JP2002530085A - Viral vectors with late transgene expression - Google Patents

Abstract

  (57) [Summary] The present invention relates to a recombinant virus comprising a therapeutic transgene operably linked to late regulatory elements. The vectors of the present invention are capable of replicating and lysing neoplastic cells. The vector may optionally include modifications to the genome to provide additional therapeutic, conditional replication or functional targeting. The invention also provides pharmaceutical formulations of such vectors. The invention further provides methods for using such vectors. The present invention further provides a method for preparing this vector.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION The genome of adenovirus has been well characterized. This information is used for exogenous DN
It has been used to design a recombinant adenovirus that can act as a vector for introducing A into target cells. Many of these viral vectors contain modifications to the early gene products to confer specific activities on the vector. For example, the early genes E1 and E2 are transcribed early after infection. The products of these genes are responsible for suppressing the ability of infected cells to respond to infection and promote viral replication in the host.

In particular, the immediate-early gene E1a is transcribed rapidly after infection. E1a products can immortalize primary cells in vivo. E1a has also been shown to bind to the cellular protein p105RB, the product of the retinoblastoma gene. The E1b gene cannot transform cells by itself, but cooperates with E1a to stably transform cells. Both E1a and E1b are required for complete transformation and tumor formation in animals. The E1a gene has various functions. The full spectrum of activity of the adenovirus E1a region is described in Bayley, S .; And M
ymryk, J.M. (1994) Intl. J. of Oncology 5: 4
25-444.

[0003] The E1b gene is known to interact with host cell proteins. In particular, two proteins, p19 and p55, are differential in the E1b sequence (differ
ential) produced by splicing. The p55 protein is p5
It is well characterized as interacting with three gene products. p53 is a well-characterized protein and has been shown to activate the apoptotic pathway of programmed cell death (PCD) when present at sufficient intracellular concentrations. Apoptosis induced by p53 has been shown to occur in response to a wide variety of cytotoxics, including irradiation, DNA damaging agents, and the like. By binding to p53, p55 prevents the formation of active p53 phosphorylated tetramers. p5
Since 3 is sequestered by p55, the primary apoptotic pathway is not initiated, and infected cells undergo uncontrolled replication in response to E1a 12S and 13S proteins in combination with RB activity.

[0004] E1a and E1b cooperate in their activity. In particular, E1a has been shown to induce p53-dependent and p53-independent apoptosis. In addition to the induction of other early viral genes, E1a advances resting cells to the S phase required for viral replication. This initial pressure on the resting cells to enter S phase by the virus is thought to induce an apoptotic response from the cells. Adenovirus binds to p53 and delays induction of apoptosis.
It responds by expressing the K protein, so that the virus can replicate before the death of its host. Adenoviruses also produce E1B 19K proteins that also protect or delay E1a-mediated apoptosis.

[0005] An alternative to this type of selectively replicating vector is the use of replication deficient adenovirus vectors, which involve extensive removal of E1 function. In particular, vectors containing E1, E3 removal and partial E4 deletion have been used for delivery of exogenous transgenes.
Such vectors have been used to deliver the p53 gene to target cells. It has been shown that exogenously administered wild-type p53 expression in p53-deficient (p53 mutated or p53-eliminated) tumor cells can induce p53-mediated apoptosis in tumor cells. Such viral vectors for p53 delivery are currently under development at Schering Corporation. Also, these vectors have demonstrated acceptable toxicity profiles and therapeutic effects for human therapeutic applications, and are in Fuse II clinical trials in humans for the treatment of p53-related malignancies.

[0006] Replication-defective and selectively replicating vectors have, at least in theory, design drawbacks that are of concern to clinicians. Replication-defective vectors have a more theoretically interesting safety profile because they do not grow uncontrollably in patients. However, effective tumor removal requires that substantially the majority of the tumor cells to be infected become infected, so a substantially molar excess of vector is usually used to ensure therapeutic efficacy. It is. Selectively replicating vectors appear to be more problematic from a safety point of view because of their ability to replicate and potentially mutate in a patient to form a fully replicating competent vector. However, exploiting the natural ability of the virus to grow under certain conditions allows these vectors to spread to surrounding tumor cells. Since these vectors themselves can replicate,
Lower initial doses of such vectors are required. This is preferred for immunological reasons and for economic reasons in the production of such medicaments.

To facilitate an understanding of the present invention, a brief overview of the life cycle of an adenovirus, a typical virus used for delivery of exogenous transgenes, is provided. The replication cycle of adenovirus in human cells can be divided into early and late, delimited by the onset of viral DNA replication. The initial phase begins when viral particles attach to cells via interactions between the virion fiber domain and cell surface receptors. Virions migrate into cells by either endocytosis or direct penetration of the cytoplasmic membrane, and are transported to the nucleus where most capsids are released. In the nucleus, the virion core protein is removed, resulting in a viral chromosome that lacks almost all of the virion protein. Expression of the viral genome is temporarily coordinated, and approximately one hour after infection.
The time starts from the E1a region. Other early genes E1b, E2, E3, and E4
Is first expressed 1.5-2 hours after infection, shortly after E1a. Many protein products encoded by early genes are required for viral DNA replication, while others prepare the DNA synthesis machinery of infected cells for efficient viral DNA replication. Several early virally encoded proteins have been implicated in protecting infected cells from immune surveillance.

[0008] The late stage of infection begins about 7 hours after infection, with the onset of DNA replication. In the native adenovirus, the messenger RNA of all late gene products is spliced from primary RNA transcribed from the major late promoter (MLP). The MLP is located at position 16.5 on the r-chain. The major late promoter is active to a limited extent early in infection, but transcription does not progress beyond map position 39. During the late stages of the viral life cycle, MLP is fully activated and continues to map location 99. Each of the late primary RNA transcripts has five different mRNs
A, processed into one of L1 to L5. All of these mRNAs contain a common tripartite, 203 nucleotide leader sequence. Late mRNA encodes proteins necessary for capsid components and virion assembly and viral chromosome packaging. Virus DN
A replication requires terminal proteins for initiation and proceeds by a semi-conservative mechanism. With the onset of replication, efficient transcription of the late gene family from the major late promoter begins, and reaches a maximum at about 18 hours post-infection.
During the late phase, viral proteins block cellular DNA and protein synthesis, so that perhaps maximum viral macromolecular synthesis can occur. Intermediate gene expression, which actually begins during the early days, peaks between 8 and 12 hours after infection. Virion assembly and viral genome packaging begin approximately 24 hours after infection. Infected cells are destroyed due to attrition and lysis, and about 10,000 per cell
Produces virions.

SUMMARY OF THE INVENTION The present invention provides a conditionally replicating recombinant virus comprising a therapeutic transgene under the control of late regulatory elements. The present invention further provides pharmaceutical formulations and methods of using the same. The present invention further provides a recombinant producer cell that can complement a packaging gene that is missing or deleted in a vector. The invention also provides methods for making such vectors and formulations.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a replication competent recombinant virus comprising a therapeutic transgene operably linked to late regulatory elements.

I. Replication Competent Recombinant Virus: The term “replication competent” refers to a virus capable of replicating its genome in mammalian cells and packaging the replicated viral genome into infectious particles. Made for It should be noted that the term replication competent generally does not apply to viruses that can only grow in cells that have been modified to provide the function of the deleted virus in trans.

The term “recombinant virus” refers to any obligate intracellular bacterium whose genome has been modified by conventional recombinant DNA technology, is capable of infecting mammalian cells, has no protein synthesis function or energy production machinery. Point to. The viral genome can be RNA or DNA contained in the coating structure of proteins and lipid membranes. The terms virus and viral vector are used interchangeably herein. Viruses useful in the practice of the present invention include recombinantly enveloped or non-enveloped DNA and RNA viruses, preferably Baculoviridae, Parvoviridae, Picornoviridae, Herpesvirus Family, Poxviridae, Adenoviridae, or Picornaviridae. Chimeric virus vectors that take advantage of the advantageous elements of the properties of each parent vector (eg, Feng et al. (1997) Nature Biotechnol).
15: 866-870) may also be useful in the practice of the present invention. Minimal vector systems in which the viral backbone contains only the sequences necessary for the packaging of the viral vector, and optionally a transgene expression cassette, can also be produced in accordance with the practice of the present invention. While it is preferred to employ viruses from commonly treated species, in some cases it may be advantageous to use vectors from different species that have favorable pathogenic characteristics.
For example, an equine herpesvirus vector for human gene therapy is described in WO 98/27216 published August 5, 1998. The vector is described as being useful for human therapy because equine virus is not pathogenic to humans. Similarly, sheep adenovirus vectors can be used for human gene therapy because they are said to evade antibodies to human adenovirus vectors. Such a vector is described in WO97, published April 10, 1997.
/ 06826.

[0013] In a preferred practice of the invention, the virus is an adenovirus. The term "adenovirus" is synonymous with the term "adenovirus vector" and refers to viruses of the adenoviridae family. The term adenoviridae
Collectively refers to animal adenoviruses of the genus Mastadenovirus, including, but not limited to, humans, cows, sheep, horses, dogs, pigs, mice, and simian adenovirus subgenus. In particular, human adenoviruses include the AF subgenus and its individual serotypes, and human adenoviruses 1, 2, 3, 4, 4a, 5, 6, 7, 8, 9, 10, 1
1 (Ad11A and Ad11P), 12, 13, 14, 15, 16, 17, 1
8, 19, 19a, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 34a, 35, 35p, 36, 37, 3
8, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and 9
Including, but not limited to, Type 1. The term bovine adenovirus includes, but is not limited to, bovine adenovirus types 1, 2, 3, 4, 7, and 10. The term canine adenovirus refers to dog 1 (strain CLL, Glaxo, RI261, Utre
ct, Toronto 26-61) and type 2. The term equine adenovirus includes, but is not limited to, horses 1 and 2. The term porcine adenovirus includes but is not limited to porcine types 3 and 4. The term recombinant adenovirus also includes chimeric vectors (or even multimeric vectors), ie, vectors constructed with complementary coding sequences from more than one virus subtype. See, for example, Feng et al., Nature Bio.
See technology 15: 866-870.

In a preferred practice of the invention, the recombinant adenovirus vector comprises an adenovirus
Obtained from the family Irus. Particularly preferred viruses are human adenovirus type 2 or
Is obtained from type 5. In the preferred practice of the invention as exemplified herein
Thus, preferred vectors are from the human adenoviridae family. More preferred is hi
This is a vector derived from the adenovirus subgroup C. Most preferred is human
Adenovirus vectors from adenovirus serotypes 2 and 5. The present invention
In a most preferred implementation of the invention, the virus is human adenovirus type 5dl309, dl 327,dl520, or wild-type adenovirus.

II. Late Regulatory Elements The term “late regulatory element” refers to a regulatory element that drives transcription of a therapeutic transgene at a later time than the element that induces initial viral replication.
Characteristically, these promoter elements are found to drive expression of packaging and other proteins later in the viral life cycle. An example of such a late regulatory element when the parent vector is an adenovirus is the adenovirus major late promoter (MLP). Other viral vector systems also have a late temporally regulated promoter. In baculovirus vectors, the AcNPV basic gene promoter and the polyhedrin gene promoter can be used (Sridhar et al. (1993) FEBS Lett.
. 315: 282-286). In herpes simplex virus, a latent activating promoter may be used. See, for example, Rivera-Gonzalez et al. (1994) Vi.
rology 202: 550-564 and Imbal et al. V
irol. 66: 5453-5463. For human papillomavirus, the HPV31b promoter may be used (Ozbun and Meyers (
1998) J. Amer. Virol. 72: 2715-22). In parvovirus, P
39 promoters. In vaccinia virus, it is the p11 promoter. In a preferred practice of the invention, the virus is obtained from the adenoviridae and the late regulatory element is the adenovirus major late promoter. The major late promoter is well characterized in the art and is at approximately map position 16.5 of the adenovirus type 2 genome.

III. Operably linked: The term “operably linked” refers to the joining of polynucleotide elements in a functional relationship. When in a functional relationship with another nucleic acid sequence, the nucleic acid sequence
"Operably linked". For example, a promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the coding sequence. Operably linked means that the nucleotide sequences being linked are typically contiguous. However, enhancers generally function when they are a few kilobases away from the promoter, and because the intron sequences can be of various lengths, some polynucleotide elements may be operably linked but not directly adjacent. And can function even in trans from different alleles or chromosomes.

IV. Therapeutic Transgene: The term “therapeutic transgene” refers to a nucleotide sequence whose expression in a target cell causes a therapeutic effect. The term therapeutic transgene includes but is not limited to a tumor suppressor gene, an antigenic gene, a cytotoxic gene, a cytostatic gene, a prodrug activating gene, an apoptotic gene, a pharmaceutical gene or an anti-angiogenic gene . The vectors of the present invention may be used to produce one or more therapeutic transgenes in tandem through the use of an IRES element or through an independently regulated promoter.

A. Tumor Suppressor Gene The term “tumor suppressor gene” refers to a nucleotide sequence whose expression in a target cell can suppress a neoplastic phenotype and / or induce apoptosis. Examples of tumor suppressor genes useful in the practice of the present invention include the p53 gene, APC gene, DPC-4 gene, BRCA-1 gene, BRCA-2 gene, WT-1 gene, retinoblastoma gene (Lee et al. (1987) ) Nature
329: 642), MMAC-1 gene, adenomatous polyposis coli protein (adenomatous polyposis coli protein).
n) (Albertsen et al., US Pat.
No. 783,666), a deleted gene in colon carcinoma (the deleted).
in colon carcinoma (DCC), MMSC-2 gene,
NF-1 gene, chromosome 3p21.3. Nasopharyngeal carcinoma tumor suppressor gene (Cheng et al., 1998, Proc. Nat. Acad. Sci. 95:
3042-3047), MTS1 gene, CDK4 gene, NF-1 gene, N
Includes the F2 gene and the VHL gene.

B. Antigenic Gene The term “antigenic gene” refers to a nucleotide sequence whose expression in a target cell causes the production of a cell surface antigenic protein that is recognizable by the immune system. Examples of antigenic genes include carcinoembryonic antigen (CEA), p53 (Levine, A. et al.
, PCT International Publication No. WO 94/02167, published February 3, 1994). Antigenic genes can be fused to MHC class I antigens to promote immune recognition.

C. Cytotoxic Gene The term "cytotoxic gene" refers to a nucleotide sequence whose expression in a cell causes a toxic effect. Examples of such cytotoxic genes include nucleotide sequences encoding Pseudomonas exotoxin, ricin toxin, diphtheria toxin, and the like.

D. Cytostatic Gene The term “cytostatic gene” refers to a nucleotide sequence whose expression in a cell causes cell cycle arrest. Examples of such cytostatic genes are p21, retinoblastoma gene, E2F-Rb gene, P16, p15, p1
Genes encoding cyclin-dependent kinase inhibitors such as p8 and p19 have been described by Brunellec et al. (PCT Publication No. W, published May 9, 1997).
Growth arrest-specific homeobox (such as described in O97 / 16459, and PCT Publication No. WO 96/30385 published October 3, 1996).
GAX) gene.

E. Cytokine Gene The term “cytokine gene” refers to a nucleotide sequence whose expression in a cell produces a cytokine. An example of such a cytokine is GM-CS
F, interleukins, especially IL-1, IL-2, IL-4, IL-12, IL
-10, IL-19, IL-20, alpha, beta, and gamma subtypes of interferons, particularly interferon α-2b and interferon α
And fusions such as -2α-1.

F. Chemokine Gene The term “chemokine gene” refers to a nucleotide sequence whose expression in a cell produces a chemokine. The term chemokine refers to a group of structurally related low molecular weight cytokine factors secreted by cells that have mitogenic, chemotactic, or inflammatory activity. They are predominantly cationic proteins of 70 to 100 amino acid residues and share four conserved cysteine residues. These proteins can be divided into two groups based on the spacing of the two amino-terminal cysteines. In the first group, the two cysteines are separated by one residue (CxC), while in the second group they are adjacent (CC) "CxC" chemokines. Examples of members of are platelet factor 4 (PF4), platelet basic protein (PBP), interleukin-8
(IL-8), melanoma growth stimulating protein (MGSA), macrophage inflammatory protein 2 (MIP-2), mouse Mig (m119), chicken 9E3 (
Or pCEF-4), porcine alveolar macrophage chemotactic factors I and II (AM
CF-I and -II), pro-B cell growth stimulating factor (PBSF), and IP10
Including, but not limited to. Examples of members of the “CC” group include monocyte chemoattractant protein 1 (MCP-1), monocyte chemoattractant protein 2 (MCP-2), monocyte chemoattractant protein 3 (MCP-3), monocyte Chemotactic protein 4 (MCP-4), macrophage inflammatory protein 1α (MIP-1-α), macrophage inflammatory protein 1β
(MIP-1-β), macrophage inflammatory protein 1γ (MIP-1-γ),
Macrophage inflammatory protein 3-α (MIP-3-α), macrophage inflammatory protein 3β (MIP-3-β), chemokine (ELC), macrophage inflammatory protein 4 (MIP-4), macrophage inflammatory protein 5 (MIP-5
), LD78β, RANTES, SIS-ε (p500), thymus and activation regulating chemokines (TARC), eotaxin, I-309, human protein HC
Including but not limited to C-1 / NCC-2, human protein HCC-3, and mouse protein C10.

G. Pharmaceutical Protein Gene The term “pharmaceutical protein gene” refers to a nucleotide sequence whose expression causes the production of a protein having a pharmacological effect in a target cell. Examples of such pharmaceutical genes include proinsulin genes and analogs (described in PCT International Publication No. WO 98/31397), growth hormone genes, dopamine, serotonin, epidermal growth factor, GABA, ACTH, NGF, VEGF (target Increasing blood perfusion into tissues, inducing angiogenesis, PCT Publication No. WO 98/32859 published July 30, 1998), including thrombospondin, etc. (H. Pro-apoptotic gene :) The term "refers to a nucleotide sequence whose expression causes programmed cell death of a cell. An example of a pro-apoptotic gene is p5
3, adenovirus E3-11.6K, adenovirus E4orf4 gene, p
53 pathway genes, and genes encoding caspases. The p16 gene is also apoptotic in Rb positive, p16 negative, p53 wild type tumors. (Frizzle et al. (1998) Oncogene 16: 3087-9.
5 and Sandig et al. (1997) Nature Medicine 3: 3.
13). I. Prodrug Activating Genes: The term "prodrug activating gene" refers to a therapy whose expression renders a non-therapeutic compound susceptible to killing of cells by external factors, or causing a toxic state to the cells. Refers to a nucleotide sequence that causes the production of a protein that can be converted to a target compound. One example of a prodrug activation gene is the cytosine deaminase gene. Cytosine deaminase converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), a potent antitumor agent. Lysis of the tumor cells provides localized release of cytosine deaminase that can convert 5FC to 5FU at the localized point of the tumor, causing killing of many surrounding tumor cells. This causes killing of many tumor cells without having to infect these cells with adenovirus (the so-called "bystander effect"). In addition, the thymidine kinase (TK) gene (see, eg, Woo et al., US Pat. No. 5,631,236, issued May 20, 1997 and Freeman et al., February 11, 1997).
(See U.S. Patent No. 5,601,818, issued on Jan. 5, 2009), wherein cells expressing the TK gene product are susceptible to selective killing by administration of ganciclovir.

J. Anti-angiogenic Gene: The term “anti-angiogenic” gene refers to a nucleotide sequence whose expression causes extracellular secretion of anti-angiogenic factors. Anti-angiogenic factors include vascular endothelial cell growth factor (V) such as angiostatin and Tie2.
Inhibitor of EGF) (PNAS (USA) (1998) 95: 8795-8)
800), and endostatin.

K. Modifications of Therapeutic Genes: Modifications and / or deletions to the above-referenced genes to encode functional subfragments of a wild-type protein may be used in the practice of the present invention. It will be readily apparent to one skilled in the art that it can be easily adapted. The term "modification" refers to a change in the genomic structure of a recombinant adenovirus vector. Such modifications include deletions and / or changes in the amino acid coding sequence to produce a protein that lacks binding to its substrate.

For example, reference to the p53 gene includes not only wild-type proteins but also modified p53 proteins, allelic variants thereof, or proteins from other mammalian species. Wild type p53 sequences are well known in the art. Mouse p53, pig p5
3, Other mammalian p53 molecules, such as horse p53, bovine p53, dog p53, etc., are known in the art and can be incorporated into the practice of the invention. "Modified p
The term "53 protein" refers to a modification to the primary, secondary, tertiary, or quaternary structure of a p53 protein that retains the function of the p53 protein. Examples of such modified p53 proteins include modifications to p53 to increase nuclear retention, deletions such as the Δ13-19 amino acid to remove the calpain consensus cleavage site, modifications to the oligomerization domain (Bracco et al. , PCT Published Application No. WO
97/0492 or as described in US Patent No. 5,573,925). Alternatively, the p53 sequence can be modified to replace the endogenous tetramerization domain with a leucine zipper oligomerization domain.

The therapeutic gene may be a signal peptide or a nuclear localization signal (NLS)
It will be readily apparent to those skilled in the art that by including a targeting molecule such as can be secreted into the medium or localized to a specific subcellular location. Therapeutic transgene and VP, a herpes simplex virus type 1 (HSV-1) structural protein
Fusion proteins with 22 are also included in the definition of therapeutic gene. When synthesized in infected cells, the fusion protein containing the VP22 signal is transported out of the infected cells and efficiently enters surrounding non-infected cells, ranging from about 16 cells in diameter. This system is particularly useful in connection with transcriptionally active proteins (eg, p53) because the fusion protein is efficiently transported to the nucleus of the surrounding cells. For example, Elliot
t, G. And O'Hare, P .; Cell. 88: 223-233: 1997
Marshall, A .; And Castellino, A .; Research
News Briefs. Nature Biotechnology. Fifteen
: 205: 1997; O'Hare et al., PC published February 13, 1997.
See T Publication No. WO 97/05265. A similar targeting moiety from the HIV Tat protein has also been described by Vives et al. (1997) J. Am. Biol. Chem. 2
72: 16010-16017.

V. Further Modifications to Viruses: The present invention also includes targeting modifications, modifications to allow the vector to replicate selectively in a particular cell type or phenotypic state, controlled expression characteristics, suicide. Provided is a recombinant adenovirus comprising further modifications to the viral genome, such as further modifications to enhance gene or cytotoxicity. However, this does not imply that other modifications to the viral genome may not be included, or that multiple such modifications may not be used.

A. Selective Replication: The term “selectively replicate” refers to the replication of one phenotypic state relative to another phenotypic state in one cell type relative to another cell type. A recombinant viral vector that can replicate preferentially in a cell or in a given cell type in response to an external stimulus. Selective replication is achieved through the use of replication control elements. The term "replication regulatory element" is used in one cell type relative to another cell type.
In cells in one phenotypic state compared to another phenotypic state (cell state specific)
Or a DN inserted into the viral genome to produce a recombinant viral vector that replicates selectively in a given cell type (inducible) in response to external stimuli.
Refers to modifications to the A sequence or the viral genome. Examples of such replication control elements include cell type-specific promoters, cell state-specific promoters, and inducible promoters.

1. Cell Type Specific Promoter: Cell type specific replication is when the virus is selected from the adenovirus genome.
This can be achieved by linking a cell type specific promoter to an early viral gene such as the El, Ela, E2 or E4 gene. The term "cell type-specific promoter" refers to a promoter that is differentially activated as a result of cell cycle progression or in different cell types. Examples of a cell type-specific promoter include a cell cycle regulatory gene promoter, a tissue-specific promoter or a pathway responsive promoter. In a preferred practice of the invention, the cell type specific promoter is linked to the E4 gene instead of the E1 gene. This is NF-
Factors such as IL6 can replace E1a in regulating E1a-responsive promoters in adenoviruses that lack E1a function (Sperge
(1992) J. L. et al. Virol. 66: 1021-1030).

(A. Cell Cycle Regulatory Gene Promoter :) The term “cell cycle regulatory gene promoter” describes a promoter of a gene that is substantially activated when entering the S phase. Examples of such promoters include promoters regulated by E2F (eg, DHFR, DNA
Polymerase α, thymidylate synthase, c-myc and b-myb promoters).

(B. Tissue Specific Promoters) Tissue specific promoters are well known in the art and are preferentially active in smooth muscle (α-actin promoter), pancreas specific (Palmit
(1987) Cell 50: 435), liver-specific (Rovet et al. (1)
992) J. Biol. Chem. 267: 20765; Lemaigne et al. (
1993). Biol. Chem. 268: 19896; Nitsch et al. (1
993) Mol. Cell. Biol. 13: 4494), stomach-specific (Kova
Rik et al. (1993) J. Am. Biol. Chem. 268: 9917), pituitary-specific (Rhodes et al. (1993) Genes Dev. 7: 913), prostate-specific (U.S. Pat. No. 5,698,443, Henderson et al., Issued December 16, 1997). Includes promoter.

(C. Pathway-responsive promoter) The term “pathway-responsive promoter” refers to a DNA sequence that binds to a protein in normal cells and causes nearby genes to transcriptionally respond to the binding of the protein. . Such a promoter can be made by incorporating a responsive element, a sequence to which the transcription factor binds. Although such responses are generally inducible, there are some cases where increasing protein levels decrease transcription. Pathway-responsive promoters can be naturally occurring or synthetic. Pathway responsive promoters are typically constructed for a functional protein that is a pathway or target. For example, a naturally occurring p53 pathway responsive promoter contains transcriptional control elements that are activated by the presence of functional p53, such as the p21 promoter or the bax promoter. Alternatively, a synthetic promoter containing a p53 binding site upstream of a minimal promoter (eg, the SV40 TATA box region) can be used to create a synthetic pathway responsive promoter.
Synthetic pathway responsive promoters are generally constructed from one or more copies of a sequence that matches a consensus binding motif. Such a consensus DNA binding motif can be easily determined. Such consensus sequences are generally arranged as direct repeats or head-to-tail repeats separated by a few base pairs. Elements containing head-to-head repeats (eg, AGGTCATGACCT) are called palindromes or inverted repeats, and those with tail-to-tail repeats are called everted repeats.

(I. Examples of Pathway Promoters) Examples of pathway responsive promoters useful in the practice of the present invention include synthetic insulin pathway responsive promoters containing a consensus insulin binding sequence (Jacob et al. (1995) J. Biol. Chem. 270: 27773-27779), a cytokine pathway responsive promoter, a glucocorticoid pathway responsive promoter (Lange et al. (1992) J. Biol. Chem. 267: 15673).
80), IL1 and IL6 pathway responsive promoters (Won K.-A and Baumann H. (1990) Mol. Cell. Biol. 10: 396.
5-3978), T3 pathway responsive promoter, consensus motif: 5′A
Thyroid hormone pathway responsive promoter including GGTCA3 ′, TPA pathway responsive promoter (TRE), TGF-β pathway responsive promoter (Groten
Dorst et al. (1996) Cell Growth and Differen.
7: 469-480). In addition, natural or synthetic E2F pathway responsive promoters may be used. One example of an E2F pathway responsive promoter is described in Parr et al. (1997) Nature Med.
icene 3: 1145-1149. It is four E2F
An E2F-1 promoter containing a binding site has been described and has been reported to be active in tumor cells with fast cycling. Examples of other pathway responsive promoters are well known in the art and are available on the Internet at http: // www. eimb.
rssi. Database of T accessible via ru / TRRD
ranscription Regulatory Regions on E
ukaryotic Genomes.

(Ii. Preferred Pathway Promoters) In a preferred practice of the invention as exemplified herein, the vector comprises:
It includes a synthetic TGF-β pathway responsive promoter that is active in the presence of a functional TGF-β pathway, such as a promoter including an SRE pathway responsive promoter and a PAI pathway responsive promoter. PAI is plasminogen activator-I
Refers to a synthetic TGF-β pathway responsive promoter containing a sequence that is highly responsive to TGF-β, isolated from the promoter region. A PAI pathway responsive promoter can be isolated as a 749 base pair fragment that can be isolated from plasmid p800luc (Zonneveld et al. (1988) PNAS 85: 5525-).
5529, and GenB with accession number J03836.
ank). SRE refers to four Smad-4 DNA binding sequences (G
TCTAGAC, Zawel et al. (1988) Mol. Cell 1: 611-6
17) (synthetic TGF-β response element).
The SRE response element can be made by annealing a complementary oligonucleotide encoding the Smad-4 binding sequence and cloning into the plasmid pGL # 3-promoter luciferase vector (commercially available from ProMega).

(I. p53 Pathway Promoter :) Similarly, “p53 pathway responsive promoter” refers to a transcription control element that is active in the presence of a functional p53 pathway. The p53 pathway responsive promoter is p21
Alternatively, it may be a naturally-occurring transcription control region that is active in the presence of a functional p53 pathway, such as the mdm2 promoter. Alternatively, the p53 pathway responsive promoter can be a synthetic transcription control region that is active in the presence of a functional p53 pathway, such as an SRE pathway responsive promoter and a PAI-RE pathway responsive promoter. p53-CON refers to two synthetic p53s upstream of the SV40 TATA box.
Consensus DNA binding sequence (Funk et al. (1992) Mol. Cell. Bi.
ol. 12: 2866-2871), and describes a p53 pathway responsive promoter containing a synthetic p53 responsive element, constructed by inserting the same. RGC refers to a synthetic p53 pathway responsive promoter using tandem p53 binding domains identified in the ribosomal gene cluster. The p53CON and RGC response elements can be constructed by annealing complementary oligonucleotides, and the p53 responsive promoter can be constructed by cloning into a plasmid pGL3-promoter luciferase vector (commercially available from ProMega). .

D. Tissue-Specific Promoters Operably Linked to Early Genes Tissue-specific promoters are well known in the art and are selectively active in smooth muscle (α-actin promoter), pancreas Specific promoter (P
almiter et al. (1987) Cell 50: 435), liver-specific promoter (Rovet et al. (1992) J. Biol. Chem. 267: 20765).
Lemaigne et al. (1993) J .; Biol. Chem. 268: 1989
6; Nitsch et al. (1993) Mol. Cell. Biol. 13: 4494
), A stomach-specific promoter (Kovarik et al. (1993) J. Biol. Ch.).
em. 268: 9917), a pituitary-specific promoter (Rhodes et al. (19
93) Genes Dev. 7: 913), and a prostate-specific antigen promoter.

2. Cell State Specific: Examples of different phenotypic states include a neoplastic phenotype relative to a normal phenotype in a given cell type. Selective replication is achieved through the use of viral replication control elements. The term viral replication control element refers to a DNA sequence that has been genetically engineered into a vector of the invention so that the virus can preferentially replicate the viral genome in a particular type of target cell. Again, these cell state specific promoters can be linked to an early gene such as E1, E2, or preferably E4 to achieve selective replication in response to a particular phenotypic state.

(A. Tumor-specific :) To achieve adenovirus expression in tumor cells, a tumor-specific promoter that drives early gene expression may be employed. "Tumor-specific promoter"
The term refers to a promoter that is active in tumor cells and inactive in non-transformed cells. Examples of tumor-specific promoters include α-fetoprotein promoter, tyrosinase promoter. The use of tumor-specific promoters to achieve conditional replication of adenovirus vectors has been described in 1995
Co-pending US patent application Ser. No. 08 / 433,798 filed May 3, 1996, and International Patent Application No. PCT / US96 / 06199, published on Nov. 7, 1996 as International Publication No. WO 96/34969. No. All of these teachings are incorporated herein by reference.

For example, the α-fetoprotein promoter can be used to replace the endogenous E4 promoter and achieve higher selectivity in conditional replication. Other factors such as NF-IL6 may substitute for E1a in regulating E1a-responsive promoters in adenoviruses lacking E1a function (S
pergel et al. (1992) J. Mol. Virol. 66: 1021-1030). And this can be avoided by replacing the E4 promoter with a tumor-specific promoter.

(B. Repressors of Viral Replication :) Pathway-responsive promoters can be used to drive viral replication in the presence of a functional pathway, but, instead, repress viral expression to regulate expression. A pathway responsive promoter can be used to drive expression of the presser. The term "repressor of viral replication" refers to a protein that, when expressed in a given cell, essentially suppresses viral replication. As will be appreciated by those skilled in the art, the repressor of viral replication will depend on the nature of the parent adenoviral vector from which the recombinant vector of the present invention is derived. For example, in the case of an adenovirus vector or other DNA oncovirus, European Patent Application No. 9410 published December 6, 1995
E2F as described in 8445.1 (Publication No. 0 685 493 A1)
-Rb fusion constructs may be utilized. The E2F-Rb fusion protein is a human E-fused to the Rb growth suppression domain (amino acids 379-928 of the wild-type Rb protein).
It consists of the DNA binding domain of the 2F transcription factor protein and the DP1 heterodimerization domain (amino acids 95-286 of wild-type E2F). The E2F-Rb fusion protein is a strong repressor of E2F-dependent transcription and
Stop at The DNA binding domain is located at amino acids 128-193, and the dimerization domain is located at 194-289. The sequence of the human E2F-1 protein is shown in Genbank under accession number M96577, deposited on August 10, 1992.
k. E2F from other E2F family members, or sequences of E2F from other species, may be used when constructing vectors for use in other species. If the recombinant virus is based on an adeno-associated virus, rep
Proteins and their derivatives are effective repressors of virus replication in the absence of adenovirus infection. In situations in which the virus is derived from the herpes simplex virus, the ICPO-NX protein (Liun et al. (1998) J. Virol. 72: 7785-7795), which is a deletion of the very early protein ICPO, can be used for efficient replication of virus replication. Can be used as a presser. Similarly, any protein with dominant negative activity can be used as a repressor of viral replication.

These modifications may be combined with the cell cycle regulatory gene promoter described above.
For example, an E2F pathway responsive promoter can be used to drive expression of a modified E1a coding sequence. Since the E2F-Rb fusion protein represses both the E2 and E2F response elements, the use of a p53 pathway responsive promoter to drive the expression of the E2F-Rb fusion protein uses E1 function and E1
Achieve suppression of both functions. In p53 deficient tumor cells, the p53 response element is inactive and E2F-Rb is not expressed. Consequently, the presence of E2F in tumor cells enhances expression of E1a and the inability to repress the E2 promoter allows viral replication to proceed.

3. Inducible Promoter The term “inducible promoter” refers to a therapeutic agent that, under certain conditions and / or in response to external chemical or other stimuli (or alone), is therapeutic. Refers to a promoter that promotes transcription of a transgene. Examples of inducible promoters are known in the scientific literature (eg, Yoshida and Hamada (1997) Bio).
chem. Biophys. Res. Comm. 230: 426-430; Ii
(1996) J. D. et al. Virol. 70 (9): 6054-6059; Hwa
ng et al. (1997) J. Am. Virol. 71 (9): 7128-7131; Lee
(1997) Mol. Cell. Biol. 17 (9): 5097-5105
And Dreher et al. (1997) J .; Biol. Chem. 272 (46)
: 29364-29371). Examples of irradiation-inducible promoters are E
Induced by ionizing radiation, such as the gr-1 promoter (Mano
(1998) Human Gene Therapy 9: 1409-1.
7; Takahashi et al. (1997) Human Gene Therapy.
8: 827-833; Joki et al., Human Gene Therapy (
(1995) 6: 1507-1513; Bootman et al. (1994) 138, Supplement S68-S71; and Ohno, T (1995) Protein Nucleic Acid Enzyme 40.
: 2624-2630), X-ray inducible promoters such as the XRE promoter (Boothman et al. (1994) Radiation Research).
h 138 (Suppl. 1): described in S68-S71), and Clostr
UV-inducible promoters such as those isolated from ium perfringens (Garnier and Cole (1988) Mol. Microb
iol. 2: 607-614).

B. Alternative Modifications to the Viral Genome: 1. Deletion of E1Bdl55K: As indicated above, the E1b55K protein binds to p53. as a result,
To enhance the effects of p53 introduced by viral vectors, 1997
McCormick, US Pat. No. 5,677,1 issued Oct. 14, 2016.
It is preferred to introduce the description of the E1Bdl55K mutation described in No. 78 to remove p53 binding. The teachings of which are all incorporated herein by reference.

In a preferred practice of the invention as exemplified herein, the virus is
P53 under the control of the MLP promoter containing a deletion of nucleotides 2247-3272 of the adenovirus genome to remove the function of the E1b55K protein
Is a recombinant adenovirus vector encoding

(2. Modification of E4) Further, modifications that increase the efficacy of the vector of the present invention include, but are not limited to, changes at E1b. The vectors of the present invention may be used to increase cytotoxicity (Mu
ller et al. (1992) J. Mol. Virol. 66: 5867-5878), or may be modified to introduce mutations in E4 to include up-regulation of viral cytopathic proteins such as E4orf4 or E3 11.6K protein. For example, the E4 region of the adenovirus genome is involved in viral DNA replication, cessation of host protein synthesis, and viral assembly. E4o
rf6 is sufficient for DNA replication and late protein synthesis in immortalized cells. However, in non-dividing cells, E4orf6 / 7 appears to be required for replication. As a result, removal of E4orf6 / 7 resulted in immortalized cells (
(I.e., tumor cells) to help limit virus replication and can be incorporated into the vectors of the invention.

In addition, deletion of E4 has been shown to reduce the immunogenicity of the vector (Wan
g et al. (1997) Gene Therapy 4: 393-400;
u et al. (1997) J. Am. Virol. 71: 4626-37) is the retained E4
Can affect transgene expression persistence, depending on the open reading frame of the gene and the promoter used to drive transgene expression (Arm
entano et al. (1997) J. Am. Virol. 71: 2408-2416). E
The 4 region also encodes a protein (E4orf6) that can bind to p53 and inactivate its transcriptional activity (Dubner et al. (1996) Sc
issue 272: 1470-73). Thus, it may be desirable to modify the E4 region to delete an open reading frame with properties that are not desirable for a particular viral construct, while retaining an open reading frame with desirable properties. For example, in the conditional replication virus described herein as E1Bdl55K-MLP-p53, the E4orf6 region is deleted to reduce p53 inactivation, while retaining E4orf3 and maintaining p53 continuity. It may allow expression and replication of the virus. Either E4orf6 or E4orf3 should be retained to preserve the replication capacity of the virus.

Another example of an E4-deleted adenovirus vector is described in Gregory et al., US Pat. No. 5,670,488, issued Sep. 23, 1997.

3. Deletion of E1 to Achieve Selective Replication in Fast-Dividing Cells To achieve selective replication of adenovirus in fast-dividing cells, the adenovirus must have one or more p300s. To produce an E1a gene product that lacks binding to a protein family member and one or more Rb protein family member proteins but retains the transactivating function of the E1a CR3 domain, Modifications and deletions from about 219 to about 289 amino acids of the E1a 289R protein (or about amino acid 173 to about amino acid 243 of the E1a 243R protein) may be included. In a preferred practice of the invention, the deletion of binding to a p300 family member introduces a deletion corresponding to amino acids 4-25 of the E1a 243R and E1a 289R proteins or amino acids 38-60 of the E1a 243R and E1a 289R proteins. Is achieved by doing In a preferred practice of the invention, the loss of binding to a pRb family member comprises the E1
amino acids 111- of the a243R and E1a289R proteins
Achieve 123. Alternatively, loss of binding to a pRb family member is
Amino acids 124 of 1a 243R and E1a 289R proteins
-127 can be achieved.

4. Modification of E3: The E3 region of adenovirus encodes a protein that helps to avoid removal by the immune system of adenovirus-infected cells (Wold et al. (1995) Curr.
Top. Microbiol. Immunol. 199: 237-274). Up-regulation of this region and its subfragments has been shown to prevent or reduce the immune response to virus-infected cells and result in long-term gene expression (Ilan et al. (1997) PNAS 94: 2587-2592; Bu).
(1997) J. Nder et al. Virol. 71: 7623-28). Thus, modifications to the E3 region (or a subcomponent thereof) that overexpresses the protein (eg, by up-regulating the E3 region with a strong constitutive promoter such as CMV) may result in immune-mediated immunization of infected cells. Due to its ability to avoid or delay removal, it may be desirable to allow a greater degree of virus replication.

B. Targeting Modifications: The present invention provides recombinant viruses that include "targeting modifications" to achieve selective targeting of the virus to particular cell types. The term "targeting modification" refers to a modification to the viral genome designed to cause selective infectivity of a particular cell type. Cell type specificity or cell type targeting can also be achieved by modification of the viral envelope proteins in vectors derived from characteristically widely infectious viruses, such as adenoviruses. For example, cell targeting involves using an adenovirus vector to achieve expression of modified knob (knob) and fiber domains that have specific interactions with unique cell surface receptors, using the viral genome to express it. Achieved by selective modification of the knob and fiber coding sequences. Examples of such modifications are described in Wickham et al. (1997) J.
. Virol. 71 (11): 8221-8229 (incorporation of RGD peptide into adenovirus fiber protein); Arnberg et al. (1997) Vi.
rology 227: 239-244 (achieving tropism for the eye and genital tract,
Modification of the adenovirus fiber gene); Harris and Lemoine
(1996) TIG 12 (10): 400-405; Stevenson et al. (
1997) J. Amer. Virol. 71 (6): 4782-4790; Michael
(1995) Gene Therapy 2: 660-668 (incorporation of gastrin releasing peptide fragment into adenovirus fiber protein);
And Ohno et al. (1997) Nature Biotechnology 1
5: 763-767 (Incorporation of the Protein A-IgG Binding Domain into Sindbis Virus). Other methods of cell-specific targeting have been achieved by conjugation of antibodies or antibody fragments to envelope proteins (eg,
Michael et al. (1993) J. Mol. Biol. Chem. 268: 6866-6
869; Watkins et al. (1997) Gene Therapy 4: 100.
4-1012; Douglas et al. (1996) Nature Biotechn.
org. 14: 1574-1578). Alternatively, specific moieties can be attached to the virus surface to achieve targeting (eg, Nilson
(1996) Gene Therapy 3: 280-286 (EGF binding to retroviral proteins).

VI Experimental Results Construction of A Vector The E1B dl 55K-MLP-p53 (FAMA) vector was prepared substantially according to the teachings of Example 1 herein. Briefly, the adenovirus genome is modified by introducing a deletion of base pairs 2247-3272 of the adenovirus genome such that the ability of the 55K protein cannot bind to inhibit the transcriptional activity of p53. I made it. Instead of this deletion, the polyA signal from adenovirus protein IX for E1b19K is replaced by the major late promoter and tripartite of Ad2, which drives this deletion and expression of p53 from plasmid pA / M / 53. ) Leader sequence (Wills et al.,
(1994) as described in Human Gene Therapy 5: 1079-88). The remainder of the viral sequence is wild-type Ad5.

B In Vitro Experiments The following experiments were performed to demonstrate that the major late promoter functions in a transient manner in the expression of p53 from replicating viruses. Experiments were performed substantially in accordance with the teachings of Example 2 herein to assess the time course of p53 expression in various tumor cell lines. The data is shown in Figures 1-4 of the accompanying drawings. The data shown show the transient expression of the cFAMA vector in various tumor cell lines with different genotypes, as well as increased expression levels as compared to the replication deficient virus expressing p53 (ACN53).

In one embodiment, the expression of p53 from a FAMA vector was compared to a substantially similar vector in which p53 was operably linked to a CMV constitutive promoter. Experiments were performed in Example 2 herein. c in substantial accordance with the teachings and the results are shown in FIG. 4 of the accompanying drawings. This is because the FAMA virus
Demonstrates replicating in a transient manner as compared to CMV containing the C virus.

Embodiment 2 In further experiments, performed according to d, the replication of the virus from FAMA was compared to a substantially similar FAIC vector in which this temporary major late promoter was replaced by a constitutive CMV promoter. M in temporary style
By expressing the p53 protein from the LP promoter, higher replication of the virus is achieved compared to the constitutive promoter. The result is
This is shown in FIG. 5 of the accompanying drawings.

C. In Vivo Experiments To confirm the efficacy of the vectors of the invention in animals in vivo, the vectors of the invention were evaluated in a mouse human prostate cancer model. The models and experiments were performed substantially in accordance with the teachings of Example 3 herein. The data is shown in Table 1 below and in FIG. 6 of the accompanying drawings.

[0058]

[Table 1] As can be seen from the data presented, the vectors of the present invention have demonstrated anti-tumor efficacy in vivo. Not only did the tumor not grow, but the size actually decreased (regres), as five of the six animals were tumor free at day 27 post-dose.
It should be noted that sed). This is particularly surprising in terms of the non-replicating p53 vector FTCB, where p53 is also expressed by the CMV promoter. Also, this effect is not simply due to the E1b55k deletion (as shown by cZAZA), which showed lower efficacy at 27 days post-dose.

VII Pharmaceutical Formulations The present invention further provides pharmaceutically acceptable formulations of the recombinant adenovirus in combination with a carrier. The vectors of the present invention may be formulated for administration in dosages according to conventional pharmaceutical practice with the addition of carriers and excipients. Dosage formulations can include intravenous, intratumoral, intramuscular, intraperitoneal, topical, matrix or aerosol delivery.

A Carrier The term “carrier” refers to a compound commonly used in formulating pharmaceutical compounds that is used to enhance the stability, sterility, and delivery of the therapeutic compound.
When the virus is formulated as a solution or suspension, the delivery system will be in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, eg, water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid, and the like. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances such as those required for similar physiological conditions (eg, pH adjusting and adjuvanting agents, tonicity adjusting agents, wetting agents, etc.) (eg, sodium acetate, Sodium lactate, sodium chloride, potassium chloride, calcium chloride, adsorbed monolaurate
urate), triethanolamine oleate, etc.).

(B) Delivery Enhancer The present invention further provides a pharmaceutical formulation of the recombinant adenovirus vector of the present invention having a carrier and a delivery enhancer. The terms "delivery enhancer" or "delivery enhancer" are used interchangeably herein and include one or more agents that facilitate the uptake of the virus into target cells. Examples of delivery enhancers are described in co-pending U.S. patent application Ser. It is described in. Examples of such delivery enhancers include surfactants, alcohols, glycols, surfactants, liver juice salts, heparin antagonists, cyclooxygenase inhibitors, hypertonic salt solutions, and acetate. Alcohols include, for example, aliphatic alcohols such as ethanol, N-propanol, isopropanol, butyl alcohol, acetyl alcohol. Glycols include glycerin, propylene glycol, polyethylene glycol, and other low molecular weight glycols such as glycerol and thioglycerol. Acetates such as acetic acid, gluconic acid and sodium acetate are further examples of delivery enhancers. Hyperosmotic salt solutions such as 1 M NaCl are also examples of delivery enhancers. Examples of surfactants include sodium dodecyl sulfate (SDS) and lysolecithin, polysorbate 80, nonylphenoxypolyoxyethylene (nonylphenoxypolyoxyethylene).
line), lysophosphatidylcholine, polyethylene glycol 400, polysorbate 80, polyoxyethylene ether, polyglycol ether surfactant and DMSO. Liver salts such as taurocholate, sodium tauro-deoxycholate, deoxycholate, chenodeoxycholate, glycocholate, glycochenodeoxycholate, and other conservatives (eg, silver nitrate) Can be used.
Heparin antagonist-like quaternary amines, such as protamine sulfate, are also
Can be used. Sodium salicylate, salicylic acid, and nonsteroidal anti-inflammatory drugs such as indomethacin, naproxen, diclofenac (NSAI
A cyclooxygenase inhibitor such as DS) can be used.

[0062] Delivery enhancers include compounds of Formula I:

[0063]

Embedded image Here, n is an integer of 2 to 8, X1 is a cholic acid group or a deoxycholic acid group, and X2 and X3 are each independently selected from the group consisting of a cholic acid group, a deoxycholic acid group, and a saccharide group. You. At least one of X2 and X3 is a saccharide group. The saccharide group may be selected from the group consisting of a pentose monosaccharide group, a hexose monosaccharide group, a pentose-pentose disaccharide group, a hexose-hexose disaccharide group, a pentose-hexose disaccharide group, and a hexose-pentose disaccharide group.

The term “surfactant” includes anionic surfactants, cationic surfactants, zwitterionic surfactants, and non-ionic surfactants. Exemplary surfactants include taurocholate, deoxycholate, taurodeoxycholate, cetylpyridium, benalconium chloride (benal).
konium chloride), twin detergent 3-14 surfactant, CHAP
S (3-[(3-cholamidopropyl) dimethylammoniol (dimethy)
lamoniol)]-1-propanesulfonic acid hydrate), Big CHAP
, Deoxy Big CHAP, Triton-X-100 surfactant, C1
2E8, octyl-BD-glucopyranoside, PLURONIC-F68 surfactant, Tween 20 surfactant and Tween 80 surfactant (CalB
iochem Biochemicals), but is not limited thereto.

The unit dosage formulation of the present invention comprises the recombinant adenovirus in lyophilized form of claim 1 and a solution for reconstitution of the lyophilized product with instructions for use. Product kit. The recombinant adenovirus of the present invention can be lyophilized by conventional procedures and reconstitution.

[0066] Another example of a delivery enhancer that can be used in the formulations of the present invention includes a calpain inhibitor. “Calpain inhibitor” (abbreviated as “CI”) refers to a compound that inhibits the proteolytic action of calpain-I (eg, μ-calpain). The term calpain inhibitor, as used herein, has calpain I inhibitory activity in addition to its other biological activities, or independently of its other biological activities, calpain I Including a compound having inhibitory activity,
A wide range of compounds have been shown to have activity in inhibiting the proteolytic action of carpai. An example of a calpain inhibitor useful in the practice of the present invention is N-acetyl-leu-le, also known as "calpain inhibitor 1".
Contains u-norleucinal. Additional calpain inhibitors are described in the following U.S. patents, which are incorporated herein by reference. U.S. Pat. No. 5,716,980 entitled Alcohol or al.
US Patent No. 5,714,471 entitled Peptide and peptide an;
alog protease inhibitors; US Patent No. 5,693,
No. 617, Inhibitors of the 26s protely
tic complex and the 20s proteasome c
US Patent No. 5,691,368 entitled Su
bestitated oxazolidine calpain and / or
cathepsin B inhibitors; US Pat. No. 5,679,6.
No. 80 title α-substituted hydrazides having
US Patent No. 5, calpain inhibitory activity;
No. 663,294, titled "Calpain-inhibiting peptides"
analogs of the kininogen heavy chai
n; US Pat. No. 5,661,150, Drug for neuropro
protection; US Patent No. 5,658,906 entitled Cysteine pr.
otease and serine protease inhibitor
s; U.S. Pat. No. 5,654,146 entitled Human ice homolog
U.S. Pat. No. 5,639,783 entitled Ketone derivatives.
U.S. Pat. No. 5,635,178 entitled Inhibition of comp;
element mediated inflammation responses
e using monoclonal antibodies specialif
ic for a component forming the C56-9
complex who inhibit the platelet
or endothelial cell activating function
ion of the C56-9 complex; U.S. Patent No. 5,629,
No. 165, Neural calcium-activated neutr
al proteinase inhibitors; US Patent No. 5,622,
No.981 Use of metabolic receptor a
gonists in progressive neurodegenera
five diseases; US Patent No. 5,622,967, Quino
Lone carboxamide Calpain inhibitors;
U.S. Pat. No. 5,621,101 titled Protein kinase inhi
bits for treatment of neurological
disorders; U.S. Patent No. 5,554,767 entitled Alpha-me
rcaptoacrylic acid derivatives haven
g calpain inhibition activity; U.S. Pat. No. 5,550,108 titled Inhibition of completion.
mediated inflation response; US Pat. No. 5,541,290, Optically pure calpain i
nhibitor compounds; U.S. Pat. No. 5,506,243; Sulfonamide derivatives; U.S. Pat. No. 5,498,7.
No. 28, Derivatives of L-tryptophanal a
nd their use as medicals; U.S. Pat.
No. 8,616, Cysteine protease and serine
PROTEASE INHIBITORS; U.S. Patent No. 5,461,146, Selected protein kinase inhibitors
for the treatment of neurological d
isorders; US Patent No. 5,444,042, Method of
treatment of neurodegeneration with
US Pat. No. 5,424,325 entitled aminoketone derivatives; US Pat. No. 5,422,3
No. 59, α-aminoketone derivatives; U.S. Pat. No. 5,416,117, Cycloproponeone derivatives.
es; US Patent No. 5,395,958 entitled Cyclopropene der.
ivatives; U.S. Patent No. 5,340,922, Neural cal.
cium-activated neutral proteinase in
hibitors; U.S. Patent No. 5,336,783 entitled Calpain in
hibitor cystamidin A and its product
ion; US Patent No. 5,328,909 entitled Cyclopropenone
and US Patent No. 5,135,916, entitled Inhi.
bition of completion mediated inflam
material response. The use of calpain inhibitors in gene therapy protocols was filed on October 15, 1998 by Atencio et al.
Co-pending US patent application Ser. Nos. 09 / 172,685 and 60/104.
321. In a preferred practice of the invention, the calpain inhibitor is Boehringer-Mannheim (Indianapolis).
s, IN).

(IIX. Therapeutic Applications :) (A. Antineoplastic Applications :) The present invention provides for the administration of the recombinant viral vectors of the present invention to neoplastic cells.
A method for removing neoplastic cells in a mammalian organism is provided. The term "neoplastic cell" is a cell that displays an abnormally growing phenotype characterized by the independence of normal cell growth controls. Because neoplastic cells do not necessarily replicate at any given point in time, the term neoplastic cell refers to cells that can be actively replicating or cells that can be temporarily non-replicating quiescent (G1 or G0). Including. The localized population of neoplastic cells is referred to as a neoplasm. Neoplasms can be malignant or benign. Malignant neoplasms are also referred to as cancer. The term cancer is used herein interchangeably with the term tumor. Neoplastic transformation refers to the conversion of a normal cell to a neoplastic cell, often a tumor cell. The term "mammalian organism" refers to humans, pigs, horses,
Including, but not limited to, cows, dogs, cats.

The present invention provides a method for eliminating neoplastic cells in vivo in a mammalian organism by administration of a pharmaceutically acceptable formulation of the recombinant adenovirus described above. The term “elimination” means a substantial reduction in the viable neoplastic cell population so as to alleviate the physiological disease of the presence of the neoplastic cell. The term "substantially"
By means a reduction in the population of viable neoplastic cells in a mammalian organism of more than about 20% of the pre-treatment population. The term "viable" means having the characteristics of uncontrolled proliferation and regulation of the cell cycle of neoplastic cells. The term "viable neoplastic cell" is used herein to distinguish viable neoplastic cells from neoplastic cells that can no longer replicate. For example, a tumor mass may remain after treatment, but a population of cells containing the tumor mass may die. Even though some tumor masses may remain, these dead cells have been removed and lack the ability to replicate.

In a preferred practice of the invention, exemplified herein, a recombinant adenovirus containing a deletion of E1B-55K gene function and expressing a tumor suppressor gene from the adenovirus major late promoter is a venous virus. It is formulated with a pharmaceutically acceptable carrier for administration by injection, intraperitoneal injection or intratumoral injection. The appropriate dose and method of administration of the vector to be administered to the mammalian organism in need of treatment is
It is determined by those skilled in the art in view of the degree of metastasis of the primary tumor, the delivery enhancer contained in the formulation, the degree to which the immunological response is suppressed, and the like. Each of these latter factors reduces the dosage of the vector provided to the mammalian organism in need of treatment. In a preferred practice of the invention, a dosage of approximately 1 × 10 ⁵ to 1 × 10 ¹³ particles (preferably 1 × 10 ⁶ to 1 × 10 ¹¹ particles, most preferably 1 × 10 ⁷ to 1 × 10 ¹⁰ particles) is used. Administered to a mammalian organism in one or more dosages in a treatment regime. A typical course of treatment is daily administration of a pharmaceutically acceptable formulation of a vector of the invention for 3 to 10 days or more, preferably 5 to 8 days or more. In a preferred embodiment, the tumor suppressor gene is a wild-type p53 gene. In another preferred embodiment, the tumor suppressor gene encodes a VP22-p53 fusion protein.

[0070] In a further preferred practice of the invention, the pharmaceutically acceptable carrier comprises a delivery enhancer. In a further preferred practice of the invention, the delivery enhancer is a calpain inhibitor. In the most preferred practice of the invention, exemplified herein, the recombinant adenovirus vector E1B- dl 55K-MLP-p53 is capable of reducing the calpain inhibitor n-acetyl-leu-leu-norleucinal (calpain inhibitor 1) to approximately 1 Formulated in a carrier solution further comprising a concentration of ５０50 μM. In such an example, the daily dosage could be reduced by 1-2 logs as compared to a formulation without such a delivery enhancer.

It is preferred to use an adenoviral vector endogenous to the type of mammal being treated. In general, it is preferred to use a virus from the species to be treated, but in some cases it may be advantageous to use a heterologous vector that possesses favorable pathogenic characteristics. For example, it is reported that sheep adenoviral vectors can be used in human gene therapy to minimize the characteristics of the human adenoviral vector's immune response (WO 97/06826).
(Released on April 10, 1997). By minimizing the immune response, rapid systemic clearance of the vector is avoided, resulting in greater persistence of the vector.

Although the present invention provides a method of using a recombinant adenovirus alone, the recombinant adenovirus of the present invention and its formulation may be used in combination with a conventional chemotherapeutic agent or treatment regime. . Examples of such chemotherapeutic agents include inhibitors of purine synthesis (eg, pentostatin, 6-mercaptopurine, 6 thioguanine, methotrexate) or inhibitors of primidine synthesis (eg, Para (Pala), azaribine), ribonucleotides To deoxyribonucleotides (eg, hydroxyurea), inhibitors of dTMP synthesis (5-fluorouracil), DNA damaging agents (eg, radiation, bleomycin, etoposide, teniposide, dactinomycin, daunorubicin, doxorubicin, mitozantrone, alkylating agents , Mitomycin, cisplatin, procarbazine) and inhibitors of microtubule function (eg, vinca alkaloids and colchicine) and farnesyl protein transferase Peptidase inhibitors, and the like. A chemotherapeutic treatment regime refers to a non-chemical procedure designed primarily to remove neoplastic cells (eg, radiotherapy). In such cases, the daily dosage during the course of treatment is reduced compared to the dosage provided without such chemotherapeutic agents.

It has been observed that the immune system can recognize and remove recombinant viral vectors. This effectively reduces the amount of adenovirus reaching the target cells,
In many cases, it will be preferable to administer the compounds of the invention in combination with an immunosuppressant (eg, etoposide). In a preferred practice of the invention, the immunosuppressive agent is administered beforehand, preferably about one week prior to introduction of the recombinant viral vector of the invention, to eliminate the humoral immune response to the viral particles. In a preferred practice of the invention, the pharmaceutically acceptable formulation of the vector of the invention is administered intratumorally following administration of the immunosuppressant for a period of about 1 day to about 2 weeks prior to administration of the vector. Is done. The vector is preferably an adenovirus vector and E1
It further includes a deletion of the B-55K protein and includes an expression cassette that expresses the p53 tumor suppressor gene from the adenovirus major late promoter. In a preferred practice of the invention, the immunosuppressant is etoposide and it is administered daily for about 1 to 7 days (preferably 3 to 7 days) prior to vector administration. In such instances, the daily dosage during the course of treatment is reduced as compared to those dosages provided in the absence of such immunosuppressants.

The present invention also provides a method for excision of said neoplastic cells in a population of normal cells contaminated by neoplastic cells ex vivo by administration of a recombinant adenovirus of the invention to said population. provide. Examples of the application of such methods are currently used in ex vivo applications, such as purging of autologous stem cell products commonly known as bone marrow purging. The term “stem cell product” refers to myectomy (my)
Oabtive) refers to a population of hematopoietic progenitor and stem cells that allows long-term reconstitution of hematopoietic function in patients undergoing therapy. Stem cell products are conventionally obtained by apheresis of mobilized or unmobilized peripheral blood. Apheresis is conventionally known as COBE Spectra Apheresis System.
m (COBE International, 1185 Oak Street)
, Lakewood, CO .; Using commercially available apheresis equipment (commercially available from Co., Ltd.). "3-log purge (3
-Log purge) (i.e., removal of about 99.9% of the tumor cells from the stem cell product) and most preferably "5-log purge".
It is preferred to optimize the processing conditions in order to achieve "" (ie removal of about 99.999% of the tumor cells from the stem cell product). In a preferred practice of the invention, a 100 ml volume of the stem cell product is treated with a concentration of about 1 × 10 ⁶ to 1 × 10 ¹⁰ particles / ml of the recombinant adenovirus of the invention at 37 ° C. for about 4 hours. .

B. Replenishment of Dendritic Cells The present invention provides a method for recruiting immature dendritic cells to the site of a tumor and replacing the dendritic cells with a localized high concentration of tumor specific to the tumor present in the patient. Provided is a recombinant viral vector that can be exposed to an antigen. The vectors of the present invention are specifically engineered to induce killing of tumor cells. Lysed tumor cells (or apoptosed)
A) Apoptotic bodies produced by tumor cells) provide dense localized concentrations of tumor-specific proteins. By introducing a gene encoding a dendritic cell chemoattractant, immature dendritic cells capable of engulfing tumor antigens are recruited to sites of lysed tumor cells, thereby recruiting tumor antigens. Swallow and present these antigens to the immune system. The term "dendritic cell chemoattractant" refers to a chemotactic chemokine that can attract and / or direct the migration of dendritic cells to a particular location. Specific chemokines (fMLP (formyl peptide of representative bacterial origin), C5a and CC chemokine monocyte chemoattractant protein (MCP) -3
, Macrophage inflammatory protein (MIP) -1α / LD78, and RAN
TES) has been demonstrated to be involved in dendritic cell recruitment and chemotaxis migration. Sozzani et al. (1995) J. Mol. Immunol. 1995 155 (7)
: 3292-5. Xu et al. Reported that all CC chemokines (MCP-1, MCP-2
, MCP-3, MIP1α, MIP-1β, and RANTES)
This suggests that it induced migration of DC-rich cells cultured with or without L-4. Xu et al., (1996) J. Am. Leukoc. Biol. 60 (3): 36
5-71. Greaves et al. (1997) J. Am. Exp. Med. 186 (6):
837-44 show that MIP-3-α specifically interacts with CC chemokine receptor 6 expressed on dendritic cells capable of directing the migration of dendritic cells. In a preferred practice of the invention, the dendritic cell chemoattractant is MIP-3-α
It is. The dendritic cell chemoattractant, if released upon cell lysis, can be expressed in an intracellular form or in a secreted form by use of a signal peptide.
When dendritic cell chemoattractants are expressed, dendritic cells then swallow tumor antigens and apoptotic bodies, mature and migrate through existing pathways to lymphocytes,
Then, the tumor antigen is presented to T cells. The resulting T cells can then recognize and kill the tumor cells. Suitable dosing regimes in such cases are in accordance with representative antineoplastic dosing formulations and regimens as described above.

IX. Diagnostic Applications In addition to the therapeutic applications described above, the vectors of the present invention are also useful for diagnostic purposes. For example, a vector of the invention may incorporate a reporter gene in place of a therapeutic gene expressed during viral replication, where the term `` reporter gene '' refers to a product that can be detected alone or in combination with additional elements. A gene capable of producing a signal. Examples of reporter genes include β-galactosidase gene, luciferase gene, green fluorescent protein gene, X-ray or magnetic field imaging system (magnetic field imaging system).
) (MRI) include nucleotide sequences encoding proteins that can be detected by an imaging system. Alternatively, such vectors can also be used to express cell surface proteins that can be recognized by binding molecules such as fluorescently labeled antibodies. Examples of in vivo applications include X-ray, CT scan, or imaging applications such as magnetic resonance imaging (MRI).

(X. Method of Making Composition) The present invention further provides a method of producing a recombinant adenovirus comprising a modification to the above-described packaging domain. The above method comprises the following steps: a. Infecting the producer cells with the recombinant virus; b. Culturing the infected producer cell described above under conditions permitting replication of the viral genome in the producer cell; c. Collecting the producer cells; and d. Purifying the recombinant adenovirus.

The term “infect” means exposing a recombinant adenovirus to a producer cell under conditions that facilitate infection of the producer cell with the recombinant virus. In cells infected by a given copy of the virus, the activities required for virus replication and virion packaging are cooperative. Therefore, it is preferred that the conditions be adjusted such that the producer cells are significantly more likely to be multiply infected with the virus. An example of a condition that enhances virus production in a producer cell is an increase in virus concentration at the stage of infection. However, it is possible that the total number of viral infections per producer cell can be excessive, resulting in a toxic effect on the cells. As a result, efforts should be made to maintain viral particle infection at a virus concentration in the range of 10 ⁶ to 10 ¹⁰ per ml (preferably about 10 ⁹ ). Chemical agents can also be used to increase the infectivity of the producer cell line. For example, the invention provides a method for increasing the infectivity of a producer cell line relative to viral infectivity by including a calpain inhibitor. As an example of a calpain inhibitor useful in the practice of the present invention, calpain inhibitor 1 (also known as N-acetyl-leucyl-leucyl-norleucinal, Boehringer
Commercially available from Mannheim).

The term “producer cell” means a cell that can facilitate the replication of the viral genome of the recombinant adenovirus to be produced and can complement the packaging defect of the recombinant adenovirus. Various mammalian cell lines are publicly available for culture of recombinant adenovirus. For example, the 293 cell line (Graham
And Smiley (1977) J. Mol. Gen. Virol. 36: 59-72)
Has been engineered to compensate for defects in E1 function and is the preferred cell line for current vector production. In a similar manner, cell lines can be developed that incorporate viral sequences that are stably integrated into the viral genome. See, for example, Cunningham and Davidson ((1997), V.
irol 197: 116-124) showed that overlapping cosmids could be used to complement the viral function deleted for the helper virus vector. A similar approach can be used to complement adenovirus and other viral elements. Additional genes, such as those encoding drug resistance, can be included to allow selection or screening for the presence of the recombinant complementing vector. Such additional genes can include, for example, genes encoding neomycin resistance, multidrug resistance, thymidine kinase, β-galactosidase, dihydrofolate reductase (DHER), and chloramphenicol acetyltransferase. Examples of other producer cell parental cell lines that can be used include HeLa cells, PERC. 6 cells (publication WO / 97 /
00326, described in application PCT / NL96 / 00244) and A549-E
One cell line (described in International Patent Application No. PCT / US97 / 810039 (published on February 23, 1998) and International Publication No. WO98 / US3473).

The term “culturing under conditions that permit replication of the viral genome” means maintaining the conditions for infected producer cells to permit growth of the virus in the producer cells. It is preferable to control the conditions so as to maximize the number of virus particles produced by each cell. As a result, the temperature,
It is necessary to monitor and control reaction conditions such as dissolved oxygen, pH, etc. CelliGen Plus Bioreactor (New Brun
switch Scientific, Inc. 44 Talmadge Roa
Commercially available bioreactors (commercially available from Edison, NL) have facilities to monitor and maintain such parameters. Optimization of infection and culture conditions will vary somewhat, but the conditions for efficient replication and production of the virus will vary according to the known characteristics of the producer cell line, the characteristics of the virus, the type of bioreactor, etc. Can be achieved by: When 293 cells are used as producer cells, the oxygen concentration is preferably maintained at about 50% to about 120% dissolved oxygen, preferably 100% dissolved oxygen. The reaction is collected when the concentration of virus particles begins to plateau (as determined by conventional methods, such as HPLC using a Resource Q column).

The term “harvesting” means collecting cells containing the recombinant adenovirus from the culture medium. This can be achieved by conventional methods, such as by means of differential centrifugation or chromatography. At this stage, the harvested cells can be stored or further processed by lysing and purifying to isolate the recombinant virus. For storage, the harvested cells are at physiological p
Buffered at H or near physiological pH and frozen at -70 ° C.

The term “lyse” refers to the rupture of a producer cell. Lysis can be achieved by various means well known in the art. If it is desired to isolate the virus particles from the producer cells, the cells are lysed using various means well known in the art. For example, mammalian cells can be lysed under low pressure (100-200 psi differential pressure) conditions or by conventional freeze-thaw methods. Exogenous free DN
A / RNA is removed by degradation using DNAse / RNAse.

The term “purify” refers to the isolation of a substantially pure population of recombinant virus particles from lysed producer cells. Conventional purification techniques such as chromatographic methods or fractional density gradient centrifugation methods can be used. In a preferred practice of the invention, the virus is Huygh et al. (1995) Human Ge.
ne Therapy 6: 1403-1416 (as described in co-pending US patent application Ser. No. 08 / 400,793, filed Mar. 7, 1995), purified by column chromatography. .

Further methods and procedures for optimizing the production of the recombinant adenovirus of the present invention are described in co-pending US patent application Ser. No. 09 / 073,076, filed May 4, 1998.

[0085] The purified virus is then mixed with a suitable excipient and carrier or delivery enhancing agent. Solutions are sterilized for individual packaging and placed in vials for storage.

Alternatively, the virus is lyophilized for storage.
And delivery enhancers, buffers, preservatives, cryoprote
ctant) and / or in a solution containing a carrier. The lyophilized virus and reconstituted solution can be packaged together as a kit for consumption by the general user with instructions for proper handling and administration.

EXAMPLES As will be apparent to those skilled in the art to which the invention pertains, the present invention may be practiced without departing from the spirit or essential characteristics of the invention, particularly in forms other than those disclosed below. Can be implemented. Accordingly, the specific embodiments of the present invention described below are to be considered illustrative and non-limiting. The scope of the present invention is not limited to the examples provided below, but is as set forth in the appended claims.

Example 1 Construction of E1B dl 55K-MLP-p53 (cFAMA) E1B55K-MLP-p53 (cFAMA) adenovirus was purified from Deng and Nickoloff (1992) Anal. Biochem 200, 81-
Prepared using 88 oligonucleotide site-directed mutagenesis techniques. All reagents, bacterial strains, and vectors used for mutagenesis were
mer Site-Directed mutagenesis kit (Cl
ontech, Palo Alto, CA). The Ad5 region containing the sequence to be mutated was cloned into the Clontech plasmid pEG.
Inserted into FP-1 and named pXB-E1B. Two primers (1
) Anneal Ad5 sequences 2236-2260 to convert G2247 to thymidine, T2
To change 248 to cytosine, and (2) the Ad5 sequence 3255-3284
To change T3272 to cytosine. A third primer is designed to delete the HindIII site in the original vector sequence and provides a method for selection.

For the mutagenesis reaction, the mutagenized oligonucleotide was first phosphorylated at the 5 ′ end and then annealed to the denatured pXB-E1B template DNA. The annealed primer / template reaction was incubated with T4 DNA polymerase, T4 DNA ligase and deoxyribonucleotides (dATP, dCTP, dGTP and dTTP) to synthesize complementary mutant strands. This complementary strand synthesis reaction was then transformed into a bacterial strain, BMH71-18 mutS. This bacterial strain is defective for mismatch repair, preventing unwanted repair of the mutated strand. This transformant was transformed with Hind to cut any parent plasmid strand.
It was digested using dIII and retransformed into DH5α for amplification. Potential E1B mutants were then screened by restriction analysis to confirm the desired mutation.

Using this procedure, a restriction enzyme nuclease cleavage site was introduced within the E1B 55K coding region. This first site was introduced by modifying positions 2247 and 2248 of the wild-type Ad5 genome. Here, guanine ²²⁴⁷ was replaced with thymidine (respectively) and thymidine ²²⁴⁸ was replaced with cytosine, introducing an EcoR1 cleavage site. This results in a modification of the E1B coding sequence at position 77 from valine to serine. A second restriction site was introduced at position 3272, where the thymidine ³²⁷² was replaced with a cytosine site (silent mutation) and an XhoI site was introduced. This new restriction site was used in a restriction digest with EcoRI and XhoI.

The cassette containing the p53 coding sequence under the control of the adenovirus major late promoter and the ternary leader sequence was removed from the plasmid pAd-MLP-p53 by EcoRI and partial XhoI digestion. This plasmid is
pBR322 derivative pML2 (pB deleted for base pairs 1140-2490)
R322) and includes type 5 adenovirus sequences extending from base pairs 1-5788 except for the base pairs deleted for type 5 adenovirus base pairs 357-3327. At the Ad5 357-3327 deletion site, the type 2 adenovirus major late promoter, type 2 adenovirus ternary leader DNA and human p53 c
A transcription unit consisting of DNA is inserted. This EcoRI / XhoI fragment is inserted into the EcoRI and XhoI sites introduced into the E1B55k coding region. The polyA sequence following wtAd5 pIX was amplified by PCR (Ad
5 sequences 4001 to 4368). Primers used for amplification included sequences to introduce an EcoRI site at the 5 'end of the Ad5 sequence and a SacII site at the 3' end of the Ad5 sequence. This fragment was then inserted into the EcoRI-SacII site just behind the E1B19k coding sequence. This SacII
The site was contained in the previously inserted MLP-p53 cassette and was upstream of the MLP promoter sequence. The resulting E1B mutation results in a sequence encoding the first 76 amino acids of the E1B55K protein, followed by a sequence encoding 11 missense amino acids resulting in a non-functionally deleted E1B protein.

Construction of the E1B dl 55K-MLP-p53 adenovirus was performed by the method of McGory et al., (1988) Virology 163, 614-617.
This was done by using homologous recombination in the adenovirus E1 region, including cell lines. This method uses two fragments of DNA, one with the E1B55k deletion /
A transfer plasmid containing the MLP-p53 cassette, and the other is Ad5 base pair 9
Other Ad5 viral DNA, including the wtAd5 genome ("Ad5 large fragment") up to the 18-3'ITR (base pair 35935) is required. The transfer plasmid used, pXC1-E1B55-2A, contains the wtAd5 sequence 22-2246.
including. For recombination to produce adenovirus cFAMA, the virus large fragment and pXC1-E1B53-2A were co-transfected into 293 cells by calcium phosphate mediated transfection. After 5 hours, the sediment was rinsed from the cells and replaced with normal medium. Fifteen days after the first transfection, the virus "comet" was isolated, the plaque was purified twice,
The viral DNA was subsequently screened using restriction enzyme analysis and DNA sequencing. Purifying the viral stock by a double cesium chloride gradient,
And Huyghe et al. (1995) Human Gene Therapy.
6: quantified by column chromatography as described in 1403-1416.

Example 2 In Vitro Evaluation of E1B dl 55K-MLP-p53 (FAMA) Example 2. a.p53 Expressing MRC9 Cells Two different viral constructs (E1B dl 55K-MLP-p53 (cFAM)
A) and ACN53 (Wills et al., (1994) Human Gene T).
The level of p53 expression from herapy, supra)) was evaluated in MRC9. A 6-well plate was seeded with 6 × 10 ⁵ MRC9 cells per well. Infection was performed at two different concentrations (1.8 × 10 ⁸ particles / ml and 1.8 × 10 ⁹ particles / ml).
ml) in a volume of 250 ml with a 1 hour pulse. This cell,
Substantially according to the instructions provided by the manufacturer, BioRad Cat.
Collected and assayed using the 00-0006 kit. The cells were harvested by scraping at 24, 48, and 72 hours as indicated. The cells were added to a lysis buffer (50 mM Tris, 250 mM NaCl,
Resuspended in 0.1% NP40, 50 mM NaF, 5 mM EDTA) and lysed by freeze-thaw (repeated three times). The cells were stained with the BioRad dye reagent concentrate provided in the kit, and the absorbance was measured at a wavelength of 595 nm. The primary antibody was a mouse anti-p53 antibody (commercially available from Novacastra as catalog number 1801 (diluted 1: 1000)). The secondary antibody was goat anti-mouse HRP (commercially available from Amersham under catalog number NA931). This data is shown in FIG. 1 of the accompanying drawings. As can be seen from the data presented, the replicable competent MLP-p53 construct was a replication-defective CMV-p53 virus (A
CN53).

Example 2.b p53-expressing SK-HEP1 cells Experiments were performed on SK-HEP1 hepatocellular carcinoma cells, except that 6x10 plates were seeded with 7.5 x 10 ⁵ cells per well. This evaluation was made according to Example 2. Performed substantially in accordance with the teachings of a. The results are shown in FIG. 2 of the accompanying drawings.

Example 2. c. P53-Expressing NCI H358 Cells Except that the experiments were performed on NCIH358 cells, except that they were seeded at 1.5 × 10 ⁵ cells per well in a 6-well plate. This evaluation was made according to Example 2 above. Performed substantially in accordance with the teachings of a. The cells using the concentration 1.8 × 10 ⁸ particles / ml of the virus indicated the concentration of virus indicated, in 1.8 × 10 ⁹ particles / ml and 4 3, the Example 2. a. Infected like. The results are shown in FIGS. 3 and 4 of the accompanying drawings.

Example 2.d. Time course of virus replication SK-BR3 cells were cultured at a concentration of 1.8 × 10 ⁹ particles / ml for 1 hour with the following recombinant adenovirus vectors. Infection was performed using pulses.

1. Mock: uninfected cells 2. rAdcon: El and protein IX without p53 coding sequence
2. Recombinant adenovirus lacking function (Wills et al.) E1B dl 55K: Example 1 above with a non-foreign transgene cassette
4. A recombinant adenovirus containing the E1B-55K deletion according to item 4.5.5K / MLPp53: a recombinant adenovirus cFAMA prepared substantially in accordance with the teachings of Example 1 above. 6. rAd-p53: ACN53 (Wills et al.) 6.55K / CMV p53: Recombinant adenovirus cFAIC containing the E1B-55K deletion as described above, further comprising an expression cassette encoding p53 under the control of the CMV promoter. Ad5WT: wild-type adenovirus type 5 These cells were harvested approximately 48 hours after infection. The DNA was applied to an agarose gel and stained with ethidium bromide according to techniques well known in the art. The result is shown in FIG. 5 of the accompanying drawings.

Example 3. Demonstration of Efficacy of In Vivo Therapy PC-3 cells (prostate carcinoma, without p53) were placed on the flank of nude mice in subQ.
Was injected. If tumors were evident (day 11), the virus was given 5 consecutive days at 11-15 days post PC-3 cell injection at a dose of 1 × 10 ¹⁰ particles per injection.
Injected intratumorally for days. Six different viral constructs (cFAMA, cFAI)
C, FTCB, ZZCB, cZAZA and wild type Ad5 (described in Example 2 above) were compared. V-PBS refers to phosphate buffered saline containing 3% sucrose and 2 mM magnesium chloride. Tumor volume was 1, 4, 7,
Evaluations were made on days 11, 15, 19, 23 and 27. The results of this evaluation are shown in FIG. 5 of the accompanying drawings and are shown in Table 1 above.

[0099] (Example 4.E1B dl 55K-MLP- Construction of cytosine deaminase) E1B dl 55K-MLP- cytosine deaminase vector, prepared in substantial accordance with the teaching of Example 1 herein. However, the transfer plasmid containing the p53 coding sequence is replaced with a DNA sequence encoding a cytosine deaminase gene.

[0100] (Example 5.AFP-E4-E1B dl Construction of 55K-MLP-p53) AFP- E4-E1B dl 55K-MLP-p53 is a recombinant adenovirus, which is herein E1 prepared substantially in accordance with the teachings of Example 1
Same as the Bdl 55K-MLP-p53 vector. However, in this construct, the E4 gene is operably linked to an α-fetoprotein tumor-specific promoter sequence (AFP) that facilitates replication of the virus in hepatocellular carcinoma cells. Other factors, such as NF-IL6, can be substituted for E1a in regulating the E1a responsive promoter in adenovirus in the absence of E1a function (Spergel et al., (1992) J. Virol 66:10.
21-1030), and this is avoided by replacing the E4 promoter with a tumor-specific promoter.

[0102] (Example 6.AFP-E4-E1B dl Construction of 55K-MLP-IFN2a) AFP- E4-E1B dl 55K-MLP-IFN2a showed recombinant adenovirus, which is herein Similar to the AFP-E4-E1B dl 55K-MLP-p53 vector prepared substantially according to the teachings of Example 5. However, in this construct, the p53 gene has been replaced with a DNA sequence encoding interferon α-2b.

[0102] (Example 7.E1A dl 01/07-E1B dl construction of 55K-MLP-p53) E1A dl 01/07-E1B dl 55K-MLP-p53 is a recombinant adenovirus, this is, this Substantially similar to the E1B dl 55K-MLP-p53 vector prepared substantially according to the teachings of Example 1 herein. However, in this virus, the E1a gene was deleted by Jelsma et al., (1998) Vi.
modified to include the in-frame deletion mutations dl 1101 and dl 1107, as described in R. 163, 494-502, Kunkel (1).
985) Zoell as modified by PNAS 82, 488-492.
er and Smith (1984) DNA 3, 479-488, using oligonucleotide site-specific techniques. All reagents, bacterial strains and M13 vectors used for mutagenesis were Muta-Gene in vitro mutagenesis kits (commercially available from Bio Rad, Hercules, CA).
In, is provided. This M13 template D useful for mutagenesis of the E1A region
NA corresponds to BamHI and Xba in the multiple cloning sequence of M13mp19.
Includes the Ad5 sequence from nucleotides 22 to 1339 inserted between the I restriction enzyme site. The resulting bacteriophage construct, M13mp19E, was then
1A, dut ung E. Amplification in E. coli bacterial strain CJ236, which produces occasional incorporation of uracil instead of thymidine in newly synthesized DNA. For the E1 mutant construct, the oligonucleotide is
One end of the removed sequence is synthesized to constitute a sequence of either 11 or 12 nucleotides of Ad5 sense DNA.

For the mutagenesis reaction, the mutagenized oligonucleotide is first phosphorylated at the 5 ′ end and then annealed to uracil-containing M13mp11E1A single-stranded template DNA. The annealed primer / template reaction was performed with T4 DNA polymerase, T4 DNA ligase and deoxyribonucleotides (dATP, dCTP).
, DGTP and dTTP) to synthesize a complementary strand containing the E1A mutation of interest. The complementary strand synthesis reaction is then transformed into ung ⁺ wild-type host bacterial strain, MV1190. Parent M13mp19E1 containing uracil
After transformation of the A DNA strand, it cannot be replicated efficiently in MV1190. Therefore, the replicative double-stranded DNA containing the target E1A mutation is enriched. M13mp19E1A phage DNA from potential E1A mutants was first screened on both strands by restriction enzyme analysis and then by DNA sequencing to confirm the desired E1A mutation, confirming the desired E1A mutation. I do.

The construction of an adenovirus containing the E1A dl 01 / deletion has been described by McGory et al., (1
988) Virology 163, 614 to 617, using homologous recombination in an adenovirus E1 region-containing 293 cell line. This method requires two plasmids, one being the complete wt modified in Example 1 to include the E1b dl 55K deletion and the MLP promoter.
Viral plasmid containing the Ad5 genome (pXC1-E1B55-2A), and the other is transfer plasmid containing the E1A gene with dl 01/07 double E1A mutants. This transfer plasmid, pLE2, contains the 22-1774 wtAd5 sequence cloned in the tetracycline gene of pBR322 (Jelsma et al., (1988) Virology 163, 494-5).
02). E1A dl 1101 and dl 1107 E1a variants have been constructed,
E1A dl 1101 and dl 1107 from M13mp19 background
For transfer of the E1a mutant, the wild-type E1A restriction enzyme fragment in pLE2 was converted to M13mp19E1 dl 1101 and M13mp19E1A dl 110.
7 was replaced with the cognate mutant E1A fragment from pLE2E1A dl 01/0
7 was produced. For recombination to produce the adenovirus E1A dl 01/07, the viral plasmids pXC1-E1B55-2A and pLE2
E1A dl 01/07 was transformed into 2 by calcium phosphate mediated transfection.
Co-transfect 93 cells. After 5 hours, the sediment is rinsed, and the cells are overlaid with growth medium containing agarose, and viral plaques are isolated. Seven to ten days after the first transfection, viral plaques were isolated and plaques were purified twice. And subsequently, the viral DNA is screened using restriction enzyme analysis and DNA sequencing. Viral stocks were purified by a double cesium chloride gradient and analyzed by Huygh et al. (1995) Human.
As described in Gene Therapy 6: 1403-1416,
Quantify by column chromatography.

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. a. The two different concentrations (1.8 × 10 ⁶ particles / ml (upper panel) and 1.8 × 1 in MRC9 cells
Replication-defective p53 vector (ACN53) at ⁰⁹ particles / ml (lower panel)
Figure 5 is an autoradiogram of a time course study comparing p53 expression levels of replication competent MLP-p53 (Ad5dElbMLPp53) virus compared to. As can be seen from the data shown, the replication competent MLP-p53 construct caused expression at a later time than the replication defective CMV-p53 virus (ACN53). The data further shows that the level of p53 expression produced from replication competent MLP-p53 virus is significantly higher than replication defective CMV-p53 virus.

FIG. 2 shows the experimental results of Example 2. b. 1 shows the results of an experiment essentially similar to the experiment shown in FIG. 1, except that it was performed on SK-HEP1 hepatocellular carcinoma cells according to the procedure of FIG. Again, the time course experiments showed that replication competent MLP-p53
Demonstrates transient and higher expression of p53 in the construct.

FIG. 3 shows the experimental results of Example 2 herein. c. NCI H358 according to the procedure of
1 shows the results of an experiment essentially identical to the experiment shown in FIG. 1, except that it was performed on breast cancer cells. Again, time course experiments demonstrate the transient expression of p53 in a replication competent MLP-p53 construct.

FIG. 4 is a diagram showing Embodiment 2 of the present specification. d. An experiment similar to that shown in FIG. 3 except that the replication competent E1b dl 55k-CMV-p53 virus was included for comparison and that only a single dose was administered according to the procedure of FIG. The results are shown. This time course experiment demonstrates the transient expression of p53 in a replication competent MLP-p53 construct compared to an essentially similar construct in which the p53 gene is under the control of a constitutive CMV promoter. This data confirms that the MLP-p53 construct is actually expressing p53 in a transient manner later in the viral replication cycle.

FIG. 5 is a diagram showing Embodiment 2 of the present specification. d. 1.8 × 10 ⁹ particles /
A digest of viral DNA from SK-BR3 cells infected with a 1 hour pulse with the indicated virus at a concentration of ml and collected approximately 48 hours later. The results shown are replication competent wild type Ad5 (Ad5WT), replication competent E1
B dl 55K (ZAZA) virus and replication competent E1B dl 55K-
The MLP-p53 (55K / MLP53) virus demonstrates that all virus DNA replicates well, whereas the replication-defective adenovirus control (rAdcon) and the replication-defective vector encoding p53 (FTCB) do not.

FIG. 6 is a graphical representation of data obtained in vivo in a PC-3 mouse tumor model. Tumor volume is plotted on the vertical axis and days after dosing are plotted on the horizontal axis. As can be seen from the data shown, replicating virus E1
B dl 55K-MLP-p53 ( cFAMA) could be retracted tumors in human cancers in vivo mouse model. Replication competent CMV driveability p5
3 viruses also replicate viral DNA, but to a lesser extent.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 C12N 15/00 ZNAA 7/00 5/00 B (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY) , KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CZ, D , DK, DM, EE, ES, FI, GB, GD, GE, HR, HU, ID, IL, IN, IS, JP, KG, KR, KZ, LC, LK, LR, LT, LU, LV, MA, MD, MG, MK, MN, MX, NO, NZ, PL, PT, RO, RU, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UZ, VN , YU, ZA F term (reference) 4B024 AA01 BA80 CA04 CA20 DA03 EA02 EA04 FA02 GA11 GA25 HA17 4B065 AA93X AA93Y AA95X AB01 AC14 AC20 BA02 BA16 BD50 CA24 CA44 4C084 AA01 AA02 AA03 AA13 MA17 MA23 MA23 NA13 NA14 ZB262 ZC412 4C087 AA01 AA02 AA03 BC83 CA12 MA13 MA17 MA22 MA23 MA24 MA59 MA66 MA67 NA13 NA14 ZB26 ZC41

Claims (22)

[Claims] 1. A replication competent recombinant virus comprising a therapeutic transgene operably linked to a late regulatory element. 2. The virus according to claim 1, wherein said virus is an adenovirus. 3. The virus of claim 2, wherein said late regulatory element is an adenovirus major late promoter. 4. The virus according to claim 3, wherein said therapeutic transgene is a tumor suppressor gene. 5. The virus according to claim 4, wherein said tumor suppressor gene is p53. 6. The virus of claim 5, further comprising a deficiency in E1B-55K function. 7. The virus of claim 6, further comprising a replication control sequence operably linked to the early gene. 8. The virus according to claim 7, wherein said replication control sequence is a tumor-specific promoter. 9. The replication control sequence according to claim 7, wherein the replication control sequence is α-fetoprotein.
The virus according to. 10. The virus according to claim 9, wherein said early gene is an E4 gene. 11. The method of claim 11, further comprising a deficiency in the function of E1a 12S and 13S.
The virus according to claim 6 (01/07). 12. A pharmaceutical formulation comprising a replication competent recombinant virus comprising a therapeutic transgene operably linked to a late control element and a pharmaceutically acceptable carrier. 13. The formulation of claim 12, further comprising a delivery enhancer. 14. The formulation of claim 13, wherein said delivery enhancer is a calpain inhibitor. 15. The method according to claim 15, wherein the calpain inhibitor is N-acetyl-leu-1.
15. The formulation of claim 14, which is eu-norleucinal. 16. The formulation of claim 13, wherein said delivery enhancer is a surfactant. 17. A method for removing tumor cells, said method comprising contacting said tumor cells with a replication competent recombinant virus comprising a therapeutic transgene operably linked to a late regulatory element. By doing. 18. The method of claim 17, wherein said method is performed in vivo. 19. The method of claim 18, wherein said vector is administered by intraperitoneal injection, intravenous injection or intratumoral injection. 20. The method of claim 17, wherein said method is performed ex vivo. 21. The method of claim 20, wherein said method is performed to remove tumor cells from a stem cell product. 22. A cell transformed with a replication competent recombinant virus comprising a therapeutic transgene operably linked to a late regulatory element.