host cells displaying ⁇ v ⁇ 3 integrin on the cell surface e.g., MDAMB435 cells
  • host cells displaying predominantly ⁇ v ⁇ l on the cell surface e.g., 293 cells
  • transduction efficiencies can be compared to determine binding affinity.
  • a non-native amino acid e.g., ligand
  • a compound other than a cell-surface protein can bind a compound other than a cell-surface protein.
  • the ligand can bind blood- and/or lymph-borne proteins (e.g., albumin), synthetic peptide sequences such as polyamino acids (e.g., polylysine, polyhistidine, etc.), artificial peptide sequences (e.g., FLAG), and RGD peptide fragments (Pasqualini et al, J Cell. Biol, 130, 1189 (1995)).
  • a ligand can even bind non- peptide substrates, such as plastic (e.g., Adey et al., Gene, 156, 27 (1995)), biotin (Saggio et al., Biochem. J., 293, 613 (1993)), a DNA sequence (Cheng et al, Gene, 171, 1 (1996); Krook et al, Biochem. Biophys., Res. Commun., 204, 849 (1994)), streptavidin (Geibel et al., Biochemistry, 34, 15430 (1995); Katz, Biochemistry, 34, 15421 (1995)), nitrostreptavidin (Balass et al., Anal.
  • plastic e.g., Adey et al., Gene, 156, 27 (1995)
  • biotin Saggio et al., Biochem. J., 293, 613 (1993)
  • a DNA sequence Choeng et al, Gene, 17
  • non-native amino acid sequences and their substrates for use in the method of the invention include, but are not limited to, short (e.g., 6 amino acids or less) linear stretches of amino acids recognized by integrins, as well as polyamino acid sequences such as polylysine, polyarginine, etc. Inserting multiple lysines and/or arginines provides for recognition of heparin and DNA.
  • Suitable non-native amino acid sequences for generating chimeric adenoviral coat proteins are further described in U.S.
  • the adenoviral coat protein comprises a non-native amino acid sequence that binds ⁇ v ⁇ 3, ⁇ v ⁇ 5, or ⁇ v ⁇ 6 integrins.
  • native binding of the adenoviral coat protein to native adenoviral cell-surface receptors such as the coxsackie and adenovirus receptor (CAR)
  • CAR adenovirus receptor
  • the non-native amino acid sequence binds ⁇ v ⁇ 3 integrin, it does so with at least about 10- fold greater affinity than the non-native amino acid sequence binds to ⁇ v ⁇ l integrin.
  • ⁇ v ⁇ 3 integrins are upregulated in tumor tissue vasculature, metastatic breast cancer, melanoma, and gliomas.
  • Adenoviral vectors displaying ligands specific for ⁇ v ⁇ 3 integrin, such as an RGD motif infect cells with a greater number of ⁇ v ⁇ 3 integrin moieties on the cell surface compared to cells that do not express the integrin to such a degree, thereby targeting the vectors to specific cells of interest.
  • the RGD motif is flanked by one or two sets of cysteine residues.
  • the replication-deficient or conditionally-replicating adenoviral vector comprises a chimeric coat protein comprising a non-native amino acid sequence that binds ⁇ v ⁇ 6 integrins.
  • ⁇ v ⁇ 6 integrins are nearly or completely absent on normal epithelium and endothelium, and are upregulated in several carcinomas including lung, colon, and ovarian cancers.
  • ⁇ v ⁇ 6 integrin binding motif RTDLXXL (SEQ ID NO: 1), wherein X can be any amino acid
  • X can be any amino acid
  • Other ⁇ v ⁇ 6 integrin-binding motifs can be used as the non-native amino acid sequence for incorporation into the adenoviral coat protein including, but not limited to, ⁇ v ⁇ 6 integrin-binding motifs of foot and mouth virus (FMV; Jackson et al, J.
  • Tumors often comprise a heterogeneous mass of tumor cells, vasculature, and tumor matrix.
  • the interstitial tumor matrix is composed of collagen, glycosaminoglycans (GAGs), and proteoglycans.
  • GAGs glycosaminoglycans
  • proteoglycans proteoglycans.
  • an adenoviral coat protein of the replication-deficient or conditionally-replicating adenoviral vector can comprise a non-native amino acid sequence that preferentially binds the tumor matrix.
  • Suitable non-native amino acid sequences include, for example, collagen-binding motifs such as WREPSFAMLS (SEQ ID NO: 4) and WREPGRMELN (SEQ ID NO: 5) described in Hall et al, Human Gene Therapy, 11, 983- 993 (2000), or other tumor matrix-binding motifs identified by display technologies (e.g., retro viral display libraries).
  • Replication-deficient or conditionally-replicating adenoviral vectors targeted to tumor matrix components collect in the vicinity of tumor cells, thereby increasing the likelihood of tumor cell transduction.
  • the adenoviral vector comprises a chimeric virus coat protein not selective for a specific type of eukaryotic cell.
  • the chimeric coat protein differs from a wild-type coat protein by an insertion of a normative amino acid sequence into or in place of an internal coat protein sequence, or attachment of a non-native amino acid sequence to the N- or C- terminus of the coat protein.
  • a ligand comprising about five to about nine lysine residues (preferably seven lysine residues) is attached to the C-terminus of the adenoviral fiber protein via a non-coding spacer sequence.
  • the chimeric virus coat protein efficiently binds to a broader range of eukaryotic cells than a wild-type virus coat, such as described in International Patent Application WO 97/20051.
  • a tumor does not comprise a homogenous population of cancer cells, such adenoviral vectors can be preferred in some embodiments.
  • the ability of an adenoviral vector to recognize a potential host cell can be modulated without genetic manipulation of the coat protein, i.e., through use of a bi- specific molecule.
  • complexing an adenovirus with a bispecific molecule comprising a penton base-binding domain and a domain that selectively binds a particular cell surface binding site enables the targeting of the adenoviral vector to a particular cell type.
  • the adenoviral fiber protein can be modified to render it less able to interact with the innate or acquired host immune system.
  • one or more amino acids of the native fiber protein can be mutated to render the recombinant fiber protein less able to be recognized by neutralizing antibodies than a wild-type fiber (see, e.g., international Patent Application WO 98/40509 (Crystal et al.)).
  • the fiber also can be modified to lack one or more amino acids mediating interaction with the reticulo-endothelial system (RES).
  • RES reticulo-endothelial system
  • the fiber can be mutated to lack one or more glycosylation or phosphorylation sites
  • the fiber (or virus containing the fiber) can be produced in the presence of inhibitors of glycosylation or phosphorylation
  • the adenoviral surface can be mutated to lack putative heparin sulfate proteoglycan binding domains (see, e.g., Dechecchi et al, Virology, 268, 382-390 (2000) and Dechecchi et al., J Virol, 75, 8772-8780 (2001)).
  • the replication-deficient or conditionally-replicating adenoviral vector is associated at its surface with an immunologically inert molecule(s) to "mask" the adenoviral particle from recognition by antibodies and other mammalian defense/clearance mechanisms such as the RES (see, for example, Moghimi and Hunter, Critical Reviews in Therapeutic Drug Carrier Systems, 18(6), 537-550 (2001)).
  • Inert molecules ideally avoid the immune system, neutralizing antibodies, and other blood-borne proteins, scavenger cells, and the reticuloendothelium system. Inert molecules also can aid in resistance to degradative enzymes.
  • Immunologically-inert molecules include, but are not limited to, a poloxamer, a poloxamine, a poly(acryl amide), a poly(2-ethyl-oxazoline), a poly[N-(2- hydroxylpropyl)methylacrylamide], a poly(vinyl alcohol), a poly(vinyl pyrrolidone), a poly(lactide-co-glycolide), a poly(methyl methacrylate), a poly(butyl-2-cyanoacrylate), or a poly(ethylene glycol) (PEG).
  • a poloxamer a poloxamine
  • a poly(acryl amide) a poly(2-ethyl-oxazoline)
  • a poly[N-(2- hydroxylpropyl)methylacrylamide] a poly(vinyl alcohol), a poly(vinyl pyrrolidone), a poly(lactide-co-glycolide), a poly(methyl methacrylate), a
  • virion proteins can be conjugated to a lipid derivative of PEG comprising a primary amine group, an epoxy group, or a diacylclycerol group to reduce collectin and/or opsonin affinity or scavenging by Kupffer cells or other cells of the RES (see, e.g., Kilbanov et al, FEBSLett, 268, 235 (1990), Senior et al., Biochem. Biophys. Ada., 1062, 11 (1991), Allen et al., Biochem. Biophys. Ada., 1066, -29 (1991), and Mori et al, FEBSLett., 284, 263 (1991)).
  • Conjugation of immunologically inert molecules to the viral surface is known in the art.
  • PEGylation of adenovirus is described in Croyle et al, J Virol, 75(10), 4792-4801 (2001), and U.S. Patent 6,399,385 (Croyle et al.).
  • Several variations of PEG molecules are commercially available which utilize different amino acids (e.g., lysine or cysteine) for attachment to the viral surface.
  • adenoviral coat proteins can be modified to contain such attachment sites.
  • the replication-deficient or conditionally-replicating adenoviral vector of the inventive method to comprise one or more cysteine and/or lysine residues genetically incorporated into a coat protein. It also can be advantageous to incorporate non- native amino acid sequences into the adenoviral coat in order to target the replication- deficient or conditionally-replicating adenoviral vector to target cells. It is preferred that such non-native amino acid sequences do not contain attachment sites for PEG molecules, which could result in blockage of cell surface binding sites on the non-native amino acid ligand.
  • the replication-deficient or conditionally- replicating adenoviral vector is PEGylated, and the non-native amino acid sequence does not comprise a cysteine or a lysine onto which a PEG molecule could attach to the non- native amino acid sequence and impede cellular transduction.
  • This construction strategy allows PEGylation of the viral particle while retaining activity.
  • the replication-deficient or conditionally-replicating adenoviral vector comprises at least one exogenous nucleic acid.
  • Any nucleic acid not native to the adenoviral vector is "exogenous.”
  • the exogenous nucleic acid encodes a peptide that exerts a biological effect in a host cell such as, for example, a peptide that is associated with or treats a biological disorder.
  • the exogenous nucleic acid can be obtained from any source, e.g., isolated from nature, synthetically generated, isolated from a genetically engineered organism, and the like.
  • the replication-deficient or conditionally- replicating adenoviral vector comprises a nucleic acid sequence encoding TNF- ⁇ .
  • the replication-deficient or conditionally-replicating adenoviral vector comprises a nucleic acid sequence encoding a TNF- ⁇ derived from any suitable mammal.
  • the nucleic acid sequence encodes a human TNF- ⁇ . While other members of the TNF family of proteins, such as Fas ligand and CD40 ligand, have utility in treating a number of diseases, TNF- ⁇ has been proven to be an effective anti-cancer agent. The effect of TNF- ⁇ on cancer is multifactorial including the induction of apoptosis and tumor necrosis.
  • TNF- ⁇ induces adhesiveness of vascular endothelium to neutrophils and platelets and decreases thrombomodulin production (Koga et al., Am. J. Physiol, 268, 1104-1113 (1995)). The result is clot formation in the tumor neovasculature and subsequent hemorrhagic necrosis of the tumors.
  • a nucleic acid sequence encoding a human TNF- ⁇ is described in detail in U.S. Patent 4,879,226.
  • An adenoviral vector encoding a human TNF is further described in U.S. Patent 6,579,522.
  • the exogenous nucleic acid can encode an angiogenesis inhibitor that inhibits or reduces neovascularization in the mammal.
  • Angiogenesis inhibitors can, for example, inhibit cell proliferation, cell migration, vessel formation, extracellular matrix degradation, production of mediators, and the like.
  • Angiogenesis inhibitors also can be antagonists for angiogenesis-promoting agents, such that the angiogenesis-promoting factors are neutralized (see, for example, Sato, Proc. Natl. Acad. Sci. USA, 95, 5843-5844 (1998)).
  • Angiogenesis inhibitors suitable for use in the inventive method include, for instance, anti-angiogenic factors, cytotoxins, apoptotic factors, anti-sense molecules specific for an angiogenic factor, ribozymes, receptors for an angiogenic factor (e.g., soluble VEGF- Rl (flt-1), soluble VEGF-R2 (flk/kdr), soluble VEGF-R3 (flt-4), and VEGF-receptor- chimeric proteins (Aiello, Proc. Natl. Acad. Sci, 92, 10457 (1995))), an antibody that binds an angiogenic factor, and an antibody that binds a receptor for an angiogenic factor.
  • an angiogenic factor e.g., soluble VEGF- Rl (flt-1), soluble VEGF-R2 (flk/kdr), soluble VEGF-R3 (flt-4), and VEGF-receptor
  • Anti- angiogenic factors include, for instance, angiostatin, thrombospondin, protamine, vasculostatin, endostatin, platelet factor 4, heparinase, interferons (e.g., INF ⁇ ), and the like.
  • any anti-angiogenic factor can be modified or truncated and retain anti-angiogenic activity.
  • active fragments of anti- angiogenic agents i.e., those fragments having biological activity sufficient to inhibit angiogenesis
  • Anti-angiogenic agents are further discussed in U.S.
  • Patent 5,840,686 International Patent Applications WO 93/24529 and WO 99/04806; Chader, Cell Different, 20, 209-216 (1987); Dawson et al., Science, 285, 245-248 (1999); and Browder et al, J Biol. Chem., 275, 1521-1524 (2000).
  • cytotoxins and apoptotic factors include, for example, p53, Fas, Fas ligand, Fas-associating protein with death domain (FADD), caspase- 3, caspase-8 (FLICE), FAIM, Gax, SARP-2, caspase-10, Apo2L, IkB, DIkB, receptor- interacting protein (R ⁇ P)-associated ICH-l/CED-3-homologous protein with a death domain (RAIDD), TNF-related apoptosis-inducing ligand (TRAIL), DR4, DR5, a cell death- inducing coding sequence of Bcl-2 which comprises anN-terminal deletion, a cell death- inducing coding sequence of Bcl-x which comprises an N-terminal deletion, Bax, Bak, Bid, Bad, Bik, Bif-2, c-myc, Ras, Raf, PCK kinase, AKT kinase,
  • Apoptotic, cytotoxic, and cytostatic transcription factors include, for example, E2F transcription factors and synthetic cell cycle-independent forms thereof, an API transcription factor, an AP2 transcription factor, an SP transcription factor (e.g., an SP1 transcription factor), a helix-loop-helix transcription factor, a DP transcription factor (e.g., DPI, DP2, and DP3), and mutants thereof (e.g., dominant negative mutants thereof and dominant positive mutants thereof), and fragments thereof (e.g., active domains thereof), and combinations thereof.
  • E2F transcription factors and synthetic cell cycle-independent forms thereof an API transcription factor
  • an AP2 transcription factor an SP transcription factor (e.g., an SP1 transcription factor)
  • a helix-loop-helix transcription factor e.g., DPI, DP2, and DP3
  • mutants thereof e.g., dominant negative mutants thereof and dominant positive mutants thereof
  • fragments thereof e.g., active domains thereof
  • Apoptotic, cytotoxic, and cytostatic viral proteins include, for example, an adenoviral El A product, an adenoviral E4/ORF6/7 product, an adenoviral E4/ORF4 product, a cytomegalovirus (CMV) product (e.g., CMV-thymidine kinase (CMV- TK)), a herpes simplex virus (HSV) product (e.g., HSV-TK), a human papillomavirus (HPV) product (e.g., HPVX), and mutants thereof (e.g., dominant negative mutants thereof and dominant positive mutants thereof), and fragments thereof (e.g., active domains thereof), and combinations thereof.
  • CMV CMV-thymidine kinase
  • HSV-TK herpes simplex virus
  • HPV human papillomavirus
  • mutants thereof e.g., dominant negative mutants thereof and dominant positive mutants thereof
  • fragments thereof
  • Cytotoxins and apoptotic factors are particularly useful in inhibiting cell proliferation, an important angiogenic process. Suitable cytotoxins and apoptotic agents can be identified using routine techniques, such as, for instance, cell growth assays and the TUNEL assay, respectively.
  • the exogenous nucleic acid also can encode pigment epithelium-derived factor (PEDF) or a therapeutic fragment thereof.
  • PEDF also named early population doubling factor- 1 (EPC-1)
  • EPC-1 early population doubling factor- 1
  • PEDF is a secreted protein having homology to a family of serine protease inhibitors named serpins.
  • PEDF is made predominantly by retinal pigment epithelial cells and is detectable in most tissues and cell types of the body.
  • PEDF has both neurotrophic and anti-angiogenic properties and, therefore, is useful in the treatment and study of a broad array of diseases.
  • Neurotrophic factors are thought to be responsible for the maturation of developing neurons and for maintaining adult neurons. It has been postulated that neurotrophic factors can actually reverse degradation of neurons associated with, for example, vision loss.
  • PEDF neurotrophic factors function in both paracrine and autocrine fashions, making them ideal therapeutic agents.
  • PEDF has been observed to induce differentiation in retinoblastoma cells and enhance survival of neuronal populations (Chader, Cell Different, 20, 209-216 (1987)).
  • PEDF further has gliastatic activity or has the ability to inhibit glial cell growth.
  • PEDF also has anti-angiogenic activity.
  • Anti- angiogenic derivatives of PEDF include SLED proteins, discussed in International Patent Application WO 99/04806. It also has been postulated that PEDF is involved with cell senescence (Pignolo et al, J Biol. Chem., 268 (12), 8949-8957 (1998)).
  • PEDF is further characterized in U.S. Patents 5,840,686, 6,319,687, and 6,451,763, and International Patent Applications WO 93/24529, 95/33480, and WO 99/04806.
  • Viral vectors comprising an exogenous nucleic acid encoding PEDF are further described in International Patent Application WO 01/58494.
  • the exogenous nucleic acid alternatively or additionally can encode a cytokine or chemokine.
  • Cytokines are generally biological factors released by cells which regulate cell-cell interactions, cellular communication, and other cellular activity. Cytokines include, for example, interferons, interleukins, and lymphokines. Chemokines attract and promote movement of cells.
  • Cytokines include, for example, Macrophage Colony Stimulating Factor (e.g., GM-CSF), Interferon Alpha (IFN- ⁇ ), Interferon Beta (IFN- ⁇ ), Interferon Gamma (IFN- ⁇ ), interleukins (IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL- 13, IL-15, IL-16, and IL-18), the TNF family of proteins, Intercellular Adhesion Molecule-1 (ICAM-1), Lymphocyte Function-Associated antigen-3 (LFA-3), B7-1, B7-2, FMS-related tyrosine kinase 3 ligand, (Flt3L), vasoactive intestinal peptide (VIP), and CD40 ligand.
  • Macrophage Colony Stimulating Factor e.g., GM-CSF
  • IFN- ⁇ Interferon Alpha
  • IFN- ⁇ Interferon Beta
  • Chemokines include, for example, B Cell- Attracting chemokine-1 (BCA-1), Fractalkine, Melanoma Growth Stimulatory Activity protein (MGSA), Hemofiltrate CC chemokine 1 (HCC-1), Interleukin 8 (IL8), Interferon-stimulated T-cell alpha chemoattractant (I-TAC), Lymphotactin, Monocyte Chemotactic Protein 1 (MCP-1), Monocyte Chemotactic Protein 3 (MCP-3), Monocyte Chemotactic Protein 4 (MCP-4), Macrophage-Derived Chemokine (MDC), a macrophage inflammatory protein (MIP), Platelet Factor 4 (PF4), RANTES, BRAK, eotaxin, exodus 1-3, and the like.
  • BCA-1 B Cell- Attracting chemokine-1
  • Fractalkine Melanoma Growth Stimulatory Activity protein
  • HCC-1 Hemofiltrate CC chemokine 1
  • Cytokines and chemokines are generally described in the art, including the Invivogen catalog (2002), San Diego, CA.
  • the exogenous nucleic acid can be the native nucleic acid or cDNA encoding the desired peptide, although modifications and variations of a coding nucleic acid sequence are possible and appropriate in the context of the invention.
  • the degeneracy of the genetic code allows for the substitution of nucleotides throughout polypeptide coding regions, as well as in the translational stop signal, without alteration of the encoded polypeptide.
  • substitutable sequences can be deduced from the known amino acid sequence of, for example, TNF- ⁇ or the nucleic acid sequence encoding TNF- ⁇ and can be constructed by conventional synthetic or site-specific mutagenesis procedures.
  • Synthetic DNA methods can be carried out in substantial accordance with the procedures of Itakura et al, Science, 198, 1056-1063 (1977), and Crea et al., Proc. Natl. Acad. Sci. USA, 75, 5765- 5769 (1978).
  • Site-specific mutagenesis procedures are described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (2d ed. 1989).
  • the nucleic acid sequence can encode a peptide with extensions on either the N- or C-terminus of the protein, so long as the peptide retains biological activity, such as TNF- ⁇ 's tumoricidal activity described in U.S. Patents 4,650,674, 5,795,967, and 5,972,347, as well as European Patents 168,214 and 155,549.
  • nucleic acid sequence encoding a homolog of any of the peptides described here i.e., any peptide that is more than about 70% identical (preferably more than about 80% identical, more preferably more than about 90% identical, and most preferably more than about 95% identical) to the protein at the amino acid level and displays the same level of activity of the desired peptide, can be incorporated into the replication-deficient or conditionally-replicating adenoviral vector.
  • the degree of amino acid identity can be determined using any method known in the art, such as the BLAST sequence database.
  • a homolog of the protein can be any peptide, polypeptide, or portion thereof, which hybridizes to the protein under at least moderate, preferably high, stringency conditions, and retains biological activity.
  • exemplary moderate stringency conditions include overnight incubation at 37° C in a solution comprising 20% formamide, 5x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in lx SSC at about 37-50° C, or substantially similar conditions, e.g., the moderately stringent conditions described in Sambrook et al., supra.
  • High stringency conditions are conditions that use, for example, (1) low ionic strength and high temperature for washing, such as 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate (SDS) at 50° C, (2) employ a denaturing agent during hybridization, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin (BSA)/0.1% Ficoll/0.1% polyvinylpyrrolidone (PVP)/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C, or (3) employ 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 mg/ml), 0.1 % SDS, and 10% dextran sulfate at
  • the nucleic acid sequence can encode a functional portion of a desired peptide, i.e., any portion of the protein that retains the biological activity of the naturally occurring, full-length protein at measurable levels.
  • a functional TNF- ⁇ fragment produced by expression of the nucleic acid sequence of the replication-deficient or conditionally-replicating adenoviral vector can be identified using standard molecular biology and cell culture techniques, such as assaying the biological activity of the fragment in human cells transiently transfected with a nucleic acid sequence encoding the protein fragment.
  • the exogenous nucleic acid also can encode a fusion protein comprising, in part, a protein of interest paired with other, preferably functional peptide portions.
  • the exogenous nucleic acid can encode a fusion protein comprising TNF- ⁇ or a biologically- active fragment thereof fused to a ligand for a cellular receptor found in tumor cells, e.g., a ligand that binds ⁇ v ⁇ 3, ⁇ v ⁇ 5, ⁇ v ⁇ 6, or CD13.
  • the exogenous nucleic acid is desirably present as part of an expression cassette, i.e., a particular nucleotide sequence that possesses functions which facilitate subcloning and recovery of a nucleic acid sequence (e.g., one or more restriction sites) or expression of a nucleic acid sequence (e.g., polyadenylation or splice sites).
  • the exogenous nucleic acid is preferably located in the El region (e.g., replaces the El region in whole or in part) or the E4 region of the adenoviral genome.
  • the El region can be replaced by a promoter-variable expression cassette comprising an exogenous nucleic acid.
  • the expression cassette is preferably inserted in a 3 '-5' orientation, e.g., oriented such that the direction of transcription of the expression cassette is opposite that of the surrounding adjacent adenoviral genome. However, it is also appropriate for the expression cassette to be inserted in a 5 '-3' orientation with respect to the direction of transcription of the surrounding genome.
  • the replication-deficient or conditionally-replicating adenoviral vector can comprise other expression cassettes containing other exogenous nucleic acids, which cassettes can replace any of the deleted regions of the adenoviral genome.
  • an expression cassette into the adenoviral genome can be facilitated by known methods, for example, by the introduction of a unique restriction site at a given position of the adenoviral genome.
  • a unique restriction site at a given position of the adenoviral genome.
  • the exogenous nucleic acid comprises a transcription-terminating region such as a polyadenylation sequence located 3' of angiogenic peptide coding sequence (in the direction of transcription of the coding sequence).
  • Any suitable polyadenylation sequence can be used, including a synthetic optimized sequence, as well as the polyadenylation sequence of BGH (Bovine Growth Hormone), polyoma virus, TK (Thymidine Kinase), EBV (Epstein Barr Virus), and the papiUomaviruses, including human papiUomaviruses and BPV (Bovine Papilloma Virus).
  • BGH Bovine Growth Hormone
  • polyoma virus polyoma virus
  • TK Thymidine Kinase
  • EBV Epstein Barr Virus
  • papiUomaviruses including human papiUomaviruses and BPV (Bovine Papilloma Virus).
  • a preferred polyadenylation sequence is the SV40 (Human Sarcoma Virus-40) polyadenylation sequence.
  • the exogenous nucleic acid is operably linked to (i.e., under the transcriptional control of) one or more promoter and/or enhancer elements, for example, as part of a promoter- variable expression cassette.
  • promoter and/or enhancer elements for example, as part of a promoter- variable expression cassette.
  • Any suitable promoter or enhancer sequence can be used in the context of the invention.
  • Suitable viral promoters include, for instance, cytomegalovirus (CMV) promoters, such as the CMV immediate-early promoter (described in, for example, U.S.
  • HIV human immunodeficiency virus
  • RS V Rous sarcoma virus
  • MMTV mouse mammary tumor virus
  • HSV herpes thymidine kinase promoter
  • promoters derived from SV40 or Epstein Barr virus, an adeno-associated viral promoter, such as the p5 promoter, and the like.
  • the promoter is the CMV immediate-early promoter.
  • the promoter can be an inducible promoter, i.e., a promoter that is up- and/or down-regulated in response to an appropriate signal.
  • an expression control sequence up-regulated by a chemotherapeutic agent is particularly useful in cancer applications (e.g., a chemo-inducible promoter).
  • an expression control sequence can be up-regulated by a radiant energy source or by a substance that distresses cells.
  • an expression control sequence can be up-regulated by ultrasound, light activated compounds, radiofrequency, chemotherapy, and cyofreezing.
  • a preferred replication-deficient or conditionally-replicating adenoviral vector according to the invention comprises a chemo-inducible or radiation-inducible promoter operably linked to an exogenous nucleic acid encoding TNF- ⁇ .
  • the use of a radiation-inducible promoter enables localized control of TNF- ⁇ production, for example, by the administration of radiation to a cell or host comprising the adenoviral vector, thereby minimizing systemic toxicity.
  • a preferred radiation-inducible promoter for use in the context of the invention is the early growth region- 1 (EGR-1) promoter, specifically at least one of the six CArG domains of the EGR-1 promoter.
  • the region of the EGR-1 promoter likely responsible for radiation-inducibility is located between nucleotides -550 bp and -50 bp and contains six CArG domains.
  • the EGR-1 promoter is described in detail in U.S. Patent 5,206,152 and International Patent Application WO 94/06916.
  • Another suitable radiation-inducible promoter is the c-Jun promoter, which is activated by X-radiation.
  • the region of the c-Jun promoter likely responsible for radiation-inducibility is believed to be located between nucleotides -1.1 kb to 740 bp.
  • the c-Jun promoter and the EGR-1 promoter are further described in, for instance, U.S. Patent 5,770,581.
  • the promoter also can be a tissue- or cell-specific promoter, such as a tumor cell-selective promoter.
  • Tumor cell-selective promoters suitable for the replication- deficient or conditionally-replicating adenoviral vector include, but are not limited to, an E2F promoter, the DF3 (muc-1) promoter, and the telomerase reverse transcriptase promoter.
  • the DF3 promoter comprises a nucleic acid sequence of SEQ ID NO: 7
  • the telomerase reverse transcriptase promoter comprises a nucleic acid sequence of SEQ ID NO: 8.
  • the promoter also can be selective for endothelial cells associated with tumors, such as the flt-1 promoter.
  • the promoter is an E2F promoter.
  • the E2F promoter can be a promoter, or a functional portion thereof, of a gene encoding any suitable member of the E2F protein family. Suitable members of the E2F protein family include, for example, E2F-1, E2F-2, E2F-3, E2F-4, E2F-5, and E2F-6.
  • the E2F promoter comprises two imperfect palindromic nucleic acid sequences, as described in, for example, Neuman et al., Mol. Cell. Biol, 10, 6607-6615 (1994).
  • the E2F promoter is an E2F-1 promoter which comprises a nucleic acid sequence of SEQ ID NO: 9.
  • the promoter can be a chimeric promoter.
  • a promoter is "chimeric" in that it comprises at least two nucleic acid sequence portions obtained from, derived from, or based upon at least two different sources (e.g., two different regions of an organism's genome, two different organisms, or an organism combined with a synthetic sequence).
  • a sequence is "obtained” from a source when it is isolated from that source.
  • a sequence is "derived” from a source when it comprises a sequence isolated from a source but modified in any suitable manner (e.g., by deletion, substitution (mutation), or other modification to the sequence).
  • a sequence is "based upon" a source when the sequence is a sequence highly homologous to the source but obtained through synthetic procedures (e.g., polynucleotide synthesis, directed evolution, etc.).
  • the two different nucleic acid sequence portions exhibit less than about 40%, more preferably less than about 25%, and even more preferably less than about 10% nucleic acid sequence identity to one another (which can be determined by methods described elsewhere herein).
  • Any suitable chimeric promoter can be used in the inventive method.
  • the chimeric promoter is comprised of a functional portion of a viral promoter and a functional portion of a cellular promoter.
  • the chimeric promoter comprises a functional portion of a viral promoter and a functional portion of a cellular promoter that is radiation-inducible.
  • the chimeric promoter comprises a functional portion of a CMV promoter and a functional portion of an EGR-1 promoter (i.e., a chimeric "CMV/EGR-1" promoter).
  • the functional portion of the CMV promoter preferably is derived from a human CMV, and more particularly from the human CMV immediate early (IE) promoter/enhancer region (see, e.g., U.S. Patents 5,168,062 and 5,385,839).
  • the functional portion of the EGR-1 promoter preferably comprises one or more CArG domains of an EGR-1 promoter, as described in, for example, U.S. Patents 6,579,522 and 6,605,712.
  • the chimeric promoter comprises a functional portion of the CMV IE enhancer/promoter region, which functional portion has SEQ ID NO: 10, and an EGR-1 promoter comprising six CArG domains.
  • the portion of the CMV IE enhancer/promoter region functions as an enhancer for the EGR-1 promoter.
  • Chimeric promoters can be generated using standard molecular biology techniques, such as those described in Sambrook et al., supra.
  • a "functional portion” is any portion of a promoter that measurably promotes, enhances, or controls expression (typically transcription) of an operatively linked nucleic acid.
  • Such regulation of expression can be measured via RNA or protein detection by any suitable technique, and several such techniques are known in the art. Examples of such techniques include Northern analysis (see, e.g., Sambrook et al, supra, and McMaster and Carmichael, PNAS, 74, 4835-4838 (1977)), RT-PCR (see, e.g., U.S.
  • the dose of replication-deficient or conditionally-replicating adenoviral vector will depend on a number of factors, including the size of a target tissue, the extent of any side-effects, the particular route of administration, and the like. Desirably, a single dose of replication-deficient or conditionally-replicating adenoviral vector comprises at least about IxIO 5 particles (which also is referred to as particle units) to at least about IxIO 13 particles of the adenoviral vector.
  • the dose preferably is at least about 1x10 particles (e.g., about 4xl0 6 -4xl0 12 particles), more preferably at least about IxIO 7 particles, more preferably at least about IxIO 8 particles (e.g., about 4xl0 8 -4xlO ⁇ particles), and most preferably at least about IxIO 9 particles to at least about IxIO 10 particles (e.g., about 4xl0 9 -4xl0 10 particles) of the adenoviral vector.
  • 1x10 particles e.g., about 4xl0 6 -4xl0 12 particles
  • IxIO 7 particles e.g., about 4xIO 8 particles
  • IxIO 9 particles e.g., about 4xl0 9 -4xl0 10 particles
  • the dose comprises no more than about IxIO 14 particles, preferably no more than about 1x10 particles, even more preferably no more than about 1x10 particles, even more preferably no more than about 1x10 particles, and most preferably no more than about IxIO 10 particles (e.g., no more than about IxIO 9 particles).
  • a single dose of replication-deficient or conditionally-replicating adenoviral vector can comprise about lxl 0 6 particle units (pu), 2x10 6 pu, 4xl0 6 pu, lxl 0 7 pu, 2xl0 7 pu, 4xl0 7 pu, IxIO 8 pu, 2xl0 8 pu, 4x10 s pu, IxIO 9 pu, 2xl0 9 pu, 4xl0 9 pu, IxIO 10 pu, 2xl0 10 pu, 4xl0 10 pu, IxIO 11 pu, 2xlO ⁇ pu, 4xlO ⁇ pu, IxIO 12 pu, 2xl0 12 pu, or 4xl0 12 pu of the replication-deficient or conditionally-replicating adenoviral vector.
  • the volume of carrier, especially pharmaceutically-acceptable carrier, in which the replication-deficient or conditionally-replicating adenoviral vector is diluted will depend on the size of the mammal and the time period over which the dose of replication-deficient or conditionally-replicating adenoviral vector is administered, typically in a pharmaceutical composition.
  • the dose of replication-deficient or conditionally-replicating adenoviral vector is administered in a pharmaceutical composition comprising about 20 ml or more of physiologically-acceptable carrier per kilogram (kg) of mammal.
  • the pharmaceutical composition comprises about 40 ml or more of physiologically acceptable carrier/kg of mammal, more preferably about 60 ml or more of physiologically acceptable carrier/per kg of mammal.
  • the pharmaceutical composition comprises about 80 ml or more of physiologically acceptable carrier/per kg of mammal, and most preferably comprises about 100 ml or more of physiologically acceptable carrier/kg of mammal.
  • the volume of pharmaceutical composition administered to a mammal can be calculated based on the surface area of a mammal, a technique routinely used in pharmacology.
  • the pharmaceutical composition comprises about 75 ml or more (e.g., about 100 ml or more) of physiologically acceptable carrier per square meter of surface area of the mammal.
  • the pharmaceutical composition comprises about 150 ml or more (e.g., about 175 ml or more, about 200 ml or more, or . about 250 ml or more) of physiologically acceptable carrier/m 2 of surface area of the mammal.
  • the dose of the replication-deficient or conditionally-replicating adenoviral vector is administered in a pharmaceutical composition comprising 275 ml or more (e.g., 300 ml or more) of physiologically-acceptable carrier/m of surface area of the mammal. It will be appreciated that smaller volumes of carrier may be appropriate in some embodiments as described in, for example, U.S. Patent Application Publication 2003/0086903.
  • the dose of replication-deficient or conditionally-replicating adenoviral vector is administered intraperitoneally to the peritoneal cavity.
  • the dose of replication-deficient or conditionally-replicating adenoviral vector can be supplied to the peritoneal cavity using any appropriate means, such as injection or instillation.
  • a "pre-dose" of a substance which saturates natural innate clearance mechanisms of the mammal such as an adenoviral vector.
  • the pre-dose can comprise any adenovirus or adenoviral vector constructs described herein, and preferably comprises replication-deficient or conditionally-replicating adenoviral vectors having a reduced ability to transduce mesothelial cells or hepatocytes than a wild-type adenoviral vector of the same serotype.
  • a pre-dose of adenoviral vector increases the persistence of a dose of replication-deficient or conditionally-replicating adenoviral vector by interfering or interacting with a mammal's clearance effector cells, thereby permitting a larger fraction of a dose of replication-deficient or conditionally-replicating adenoviral vectors to reach the bloodstream and remain in circulation.
  • a pre-dose of adenoviral vector can provoke a tolerance in the mammal to the replication-deficient or conditionally-replicating adenoviral vector.
  • the pre-dose of adenoviral vector can comprise any suitable number of adenoviral particles in any suitable volume of physiologically acceptable carrier, such as the doses of adenoviral vectors and volumes of physiologically acceptable carrier described herein.
  • the pre-dose of adenoviral vector can be administered to the mammal using any route of administration, such as intravenous, intraarterial, or intraperitoneal delivery, and can occur at any time prior to the administration of the dose of replication-deficient or conditionally-replicating adenoviral vector, desirably such that the administration of the pre-dose increases the circulation time of the dose of replication-deficient or conditionally-replicating adenoviral vector.
  • the pre- dose is preferably administered about 5 minutes to about 60 minutes (e.g., about 10 minutes to about 45 minutes) prior to the administration of the dose of replication-deficient or conditionally-replicating adenoviral vector.
  • the pre-dose can be administered about 15 minutes to about 30 minutes prior to administering the dose of replication-deficient or conditionally-replicating adenoviral vector.
  • the invention provides a method of destroying tumor cells in a mammal.
  • the replication-deficient adenoviral vector comprises (a) an exogenous nucleic acid sequence which is operably linked to a tumor cell-selective promoter, (b) a fiber protein wherein a native CAR-binding site is disrupted, and (c) and a penton base protein wherein a native integrin-binding site is disrupted, as described herein.
  • Tumor cells and/or cells associated with or in close proximity to a tumor are transduced and a tumoricidal agent encoded by the exogenous nucleic acid sequence is produced, thereby destroying tumor cells in the mammal.
  • Many tumoricidal agents are described herein and identified in the art.
  • a preferred tumoricidal agent is a human TNF- ⁇ .
  • the target tissue is a solid tumor or a tumor associated with soft tissue (i.e., soft tissue sarcoma), in a human.
  • the tumor can be associated with cancers of (i.e., located in) the oral cavity and pharynx, the digestive system, the respiratory system, bones and joints (e.g., bony metastases), soft tissue, the skin (e.g., melanoma), breast, the genital system, the urinary system, the eye and orbit, the brain and nervous system (e.g., glioma), the peritoneum (e.g., ovarian cancer or peritoneal carcinomatosis), or the endocrine system (e.g., thyroid or adrenal gland) and is not necessarily the primary tumor.
  • cancers of i.e., located in) the oral cavity and pharynx, the digestive system, the respiratory system, bones and joints (e.g., bony metasta
  • Tissues associated with the oral cavity include, but are not limited to, the tongue and tissues of the mouth. Cancer can arise in tissues of the digestive system including, for example, the esophagus, stomach, small intestine, colon, rectum, anus, liver (e.g., hepatobiliary cancer), gall bladder, and pancreas. Cancers of the respiratory system can affect the larynx, lung, and bronchus and include, for example, non-small cell lung carcinoma.
  • Tumors can arise in the uterine cervix, uterine corpus, ovary vulva, vagina, prostate, testis, and penis, which make up the male and female genital systems, and the urinary bladder, kidney, renal pelvis, and ureter, which comprise the urinary system.
  • the target tissue also can be associated with lymphoma (e.g., Hodgkin's disease and Non- Hodgkin's lymphoma), multiple myeloma, or leukemia (e.g., acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, and the like).
  • lymphoma e.g., Hodgkin's disease and Non- Hodgkin's lymphoma
  • leukemia e.g., acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia,
  • the tumor can be at any stage, and can be subject to other therapies.
  • the replication-deficient or conditionally-replicating adenovirus vectors of the inventive method are useful in treating tumors (i.e., destruction of tumor cells or reduction in tumor size) that have been proven to be resistant to other forms of cancer therapy, such as radiation-resistant tumors.
  • the tumor also can be of any size.
  • the replication-deficient or conditionally- replicating adenoviral vectors of the inventive method mediate reduction of the size of initially large tumors (e.g., 42 cm (cross-sectional surface area) or 4400 cm in volume). Ideally, the inventive method results in cancerous (tumor) cell death and/or reduction in tumor size.
  • tumor cell death can occur without a substantial decrease in tumor size due to, for instance, the presence of supporting cells, vascularization, fibrous matrices, etc. Accordingly, while reduction in tumor size is preferred, it is not required in the treatment of cancer.
  • One advantage of the inventive method over previous cancer therapies is the ability to target tumor cells while better avoiding non-target tissues. Reducing native binding of the replication-deficient or conditionally-replicating adenoviral vector reduces transduction of non-target tissues such as liver, spleen, kidney, and lung, thereby providing a greater fraction of the dose of replication-deficient or conditionally-replicating adenoviral vector available for target tissue, e.g., tumor, transduction.
  • the replication-deficient or conditionally- replicating adenoviral vector can comprise a non-native amino acid sequence (i.e., ligand) incorporated into an adenoviral coat protein, such as an adenoviral fiber protein, which is specific for a cellular receptor expressed in tumor cells.
  • a non-native amino acid sequence i.e., ligand
  • an adenoviral coat protein such as an adenoviral fiber protein
  • a ratio of the level of tumor transduction by the replication-deficient or conditionally-replicating adenoviral vector compared to the level of, for example, liver transduction by the replication-deficient or conditionally-replicating adenoviral vector of at least about 0.1:1 can be achieved.
  • the ratio of the level of tumor transduction by the replication- deficient or conditionally-replicating adenoviral vector compared to the level of liver transduction by the replication-deficient or conditionally-replicating adenoviral vector is at least about 0.5:1, most preferably at least about 1:1.
  • the replication-deficient or conditionally-replicating adenoviral vector is desirably present in a pharmaceutical composition comprising a pharmaceutically acceptable carrier (e.g., a physiologically acceptable carrier).
  • a pharmaceutically acceptable carrier e.g., a physiologically acceptable carrier
  • Any suitable pharmaceutically acceptable carrier can be used within the context of the invention, and such carriers are well known in the art. The choice of carrier will be determined, in part, by the particular method used to administer the pharmaceutical composition.
  • Suitable formulations include aqueous and non-aqueous solutions, isotonic sterile solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood or other bodily fluid of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the pharmaceutically acceptable carrier is a liquid that contains a buffer and a salt.
  • the formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, immediately prior to use.
  • sterile liquid carrier for example, water
  • Extemporaneous solutions and suspensions can be prepared from sterile powders, granules, and tablets.
  • the pharmaceutically acceptable carrier is a buffered saline solution.
  • the pharmaceutical composition is formulated to protect the adenoviral vector from damage prior to administration.
  • the particular formulation desirably decreases the light sensitivity and/or temperature sensitivity of the adenoviral vector.
  • the pharmaceutical composition will be maintained for various periods of time and, therefore should be formulated to ensure stability and maximal activity at the time of administration.
  • the pharmaceutical composition is maintained at a temperature above 0° C, preferably at 4° C or higher (e.g., 4-10° C).
  • the pharmaceutical composition can be maintained at the aforementioned temperature(s) for at least 1 day (e.g., 7 days (1 week) or more), though typically the time period will be longer, such as at least 3, 4, 5, or 6 weeks, or even longer, such as at least 10, 11, or 12 weeks, prior to administration to a patient.
  • the adenoviral gene transfer vector optimally loses no, or substantially no, activity, although some loss of activity is acceptable, especially with relatively higher storage temperatures and/or relatively longer storage times.
  • the activity of the adenoviral vector composition decreases about 20% or less, preferably about 10% or less, and more preferably about 5% or less, after any of the aforementioned time periods.
  • the pharmaceutical composition preferably comprises a pharmaceutically acceptable liquid carrier, such as, for example, those described above, and a stabilizing agent selected from the group consisting of polysorbate 80, L-arginine, polyvinylpyrrolidone, ⁇ -D-glucopyranosyl ⁇ -D-glucopyranoside dihydrate (commonly known as trehalose), and combinations thereof. More preferably, the stabilizing agent is trehalose, or trehalose in combination with polysorbate 80. The stabilizing agent can be present in any suitable concentration in the pharmaceutical composition.
  • the trehalose desirably is present in a concentration of about 2- 10% (wt./vol.), preferably about 4-6% (wt/vol.) of the pharmaceutical composition.
  • the trehalose and polysorbate 80 are present in the pharmaceutical composition, the trehalose preferably is present in a concentration of about 4-6% (wt./vol.), more preferably about 5% (wt./vol.), while the polysorbate 80 desirably is present in a concentration of about 0.001- 0.01% (wt./vol.), more preferably about 0.0025% (wt./vol.).
  • the pharmaceutically acceptable liquid carrier preferably contains a saccharide other than trehalose.
  • a stabilizing agent e.g., trehalose
  • the pharmaceutically acceptable liquid carrier preferably contains a saccharide other than trehalose.
  • the pharmaceutical composition can comprise additional therapeutic or biologically active agents.
  • therapeutic factors useful in the treatment of a particular indication can be present.
  • Factors that control inflammation such as ibuprofen or steroids, can be part of the pharmaceutical composition to reduce swelling and inflammation associated with in vivo administration of the adenoviral vector and physiological distress.
  • Immune system suppressors can be administered with the pharmaceutical composition to reduce any immune response to the adenoviral vector itself or associated with a disorder.
  • immune enhancers can be included in the pharmaceutical composition to upregulate the body's natural defenses against disease.
  • a typical course of treatment for most types of cancer is radiation therapy. Accordingly, the method of the invention can further comprise administering a dose of radiation to a subject.
  • Radiation therapy uses a beam of high-energy particles or waves, such as X-rays and gamma rays, to eradicate cancer cells by inducing mutations in cellular DNA. In that cancer cells divide more rapidly than normal cells, tumor tissue is more susceptible to radiation than normal tissue. Radiation also has been shown to enhance exogenous DNA expression in exposed cells.
  • TNF- ⁇ When the nucleic acid sequence encoding TNF- ⁇ is operably linked to a radiation-inducible promoter, radiation potentiates TNF- ⁇ production and maintains therapeutic levels of TNF- ⁇ at the tumor site continuously throughout the period of radiation therapy, in addition to the additive or synergistic effect of radiation and TNF- ⁇ observed in eradicating tumor cells (see, for example, Hersh et al., Gene Therapy, 2, 124-131 (1995), and Kawashita et al., Human Gene Therapy, 10, 1509- 1519 (1999)).
  • any type of radiation can be administered to a mammal, so long as the dose of radiation is tolerated by the mammal without significant negative side-effects.
  • Suitable types of radiotherapy include, for example, ionizing (electromagnetic) radiotherapy (e.g., X- rays or gamma rays) or particle beam radiation therapy (e.g., high linear energy radiation).
  • Ionizing radiation is defined as radiation comprising particles or photons that have sufficient energy to produce ionization, i.e., gain or loss of electrons (as described in, for example, U.S. Patent 5,770,581).
  • the effects of radiation can be at least partially controlled by the clinician.
  • the dose of radiation is preferably fractionated for maximal target cell exposure and reduced toxicity.
  • Radiation can be administered concurrently with radiosensitizers that enhance the killing of tumor cells, or with radioprotectors (e.g., IL-1 or IL-6) that protect healthy tissue from the harmful effects of radiation.
  • radioprotectors e.g., IL-1 or IL-6
  • the application of heat, i.e., hyperthermia, or chemotherapy can sensitize tissue to radiation.
  • the source of radiation can be external or internal to the mammal.
  • External radiation therapy is most common and involves directing a beam of high-energy radiation to a tumor site through the skin using, for instance, a linear accelerator. While the beam of radiation is localized to the tumor site, it is nearly impossible to avoid exposure of normal, healthy tissue. However, external radiation is usually well tolerated by patients.
  • Internal radiation therapy involves implanting a radiation-emitting source, such as beads, wires, pellets, capsules, and the like, inside the body at or near the tumor site. Such implants can be removed following treatment, or left in the body inactive.
  • Types of internal radiation therapy include, but are not limited to, brachytherapy, interstitial irradiation, and intracavity irradiation.
  • a less common form of internal radiation therapy is radioimmunotherapy wherein tumor-specific antibodies bound to radioactive material is administered to a patient. The antibodies seek out and bind tumor antigens, thereby effectively administering a dose of radiation to the relevant tissue
  • the total dose of radiation administered to a mammal in the context of the invention preferably is about 5 Gray (Gy) to about 70 Gy. More preferably, about 10 Gy to about 65 Gy (e.g., about 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, 50 Gy, 55 Gy, or 60 Gy) are administered over the course of treatment. While a complete dose of radiation can be administered over the course of one day, the total dose is ideally fractionated and administered over several days. Desirably, radiotherapy is administered over the course of at least about 3 days, e.g., at least 5, 7, 10, 14, 17, 21, 25, 28, 32, 35, 38, 42, 46, 52, or 56 days (about 1-8 weeks).
  • a daily dose of radiation will comprise approximately 1-5 Gy (e.g., about 1 Gy, 1.5 Gy, 1.8 Gy, 2 Gy, 2.5 Gy, 2.8 Gy, 3 Gy, 3.2 Gy, 3.5 Gy, 3.8 Gy, 4 Gy, 4.2 Gy, or 4.5 Gy), preferably 1-2 Gy (e.g., 1.5-2 Gy).
  • radiation preferably is not administered every day, thereby allowing the subject to rest and the effects of the therapy to be realized.
  • radiation desirably is admimstered on 5 consecutive days, and not administered on 2 days, for each week of treatment, thereby allowing 2 days of rest per week.
  • radiation can be administered 1 day/week, 2 days/week, 3 days/week, 4 days/week, 5 days/week, 6 days/week, or all 7 days/week, depending on the response of the patient to therapy and any potential side effects.
  • a dose of one or more chemotherapeutics can be administered to a mammal in conjunction with administering a replication-deficient adenoviral vector comprising a nucleic acid sequence encoding TNF- ⁇ .
  • a chemotherapeutic agent can be administered before administration of the replication-deficient adenoviral vector, after administration of the replication-deficient adenoviral vector, or concurrently with the replication-deficient adenoviral vector in the same pharmaceutical composition or as a separate administration. Any suitable chemotherapeutic can be used.
  • Suitable chemotherapeutics include, but are not limited to, adriamycin, asparaginase, bleomycin, busulphan, cisplatin, carboplatin, carmustine, capecitabine, chlorambucil, cytarabine, cyclophosphamide, camptothecin, dacarbazine, dactinomycin, daunorubicin, dexrazoxane, docetaxel, doxorubicin, etoposide, floxuridine, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, mechlorethamine, mercaptopurine, meplhalan, methotrexate, mitomycin, mitotane, mitoxantrone, nitrosurea, paclitaxel, pamidronate, pentostatin, plicamycin,
  • chemotherapeutics administered to a subject will depend on the standard chemotherapeutic regimen for a particular tumor type. In other words, while a particular cancer may be routinely treated with a single chemotherapeutic agent, another may be routinely treated with a combination of chemotherapeutic agents.
  • the chemotherapeutic agent administered to a subject is selected from the group consisting of 5- fluorouracil (5-FU), cisplatin, paclitaxel, gemcitabine, cyclophosphamide, capecitabine, and/or doxorubicin. Any suitable dose of the one or more chemotherapeutics can be administered to a mammal, e.g., a human.
  • Suitable doses of the chemotherapeutics described above are known in the art, and are described in, for example, U.S. Patent Application Publication No. 2003/0082685 Al .
  • the dose preferably comprises about 50 mg per m 2 of body surface area of the patient per day (i.e., mg/m 2 /day) to about 1500 mg/m 2 /day (e.g., about 100 mg/m 2 /day, about 500 mg/m 2 /day, and about 1000 mg/m 2 /day).
  • the dose of 5-FU comprises about 100 mg/m 2 /day to about 300 mg/m 2 /day (e.g., 200 mg/m 2 /day) or about 900 mg/m 2 /day to about 1100 mg/m 2 /day (e.g., about 1000 mg/m 2 /day).
  • the dose preferably comprises about 25 mg/m 2 /day to about 500 mg/m 2 /day (e.g., about 50 mg/m 2 /day, about 100 mg/m 2 /day, or about 300 mg/m 2 /day).
  • the dose of cisplatin is about 50-100 mg/m 2 /day, most preferably 75 mg/m 2 /day.
  • the dose preferably comprises about 500 mg/m 2 /day to about 1500 mg/m 2 /day (e.g., about 700 mg/m 2 /day, about 800 mg/m 2 /day, or about 900 mg/m 2 /day). More preferably, the dose of capecitabine comprises about 800 mg/m 2 /day to about 1000 mg/m 2 /day (e.g., about 900 mg/m 2 /day).
  • chemotherapy is not administered every day, thereby allowing the subject to rest and the effects of the therapy to be realized.
  • chemotherapy desirably is administered on 5 consecutive days, and not administered on 2 days, for each week of treatment, thereby allowing 2 days of rest per week.
  • chemotherapy can be administered 1 day/week, 2 days/week, 3 days/week, 4 days/week, 5 days/week, 6 days/week, or all 7 days/week, depending on the response of the patient to therapy and any potential side effects.
  • it may be advantageous to employ a method of administering the one or more chemotherapeutics wherein a dose is continuously administered to a subject over a prolonged period of time.
  • continuous infusion of the subject with the chemotherapeutic may be desirable.
  • the duration of the administration of the dose of the one or more chemotherapeutics may be any suitable length of time.
  • Standard infusion rates for the chemotherapeutics described herein are known in the art and can be modified in any suitable manner according to the nature of the disease. For example, when 5-FU is administered, a typical infusion rate is about 96 hours per treatment week (i.e., 5 days per week).
  • Other aspects of cancer chemotherapy and dosing schedules are described in, for example, Bast et al. (eds.), Cancer Medicine, 5 t edition, BC. Decker Inc., Hamilton, Ontario (2000).
  • EXAMPLE 1 This example demonstrates the tolerability of tropism-modified adenoviral vectors as compared to adenoviral vectors having native tropism in accordance with the inventive method.
  • Adenoviral serotype 5 El E3/E4-deficient adenoviral vectors containing, in place of the deleted El region, a nucleic acid sequence encoding a human TNF- ⁇ sequence operably linked to a chimeric CMV/EGR-1 promoter construct were generated. Three groups of mice were treated with three different versions of the TNF -expressing adenoviral vector. The first group of mice received an adenoviral vector as described above containing no modifications to any of the capsid proteins (Adc v EGR TNF).
  • mice received an adenoviral vector in which the AB loop of the adenoviral fiber protein was modified to disrupt CAR binding, and in which the integrin-binding domain of the adenoviral penton base protein was disrupted (Adc M v /EGR TNF**).
  • mice received an adenoviral vector in which the RGD ligand, which binds ⁇ v ⁇ 3 and ⁇ v ⁇ 5 integrins, was inserted into the HI loop of the adenoviral fiber protein of Adc M v /EGR TNF** (Adc MV/EGR TNF**RGD) using methods described herein (see, e.g., Einfeld et al., J Virol, 75, 11284-11291 (2001)).
  • a dose of 3xl0 10 particle units (pu) of each respective adenoviral vector was administered via intraperitoneal (i.p.) injection on days 0, 3, and 7 of the therapeutic period.
  • Body weight was used as an indicator of tolerance of vector administration. Body weight loss caused by TNF- ⁇ expression was significantly reduced in the mice treated with Adc M v /EGR TNF** and Adc M v /EGR TNF**RGD as compared to mice treated with Adc M v EGR TNF or the control (see Figure 1).
  • This example demonstrates that i.p. administration of an adenoviral vector expressing TNF- ⁇ under the control of a chimeric CMV/EGR-1 promoter and modified to reduce native binding to host cell receptors is well tolerated in mice.
  • EXAMPLE 2 This example demonstrates the tolerability of the inventive method in vivo.
  • Adenoviral serotype 5 El/E3/E4-deficient adenoviral vectors containing, in place of the deleted El region, a nucleic acid sequence encoding a human TNF- ⁇ sequence operably linked to each of the following promoters were generated: an EGR-1 promoter, a human CMV/EGR-1 chimeric promoter, an E2F promoter, a human CMV promoter, an RSV promoter, a DF3 promoter, and a human telomerase reverse transcriptase (hTERT) promoter.
  • the AB loop of the adenoviral fiber protein was modified to disrupt CAR binding.
  • the integrin-binding domain of the adenoviral penton base protein was disrupted (Ad ⁇ NF F*PB*).
  • a ligand which binds ⁇ v ⁇ 3 and ⁇ v ⁇ 5 integrins to mediate viral transduction was inserted into the HI loop of the adenoviral fiber protein of AdL.F*PB* using methods described herein (see, e.g., Einfeld et al., supra).
  • Each adenoviral vector was administered via intraperitoneal (i.p.) injection to Balb/c mice at a dose of 3xl0 10 , 6xl0 10 , 1.2xl0 ⁇ , or 2.4xl0 ⁇ particle units (pu). Mice were followed for 17 days post treatment. Body weight was used as an indicator of tolerance of vector administration. Dose dependent decreases in body weight were observed in mice treated with each adenoviral vector, with the exception of the vectors expressing TNF- ⁇ under the control of the E2F promoter and the DF3 promoter. [0090] This example demonstrates that i.p. administration of an adenoviral vector expressing TNF- ⁇ under the control of an E2F promoter or a DF3 promoter and modified to reduce native binding to host cell receptors is well tolerated in mice.
  • EXAMPLE 3 This example demonstrates the efficacy of the inventive method in vivo.
  • certain adenoviral vector constructs described in Example 2 were injected into the i.p. cavity of mice at 5, 8, and 12 days after implantation of CaOv3 ovarian tumor cells.
  • the vector doses were based on the single administration maximum tolerated dose (MTD) identified in the experiments described in Example 2, and are set forth below in Table 1.
  • the MTD was not established for the adenoviral vector comprising the DF3 promoter.
  • each of the adenoviral vector constructs were injected into the i.p. cavity of athymic nude mice at 5, 8, and 12 days after implantation of CaOv3 ovarian tumor cells at the dose set forth in Table 1 (i.e., the high (H) dose), 1/3 the dose set forth in Table 1 (i.e., the mid (M) dose), or 1/10 the dose set forth in Table 1 (i.e., the low (L) dose).
  • Adenoviral vector final formulation buffer (FFB) was injected into mice as a control.
  • Tumor nodules in the peritoneal space were resected in a blinded fashion 35 days after tumor cell implantation.
  • Treatment with Ad tNF EGR, Ad ⁇ NF CMV/EGR-1, Ad ⁇ NF DF3, and Adx NF RSV produced significant anti-tumor activity at 1/3 the MTD, as measured by tumor weight (see Figure 2a).
  • This example demonstrates that intraperitoneal administration of an adenoviral vector encoding TNF operably linked to a tumor cell selective promoter and modified to reduce native binding to host cell receptors is well tolerated and effective in vivo.
  • EXAMPLE 4 This example demonstrates the tolerability of the inventive method in vivo.
  • Adenoviral serotype 5 El/E3/E4-deficient adenoviral vectors containing, in place of the deleted El region, a nucleic acid sequence encoding a human TNF- ⁇ sequence operably linked to either a human CMV/EGR-1 chimeric promoter or an E2F-1 promoter were generated.
  • the AB loop of the adenoviral fiber protein was modified to disrupt CAR binding (AdL.F*).
  • AdL.F*PB* the integrin-binding domain of the adenoviral penton base protein was disrupted.
  • a ligand which binds ⁇ v ⁇ 3 and ⁇ v ⁇ 5 integrins to mediate viral transduction was inserted into the HI loop of the adenoviral fiber protein of AdL.F*PB* to create AdCMV/EGR-1 TOF **RGD and AdE2F ⁇ NF **RGD using methods described herein (see, e.g., Einfeld et al, supra).
  • Ad T NF CMV/EGR-1 **RGD and Ad ⁇ NF E2F**RGD were administered via intraperitoneal (i.p.) injection to Balb/c mice at a dose of 6xl0 10 , 1.2xl0 ⁇ , or 2.4xlO ⁇ particle units (pu). Mice were followed for 10 days post treatment. Body weight and survival were used as indicators of tolerance of vector administration. Three out of the five mice receiving the Ad T N F CMV/EGR-1 **RGD vector died following vector administration, and those mice that did survive exhibited a decrease in body weight as a result of vector administration.
  • mice receiving the Ad ⁇ NF E2F**RGD vector died as a result of vector administration, and body weights remained stable (see Figures 3a and 3b).
  • cisplatin (1 mg/kg body weight).
  • Cisplatin was administered at 1 day, 4 days, and 7 days after adenoviral vector administration. Mice were followed for 10 days post treatment. Body weight and survival were used as indicators of tolerance of the combined treatment.
  • mice treated with the Ad T NF CMV/EGR-1 **RGD vector died following the combined treatment, and the mice that did survive exhibited a decrease in body weight as a result of treatment. None of the mice receiving the Ad ⁇ NF E2F**RGD died as a result of the administration of the adenoviral vector and cisplatin, and body weights remained stable (see Figure 4).
  • EXAMPLE 5 This example demonstrates the ability of the inventive method to efficiently deliver adenoviral vectors comprising a transgene to tumor tissue in vivo.
  • Cisplatin (1 mg/kg body weight) was administered to mice i.p. at 1 day, 4 days, and 7 days after adenoviral vector administration. Mice treated with cisplatin but not subjected to adenoviral vector administration served as an additional control.
  • mice treated with Ad ⁇ NF E2F**RGD exhibited a higher therapeutic index, as measured by percent survival at 75 days post treatment and percent body weight loss, as compared to mice treated with Ad T NF CMV/EGR-1**RGD (see Figures 6a and 6b).
  • This example establishes that the inventive method results in delivery of an adenoviral vector modified to reduce native binding to host cell receptors to tumor tissue in the peritoneal cavity, and that the therapeutic efficacy of the adenoviral vector is enhanced through the use of a tissue-specific promoter operatively linked to an exogenous nucleic acid sequence of interest.
  • This example demonstrates that intraperitoneal administration of an adenoviral vector encoding TNF operably linked to a tumor cell-selective promoter and modified to reduce native binding to host cell receptors is well tolerated and effective in vivo.
  • EXAMPLE 6 [0106] This example demonstrates the tolerability of the inventive method in a lung cancer model.
  • Adenoviral serotype 5 El/E3 E4-deficient adenoviral vectors containing, in place of the deleted El region, a nucleic acid sequence encoding a human TNF- ⁇ sequence operably linked to an EGR-1 promoter, an RSV promoter, or a DF3 promoter were generated.
  • the AB loop of the adenoviral fiber protein was modified to disrupt CAR binding, and the integrin- binding domain of the adenoviral penton base protein also was disrupted (AdL.F*PB*).
  • the RGD ligand was inserted into the HI loop of the adenoviral fiber protein of AdL.F*PB* to create AdEGR ⁇ NF **RGD, AdRSV TOF **RGD, and AdDF3 ⁇ NF **RGD using methods described herein (see, e.g., Einfeld et al., supra).
  • AdEGRTNF* *RGD AdEGRTNF* *RGD
  • AdRSVTNF* *RGD AdDF3 ⁇ NF* *RGD
  • AdDF3 ⁇ NF* *RGD was injected into the i.p. cavity of mice at 3, 6, and 10 days after implantation of RERF-LC-AI human lung squamous cell carcinoma cells (IxIO 7 cells/mouse).
  • the cell line RERF-LC-AI was originally purchased as GT3TKB (gastric cancer cell line) from RIKEN BioResource Center (Japan). According to RIKEN, the GT3TKB cell line is considered the RERF-LC- AI cell line with 99.5% confidence limits.
  • AdEGRTNF* *RGD, AdRSVTNF* *RGD, and AdDF3 ⁇ N F * *RGD were each administered at a dose per injection of 6xl0 10 pu, 1.2xlO ⁇ pu, and 1.2xlO ⁇ pu, respectively.
  • Saline injections served as a control.
  • IP lavage was performed on each mouse 14 days after tumor cell implantation, and mice were observed for changes in body weight and survival rates.
  • Body weights of mice from all treatment groups decreased shortly after vector administration, but stabilized by the end of the study period. The median survival time was 31 days for control mice. Median survival times were not determined for vector-treated animals, since approximately 80-100% of all treated mice survived more than 42 days.
  • This example demonstrates that intraperitoneal administration of an adenoviral vector encoding TNF operably linked to a tumor cell-selective promoter and modified to reduce native binding to host cell receptors is well tolerated in vivo.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Virology (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne une méthode permettant de détruire les cellules tumorales dans la cavité péritonéale d'un mammifère. Ce procédé consiste à administrer au mammifère une dose d'un vecteur adénoviral à réplication déficiente comprenant (a) une séquence d'acide nucléique exogène codant pour un TNf-α humain, lié de manière fonctionnelle à un promoteur sélectif dirigé sur les cellules tumorales, (b) une protéine fibreuse présentant une disruption du site de liaison natif du récepteur CAR, et (c) une protéine base du penton présentant une disruption du site natif de liaison de l'intégrine.
PCT/US2005/019630 2004-06-04 2005-06-03 Methode permettant une utilisation plus sure et plus efficace de vecteurs adenoviraux pour le traitement des cancers WO2005117993A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US57711704P 2004-06-04 2004-06-04
US60/577,117 2004-06-04

Publications (2)

Publication Number Publication Date
WO2005117993A2 true WO2005117993A2 (fr) 2005-12-15
WO2005117993A3 WO2005117993A3 (fr) 2006-06-15

Family

ID=35463367

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2005/019630 WO2005117993A2 (fr) 2004-06-04 2005-06-03 Methode permettant une utilisation plus sure et plus efficace de vecteurs adenoviraux pour le traitement des cancers

Country Status (1)

Country Link
WO (1) WO2005117993A2 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10420820B2 (en) * 2014-09-29 2019-09-24 Counterpoint Biomedia LLC Targeting of pharmaceutical agents to pathologic areas using bifunctional fusion polypeptides
US11077156B2 (en) 2013-03-14 2021-08-03 Salk Institute For Biological Studies Oncolytic adenovirus compositions
US11130968B2 (en) 2016-02-23 2021-09-28 Salk Institute For Biological Studies High throughput assay for measuring adenovirus replication kinetics
US11401529B2 (en) 2016-02-23 2022-08-02 Salk Institute For Biological Studies Exogenous gene expression in recombinant adenovirus for minimal impact on viral kinetics
US11813337B2 (en) 2016-12-12 2023-11-14 Salk Institute For Biological Studies Tumor-targeting synthetic adenoviruses and uses thereof
US12365878B2 (en) 2018-04-09 2025-07-22 Salk Institute For Biological Studies Oncolytic adenovirus with enhanced replication properties comprising modifications in E1A, E3, and E4

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995030007A1 (fr) * 1994-05-02 1995-11-09 University Of Washington Mutants de la thymidine kinase
US20040167088A1 (en) * 2003-02-25 2004-08-26 Genvec, Inc. Method of using adenoviral vectors with increased persistence in vivo

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11077156B2 (en) 2013-03-14 2021-08-03 Salk Institute For Biological Studies Oncolytic adenovirus compositions
US10420820B2 (en) * 2014-09-29 2019-09-24 Counterpoint Biomedia LLC Targeting of pharmaceutical agents to pathologic areas using bifunctional fusion polypeptides
US11273206B2 (en) 2014-09-29 2022-03-15 Counterpoint Biomedica Llc Targeting of pharmaceutical agents to pathologic areas using bifunctional fusion polypeptides
US11130968B2 (en) 2016-02-23 2021-09-28 Salk Institute For Biological Studies High throughput assay for measuring adenovirus replication kinetics
US11401529B2 (en) 2016-02-23 2022-08-02 Salk Institute For Biological Studies Exogenous gene expression in recombinant adenovirus for minimal impact on viral kinetics
US12281324B2 (en) 2016-02-23 2025-04-22 Salk Institute For Biological Studies Exogenous gene expression in recombinant adenovirus for minimal impact on viral kinetics
US11813337B2 (en) 2016-12-12 2023-11-14 Salk Institute For Biological Studies Tumor-targeting synthetic adenoviruses and uses thereof
US12365878B2 (en) 2018-04-09 2025-07-22 Salk Institute For Biological Studies Oncolytic adenovirus with enhanced replication properties comprising modifications in E1A, E3, and E4

Also Published As

Publication number Publication date
WO2005117993A3 (fr) 2006-06-15

Similar Documents

Publication Publication Date Title
AU2019216631B2 (en) Enhanced adoptive cell therapy
US6403370B1 (en) Oncolytic/immunogenic complementary-adenoviral vector system
KR102628234B1 (ko) 알부민-결합 모이어티를 포함하는 아데노바이러스
Wu et al. Cancer gene therapy by adenovirus-mediated gene transfer
US6472176B2 (en) Polynucleotide encoding chimeric protein and related vector, cell, and method of expression thereof
WO1998035028A9 (fr) Systeme de vecteurs adenoviraux complementaires oncolytiques/immunogenes
EP1455840B1 (fr) Traitement du sarcome humain avec vector adenoviral tnf-alpha
JP2012139220A (ja) 複製欠損アデノウイルスｔｎｆベクター
US20210069253A1 (en) Recombinant adenoviruses and stem cells comprising same
US20060140909A1 (en) Method of using adenoviral vectors with increased persistence in vivo
EP1196616B1 (fr) Vecteurs anti-cancereux de replication
AU699867B2 (en) Recombinant adenoviruses for gene therapy in cancers
AU2001271549A1 (en) Replication deficient adenoviral TNF vector
Nakamura et al. Effective gene transfer to human melanomas via integrin-targeted adenoviral vectors
SK4412001A3 (en) Selectively replicating viral vectors
AU2004260044A1 (en) Subgroup B adenoviral vectors for treating disease
WO2005117993A2 (fr) Methode permettant une utilisation plus sure et plus efficace de vecteurs adenoviraux pour le traitement des cancers
KR20080090486A (ko) 신규한 아데노바이러스를 이용한 암 치료 방법 및 조성물
Särkioja Adenoviral gene therapy for non-small cell lung cancer
AU2002342263A1 (en) Treatment of human cancer with adenoviral vector expressing TNF-alpha

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

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

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM 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
NENP Non-entry into the national phase

Ref country code: DE

WWW Wipo information: withdrawn in national office

Country of ref document: DE

122 Ep: pct application non-entry in european phase