TITLE
ADENONIRAL VECTORS
BACKGROUND OF THE INVENTION
In order to facilitate the understanding of the present invention, a bπef overview of the life cycle of the adenovirus is offered. The adenoviral rephcative cycle in human cells can be divided into the early and late phase which are punctuated by the onset of viral DNA replication The early phase beings when viral particles attach to cells through interaction between the viπon fiber domain and cell surface receptors. The viπon moves into the cell by either endocytosis or direct penetration of the cytoplasmic membrane and is transported to the nucleus where most of the capsid is shed In the nucleus, the vinon core proteins are removed yielding viral chromosomes that are almost entirely devoid of viπon proteins Expression of the viral genome is temporally coordinated and begins with the EIA region about one hour after infection, The other early genes E1B, E2, E3 and E4 are expressed soon after EIA at 1.5-2 0 hours post infection A number of the protein products encoded by the early genes are required for viral DNA replication, while other prepare the DNA synthesis machinery of the infected cell for efficient viral DNA replication Some early virally encoded proteins have been associated with protecting infected cells from immune surveillance
The late phase of infection with onset of DNA replication begins at about 7 hours post infection In the native adenovirus. the messenger RNAs for all late gene products are spliced from a primary RNA which is transcribed from the major late promoter (MLP) The MLP is located at position 16 5 on the r-strand Although the major late promoter is active to a limited extent in the early phase of infection, the transcnption does not proceed past map position 39 Dunng the late phase of the viral life cycle, the MLP is fully activated and continues to map position 99 Each late pπmary RNA transcπpt is processed into one of five different mRNAs, L1-L5 These mRNAis all contain a common tripartite leader sequence of 203 nucleotides and a common 3ι end Late mRNAs encode capsid components and proteins required for
assembly of virions and packaging of the viral chromosome. Viral DNA replication requires the terminal protein for initiation and proceed by a semiconservative mechanism. With the onset of replication, efficient transcπption of the late gene families from the major late promoter begins and attains a maximal level approximately 18 hours post infection. Duπng the late phase viral proteins block cellular DNA and protein synthesis, presumably so that maximum viral macromolecular synthesis can occur. Intermediate gene expression, which actually begins duπng the early phase, reaches a maximum between 8 -12 hour post infection. Assembly of the virion and packaging of the viral genome begins at about 24 hours after infection. Infected cells are killed because of attπtion and lyse yielding approximately 10,000 virions per cell.
The adenovirus El region, which encodes the immediate early gene EIA and the early gene E1B, plays a key role in the adenovirus life cycle and is responsible for interfeπng with the ability of the infected cell to regulate cell cycle progression, differentiation and programmed cell death (apoptosis). The EIA gene products stimulate infected cells, which are normally differentiated and quiescent, to progress into the S-phase of the cell cycle in order that viral DNA replication can occur. Normal cells usually respond to unscheduled stimulation of cell cycle progression (by EIA or other mitogenic factors) by activation of p53-dependent apoptosis. However, in the context of a viral infection the EIA products do not stimulate apoptosis because the protein products of the El region gene, E1B, are effective inhibitors of apoptosis. Therefore, duπng the early stage of viral infection the EIA and E1B gene products cooperate to bπng about a quasi-tumoπgenic state in the infected cell which is required for efficient viral DNA replication and a productive infection cycle. The full scope of activity of the adenovirus EIA region is descπbed in Bayley, S. and Mymryk, J. (1994) Intl. J. of Oncology 5:425-444. For a comprehensive review of the adenovirus biology, see Shenk, T. (1996), Fields Virology, 3rdEdιtιon: p2111-2148.
The El genomic sequence is located at the extreme left end of the 36kb adenoviral genome. The pπmary EIA mRNA is differentially spliced duπng the early phase of replication into two prominent mRNAs, called 13S and 12S, which give πse to 289R and 243R AA proteins, respectively. The 289R and 243R proteins differ only by an internal sequence of 46 amino acids that is unique to the larger protein.
Although the EIA pπmary transcπpt is spliced into to 3 other mRNAs called US, 10S, and 9S, which encode for proteins of 217R, 171R and 55R respectively, these messages and not made efficiently in the early phase of infection and it is likely that the 289R and 243R proteins carry out the pπmary functions of EIA duπng the viral life cycle. The EIB gene transcπpt is also differentially spliced to yield mRNAs of 22S, 14.5S, 14S, and 13S each of which contains two open reading frames. One of these open-reading frames is common to all of the messages and encodes a protein of 179R. Depending on the mRNA, the other open reading frames give πse to proteins of 84R, 93R, 155R and 496R. Of the EIB proteins the 176R and 496R proteins, which are also referred to as E1B19K and E1B55K respectively, are the most prominent and best characteπzed
The EIA and EIB gene products play cπtical roles in the productive infection cycle to prepare the infected cell for viral replication and to regulate viral specific processes The EIA and EIB products do not contain mtπnsic enzymatic activities, but are thought to carry out their functions by interacting with a number of cellular proteins The EIA proteins associate with a wide range of cellular proteins including p400, p300, cAMP-responsive transcription binding protein (CBP), pl30, pl07, pRb, cychn A, cdk2 and TATA-binding protein (TBP). Mapping studies have been used to compare cellular protein binding domains and functional domains m the EIA proteins. Stimulation of cell cycle progression by EIA has been mapped to three regions in the common ammo terminal domain of the 243R and 289R proteins (Howe, et al., (1990) PNAS 87-5883-5887) These regions are commonly referred to as the amino-terminal domain, conserved region 1 (CR1) and conserved region 2 (CR2). The amino- terminal domain and CR1 are required to bind a number of proteins including p300/CBP which are thought to be co-activators of gene transcπption that have been implicated m regulation of cell proliferation and differentiation. The third EIA region required for cell cycle regulation by EIA is CR2 which is required for association with the know members of the pRb family of related cell cycle regulators including pRb, pl07 and pl30 The pRb famiK members regulate the cell cycle by binding to a the E2F class of transcπption factors that in turn regulate expression of genes that are required for
cell cycle phase transitions. Binding of p300 and the pRb family members appears to inactivate the ability of these proteins to suppress cell cycle progression and this appears to be the major mechanism by which EIA induces resting cells to progress into the cell cycle. A large body of evidence has accumulated to support this hypothesis. For example, by associating with pRb the EIA proteins disrupt E2F-pRb complexes which frees E2F to stimulate gene expression that allows progression into the S-phase of the cell cycle. It is not precisely known how p300 regulates cell proliferation, but p300 is known to regulate expression of genes that are required to maintain a differentiated phenotype and that inhibition of p300 can block terminal differentiation. In addition, it is known that ElA-mutants that associate with p300, but are defective for binding pRb, are nevertheless able to stimulate phosphorylation of pRb which leads to disruption of pRb-E2F complexes and cell cycle progression (Wang, et a , (1991) Mol Cell. Bio. 11, 4253-4265)
Induction of unscheduled DNA synthesis by EIA is a cellular stress that is sensed by the infected host cell The infected cell responds by inducing apoptosis which is normally mediated by p53 The p53 gene is activated in response to a wide vaπety of cellular stresses including DNA damage, hypoxia and expression of mitogenic oncogenes including EIA Productive viral infection cannot occur if the infected cell commits programmed cell death and therefore the virus has evolved to inhibit apoptosis, at least early duπng the infectious cycle, by production of the E1B 19K and 55K products For a review of regulation of apoptosis by EIB, see White, E (1998) Seminars in Virology 8, 505-513 The E1B19K is considered to be the pπmary inhibitor of El A-induced apoptosis because E1B 19K alone blocks EIA induced apoptosis more efficiently than E1B55K alone The E1B 19K inhibits apoptosis by two different mechanisms First, E1B 19K associates with the proapoptotic Bcl-2 famih members Bax. Nb /Bik and BNIP3, and inhibit the ability of these proteins to induce apoptosis Second, the E1B19K protein can inhibit apoptosis by interacting with factors such as FADD and CED4 which normally act to activate caspases for apoptosis The E1B55K protein binds to and downregulates the ability of p53 to act as an activator of transcπption Therefore, the E1B55K protein can augment the E1B19K protein to inactivate p53-dependent apoptosis
In addition to stimulating cell cycle progression and suppressing apoptotic pathways the EIA and EIB proteins also play important viral specific roles duπng the replication cycle of the virus The EIA proteins initiate the coordinated expression of the viral genome by stimulating expression of promoters for the EIB gene in addition to the other early gene regions E2, E3 and E4 The 289R EIA protein is pπmaπly responsible for the transactivation of the early adenoviral promoters and mapping studies have shown that the 46 ammo acid unique domain of the 289R protein plays the major role in activation of the early viral promoters. The E1B55K protein also carπes out cπtical viral specific roles duπng the productive infection cycle. Adenovirus E1B55K mutants are defective for late viral protein production and shutoff of host cell protein synthesis Consequently these mutant viruses are defective for growth on a number of human cell lines (Babiss and Ginsberg, (1984) J Virol 50.202-212, Babiss et al, 1985 Mol Cell Biol 52552-2558, Pidler et α/.,(1986) Mol.Cell.Bιol.6:470- 476, Yew et al ,(1990) Virology 179 795-805) More recently it has been suggested that the E1B55K may alter cell cycle controls in infected cells (Goodrum and Ornelles (1997) J Virol 71, 548-561) and in addition E1B55K may influence viral DNA replication (Ridgway ei al, (1997) Virology 237 404-413)
Attempts have been made to exploit the ability of the E1B55K protein to bind p53 in the design of adenoviruses that selectively replicate in and kill p53 deficient cells by the elimination of E1B55K function See McCormick, United States Patent No 5,677,178 issued October 14, 1997 A particular vector, ONYX-O15 contains a deletion in the E1B55K coding sequence This prevents the expression of a E1B55K product capable of binding p53 and is claimed to result in preferential replication of the virus in p53 deficient tumor cells However, a number of reports, in addition to data presented below , have brought the replication specificity of the ElB55K-defectιve viruses for p53 deficient tumor cells into question Goodrum and Ornelles (1997) J Virol 71, 548-561 ha\ e suggested that the E1B55K proteins relieve growth restπctions imposed on
replication by the cell cycle and that the ability of E1B55K mutant viruses to replicate is not mediated by the status of p53 In addition, other studies have suggested that the interaction between p53 and E1B55K may be required for efficient viral replication (Ridgway et al (1997) Virology 237 404-413) Together
these observations suggest that E1B55K mutant viruses are growth defective in all cell types and do not preferentially target p53 defective tumor cells for selective cell killing.
Alternative to the idea of selectively replicating vector is the employment of a replication deficient adenoviral vector containing extensive elimination of El function. In particular, vectors containing elimination of El, E2, E3 and partial E4 deletions have been employed to delivery exogenous transgenes. Such vectors have been employed to deliver the p53 gene to target cells. It has been demonstrated that the expression of an exogenously administered wild type p53 m a p53 deficient (p53 mutated or p53 null) tumor cell is capable of inducing p53-medιated apoptosis in the tumor cell. Such viral vectors for the delivery of p53 are currently under development Scheπng Corporation and Introgen Corporation. Again, these vectors have demonstrated acceptable toxicology profiles and therapeutic efficacy for human therapeutic applications and are in Phase II clinical tπals in humans for the treatment of p53 related malignancies Replication deficient and selectively replicating vectors have, at least in theory, design drawbacks which are of concern to clinicians Because the replication deficient vectors will not propagate uncontrollably in the patient, they have a more theoretically appealing safety profile However, as effective tumor elimination requires the infection of the substantial majoπty of the tumor cells being infected, a substantial molar excess of vector is commonly used to insure therapeutic effectiveness
Replicating vectors are viewed as being more of an issue from a safety perspective because of their ability to replicate and potentially infect non-target (normal) cells. However, by exploiting the natural ability to the virus to propagate enables these vectors to spread to suπounding tumor cells. Since the vectors themselves are able to replicate, a lower initial dose of such vectors is required This is favorable from an immunological perspective as well as for economic reasons in the manufacture of such agents \
SUMMARY OF THE INVENTION
The present invention provides a replication competent recombinant adenoviral vector containing a p53 gene under control of a constituitive viral or cellular promoter wherein said vector contains deletions in the E1B55K and/or E1B19K proteins. The
invention further provides pharmaceutical formulations and methods of use of same. The present invention also provides method of making such vectors and formulations.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 are diagrammatic representations of the adenoviral vectors used in the expenments presented herein. Ad5WT represents a wild-type adenovirus type 5 genome. dl55K/CMVp53 descπbes a recombinant adenovirus m which the CMV-p53 expression cassette is inserted into the adenovirus type 5 genome corresponding to a deletion in nucleotides 2247-3272 of the EIB gene. ElBdl55K descπbes a recombinant adenovirus which contains a deletion nucleotides 2247-3272 of the EIB gene rAdp53 is a recombinant adenovirus containing deletions m El and protein IX function descπbed in Wills, et al. (1994) Human Gene Therapy as ACN53
Figure 2 is recovered DNA from SK-BR3 cells infected with a one hour pulse of the indicated viruses at a two different concentrations, 1.8 x 108 and 1.8 x 109 particles/ml and harvested approximately 48 hours later according to the procedures of Example 2 herein. Fragmented DNA is seen in those lanes representative of apoptosis The figure demonstrates that the FAIC vector induces substantial apoptosis in the SK-BR3 tumor cells at both concentrations of virus.
Figure 3 is recovered DNA from NCI H358 lung non-small cell carcinoma line (p53null, pRb+) cells infected with a one hour pulse of the indicated viruses at 1.8 x 109 particles/ml and harvested approximately 48 hours later according to the procedures of Example3 herein The characteristic smear of fragmented DNA is seen in those lanes representative of apoptosis The figure demonstrates that the FAIC vector induces substantial apoptosis in the NCI H358 tumor cells.
Figure 4 is an agarose gel stained with ethidium bromide of a Hindlll digest of recovered DNA from cells infected with the FAIC vector for a peπod of 48 hours according to the procedures of Example 4 herein The upper panel represents results of expenments performed with SK-BR3 tumor cells The lower panel contains data generated from a similar experiment performed in NCI H358 tumor cells The results presented demonstrate that replication competent wild-type Ad5 (Ad5WT), replication competent ElBdl55K (ZAZA) virus and replicaton competent dl55K/CMVp53 (FAIC)
virus all replicate their viral DNA well while the replication deficient adenovirus control (rAdcon) and the replication deficient vector encoding p53 (rAdp53) does not.
Figure 5 is a graphical representation of the data obtained in vivo in a PC-3 mouse tumor model according to the procedures of Example 5 herein Tumor volume is plotted on the vertical axis and days following administration is plotted on the hoπzontal axis. As can be seen from the data presented, the virus dl55K/CMVp53 (cFAIC) was able to produce tumor regression in an in vivo mouse model of human cancer.
DETAILED DESCRIPTION OF THE INVENTION The present invention provides a replication competent recombinant adenovirus containing a constituitive viral or cellular promoter operably linked to a p53 gene, wherein said vector is defective m E1B55K function.
I Replication Competent Recombinant Adenovirus:
The term "replication competent" is made m reference to a virus which is capable of replicating its genome and packaging the replicated viral genome into infectious particles in mammalian cells It should be noted that the term "replication competent" does not generally apply to virus that can only be grown in cells which have been modified to provide deleted adenoviral functions in trans.
The term "recombinant adenovirus" refers to an adenovirus modified by conventional recombinant DNA technology The term "adenovirus" is synonomous with the term "adenoviral vector" and refers to viruses of the genus adenoviridiae. The term adenoviπdiae refers collectively to animal adenoviruses of the genus mastadenovirus including but no limited to human, bovine, ovme, equine, canine, porcine, uπne and simian adenovirus subgenera In particular, human adenoviruses includes the A-F sugenera as well as the individual serotypes thereof the individual serotypes and A-F subgenera including but not limited to human adenovirus types 1, 2, 3, 4, 4a, 5, 6, 7, 8. 9, 10, 1 1 (Adl lA and Ad I IP), 12, 13,14,15,16,17,18,19, 19a. 20, 21, 22, 23. 24. 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34a, 35, 35p, 36, 37, 38, 39, 40, 41, 42. 43, 44. 45, 46, 47, 48, and 91 The term bovine adenoviruses includes but is not limited to bovine adenovirus types 1,2,3,4,7, and 10.
The term canine adenoviruses includes but is not limited to canine types 1 (strains CLL, Glaxo, RI261, Utrect, Toronto 26-61) and 2. The term equine adenoviruses includes but is not limited to equine types 1 and 2. The term porcine adenoviruses includes but is not limited to porcine types 3 and 4. The term recombinant adenovirus also includes chimeπc (or even multimeπc) vectors, i.e. vectors constructed using complementary coding sequences from more than one viral subtype. See, e.g. Feng, et al. Nature Biotechnology 15:866-870.
In the preferred practice of the invention as exemplified herein, the recombinant adenoviral vector is deπved from genus adenoviπdiae. Particularly preferred viruses are denved from the human adenovirus type 2 or type 5. In the preferred practice of the invention as exemplified herein, the preferred vector is deπved from the human adenoviπdiae More preferred are vectors deπved from human adenovirus subgroup C Most prefeπed are adenoviral vectors deπved from the human adenovirus serotypes 2 and 5 The function of the E3 region may be retained, but is preferably deleted In the most preferred practice of the invention the virus is deπved human adenovirus Type 5 ( tAd5) dl309, dl327 or dl520
II Operably Linked
The term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship A nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcπption of the coding sequence Operably linked means that the nucleotide sequences being linked are typically contiguous However, as enhancers generally function when seperated from the promoter by several kilobases and intronic seqences may be of vaπable lengths, some polynucleotide elements may be operably linked but not directly flanked and may even function in trans from a different allele or chromosome
III p53 Gene
The term "p53 gene" refers to the DNA sequence encoding the wild type p53 protein, modified p53 protein, targeted p53 proteins and p53 pathway genes. The term
"wild type p53 gene" is the DNA sequence encoding the full length p53 tumor suppressor gene The term "wild type p53 gene" includes DNA sequences encoding p53 molecules deπved from human as well as other mammalian sources such as porcine p53, equine p53, bovine p53, canine p53, etc The term p53 includes p53 molecules deπved from human as well as other mammalian sources such as porcine p53, equine p53, bovine p53, canine p53, etc
The term "modified p53 protein" refers to modifications and or deletions to the above referenced genes so as to encode functional subfragments of the wild type protein Such modifications include deletions and/or changes in ammo acid coding sequence so as to produce a protein deficient in binding to its substrate. Examples of such modified p53 proteins include modifications to p53 to increase nuclear retention, deletions such as the Δ13-19 amino acids to eliminate the calpain consensus cleavage site, modifications to the o gomeπzation domains (as descπbed in Bracco, et al PCT published application WO97/0492 or United States Patent No 5,573,925) Alternatively, the p53 sequence may be modified to replace the endogenous tetramenzation domains with a leucine zipper oligermization domain Furthermore, the p53 molecule may be truncated as descπbe in
The term "targeted p53 protein" refers to a p53 protein or modified p53 protein which has been further modifed to introduce a targeting moiety such as a signal peptide or nuclear localization signal (NLS) to the native or modified Examples of such targed p53 protines are fusion proteins of the wild-type or modified p53 protein with the herpes simplex virus type 1 (HSV-1 ) structural protein, VP22 Fusion proteins containing the VP22 signal, when synthesized in an infected cell, are exported out of the infected cell and efficiently enter suπounding non-infected cells to a diameter of approximately 16 cells wide See, e g Elliott, G & O'Hare, P Cell 88.223- 233 1997, Marshall, A & Castel no A Research News Bπefs Nature Biotechnology 15 205 1997, O'Hare et al PCT publication WO97/05265 published February 13. 1997 A similar targeting moiety denved from the HIV Tat protein is descπbed in Vives, et al (1997) J Biol Chem 272 16010-16017) and may also be employed
IV . Con sti tui ti ve Viral or Cel lul ar Promoters :
The term "constituitive viral or cellular promoters" refers to a DNA sequence generally active in a mammalian cell which is not temporally regulated in its expression pattern. Examples of viral constituitive promoters include the cytomegalovirus immediate early (CMV) promoter, Rous Sarcoma Virus Long Terminal Repeat (RSV LTR), simian virus (SV) 40. Examples of constituitive cellular promoters include the elongation factor alpha (EF) promoter, the phospho glycerate kinase (PGK) promoter and the like.
V. Defective in E1B55K Function: The term "defective in E1B55K function" refers to the introduction of modifications to the E1B55K gene so as to eliminate its ability to bind p53 and or inactivate the transcription stimulating activity of p53. This may include complete elimination of theElB55K coding sequence. As previously indicated the E1B55K protein binds to p53. Consequently, in order to enhance the effect of the p53 introduced by the viral vector it is preferred to eliminate p53 binding described in
McCormick, United States Patent No. 5,677,178 issued October 14, 1997, the entire teaching of which is herein incorporated by reference. In the preferred practice of the invention as exemplified herein, recombinant adenovirus contains a deletion of nucleotides 2247 to 3272 of the adenoviral genome to eliminate the function of the E1B55K protein.
VI. Demon strati on of Ef f i c ac y I n V i tro :
In order to demonstrate the efficacy of the vectors of the present invention, the vectors were evaluated first using in vitro experiments. The in vitro experiments presented below were designed to determine the potential of the vectors to ablate tumor cells as compared to wild type virus and several other vector constructions. The following vectors were evaluated in compaπtive studies: wild type adenovirus type 5 (Ad5wt): a mutant adenovirus (dl55K/CMVp53) which contains a deletion of nucleotides 2247 to 3272 of the adenoviral genome to eliminate production of a functional E1B55K protein and contains an expression cassette with the wild type human p53 gene operably linked to the cytomegalovirus promoter (CMV) prepared in
substantial accordance with the teaching of Example 1 herein; the mutant adenovirus ElBdl55K which contains deletion of nucleotides 2247 to 3272 of the adenoviral genome to eliminate production of a functional E1B55K protein; the replication defective adenovirus vector ACN53 (Wills, et al. (1994) Human Gene Therapy 5:1079-1088) in which the complete El region is replaced with the p53 gene under control of the CMV promoter; and a control vector which contains the El region deletion of ACN53 but does not encode a therapeutic transgene (ZZCB).
These vectors were assayed for their ability to replicate in and induce apoptosis in two tumor cell lines, the SK-BR3 and NCI H358 tumor cell lines. The procedures for these expenments are found m Examples 2 through 4 herein and the data is presented in Figure 2,3. and 4 of the attached drawings. As indicated by the data presented, the recombinant adenoviral vectors of the present invention are capable of replication and the induction of substantial apoptosis in tumor cell lines.
VII Demonstration of Efficacy in Vivo: The ability of the dJ55K/CMVp53 virus to effectively eliminate tumor cells in vitro was confirmed m an in vivo animal model The details of these expeπment are contained in Example 5 herein The results of these expenments are presented m Table 1 below and Figure 5 of the attached drawings The replication competent recombinant adenovirus expressing p53 from the CMV promoter (dJ55K/CMVp53) produced tumor cell killing and elimination equivalent to wild-type adenovirus The recombinant replication deficient adenovirus (rAdp53) did not results in tumor regression and tumor mass remained in all animals treated
Day 27 Post Initiation of Treatment, 2 Treated/saline control
Consequently, the in vitro data was confirmed in an in vivo model of a human prostate tumor. The foregoing results demonstrate the efficiency and superiority of the vectors of the present invention relative to previously described vectors. This data demonstrates that the vectors of the present invention are therapeutically viable for the ablation of tumor cells in vivo in mammals including human beings.
VIII. Additional Modifications to the Virus:
The present invention also provides replication competent recombinant adenoviruses containing additional modifications to the viral genome such as targeting modifications, modifications to make the vectors seletively replicating in particular cell types or phenotypic states, controlled expression characteristics, suicide genes or additional toxic elements to enhance cytotoxicity. However, this is not meant to imply that other modifications to the viral genome may not also be included.
The term "selectively replicating" refers to a recombinant viral vector capable of preferential replication in one cell type versus another cell type, in a cell in one phenotypic state relative to another phenotypic state, or in a given cell type in response to an external stimuli. Selective replication is achieved by the use of replication control elements. The term "replication control elements" refers to DNA sequences inserted into the viral genome or modifications to the viral genome in order to produce recombinant viral vectors which selectively replicate in one cell type versus another cell type, in a cell in one phenotypic state relative to another phenotypic state (cell state specific), or in a given cell type in response to an external stimuli (inducible). Examples of such replication control elements include cell-type specific promoter, cell state specific promoters, and inducible promoters.
Cell type specific replication may be achieved by the linkage of a cell type specific promoter to an early viral gene such as the El, Ela, E2 or E4 gene when the virus is selected from the adenovirus genome. The term "cell type specific promoter" refers to promoters which are differentially activated in as a result of cell cycle progression or in different cell types. Examples of cell-type specific promoters includes cell cycle regulatory gene promoters, tissue specific promoters or pathway responsive promoters.
The term "cell cycle regulatory gene promoters" descπbe promoters for genes which are activated substantially upon entry into S-phase. Examples of such promoters include the E2F regulated promoters (e.g. DHFR, DNA polymerase alpha, thymidylate synthase, c-myc and b-myb promoters). Tissue specific promoters are well known in the art and include promoters active preferentially in smooth muscle (α-actm promoter), pancreas specific (Palmiter, et α/.(1987) Cell 50:435), liver specific Rovet, et.al. (1992) J. Biol. Chem.. 267.20765, Lemaigne, et al. (1993) J. Biol. Chem.. 268: 19896; Nitsch, et al. (1993) Mol. Cell. Biol. 13:4494), stomach specific (Kovarik, et al. (1993) J. Biol. Chem.. 268-9917, pituitary specific (Rhodes, et al. (1993) Genes Dev. 7:913, prostate specific (United States Patent 5,698,443, Henderson, et α/.issued December 16, 1997), etc.
The term "pathway-responsive promoter" refers to DNA sequences that bind a certain protein and cause nearby genes to respond transcπptionally to the binding of the protein in normal cells. Such promoters may be generated by incorporating response elements which are sequences to which transcπption factors bind. Such responses are generally inductive, though there are several cases where increasing protein levels decrease transcπption Pathway-responsive promoters may be naturally occurnng or synthetic Pathway-responsive promoters are typically constructed in reference to the pathway or a functional protein which is targeted. For example, a naturally occurnng p53 pathway-responsive promoter would include transcπptional control elements activated by the presence of functional p53 such as the p21 or bax promoter. Alternatively, synthetic promoters containing p53 binding sites upstream of a minimal promoter (e.g the SV40 TATA box region) may be employed 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 consensus DNA binding motifs can readily be determined. Such consensus sequences are generally arranged as a direct or head-to-tail repeat separated by a few base pairs Elements that include head-to-head repeats (e.g AGGTCATGACCT) are called palindromes or inverted repeats and those with tail-to- tail repeats are called everted repeats
Examples of pathway-responsive promoters useful in the practice of the present
invention include synthetic insulin pathway-responsive promoters containing the consensus insulin binding sequence (Jacob, et al. (1995). J. Biol. Chem. 270:27773- 27779), the cytokine pathway-responsive promoter, the glucocorticoid pathway- responsive promoter (Lange, et α/.(1992) J Biol. Chem. 267: 15673-80), IL1 and EL6 pathway-responsive promoters (Won K.-A and Baumann H. (1990) Mol.Cell.Biol. 10: 3965-3978), T3 pathway-responsive promoters, thyroid hormone pathway- responsive promoters containing the consensus motif: 5' AGGTCA 3.', the TPA pathway-responsive promoters (TREs), TGF-β pathway-responsive promoters (as descπbed in Grotendorst, et al.(\996) Cell Growth and Differentiation 7: 469-480). Additionally, natural or synthetic E2F pathway responsive promoters may be used.
An example of an E2F pathway responsive promoter is descπbed m Parr, et al. (1997, Nature Medicine 3: 1145-1149) which descπbes an E2F-1 promoter containing 4 E2F binding sites and is reportedly active in tumor cells with rapid cycling. Examples of other pathway-responsive promoters are well known in the art and can be identified in the Database of Transcπption Regulatory Regions on Eukaryotic Genomes accessible through the internet at http://www.eimb.rssi.ru/TRRD.
For example, in one embodiment of the invention the vector compπses a synthetic TGF-β pathway-responsive promoter active in the presence of a functional TGF-β pathway such as the promoter containing SRE and PAI pathway responsive promoters PAI refers to a synthetic TGF-β pathway-responsive promoter compnsmg sequences responsive to TGF-β signally isolated from the plasminogen activator-I promoter region. The PAI-path way-responsive promoter may be isolated as a 749 base pair fragment isolatable from the plasmid p8001uc (as descπbed in Zonneveld, et al. (1988) PNAS 85:5525-5529 and available from GenBank under accession number J03836) SRE refers to a synthetic TGF-β response element compnsing a repeat of 4 of the Smad-4 DNA binding sequences (GTCTAGAC as descπbed in Zawel, et al (1988) Mol Cell 1 :61 1-617. The SRE response element may be generated by annealing complimentary ohgonucleoϋdes encoding the Smad-4 binding sequences and cloning in plasmid pGL#3 - promoter luciferase vector (commercially available from ProMega)
Tissue specific promoters are well known in the art and include promoters active preferentially m smooth muscle (alpha-actm promoter), pancreas specific (Palmiter, et al. (1987) Cell 50:435), liver specific Rovet, et.al. (1992) J. Biol. Chem.. 267:20765; Lemaigne, et al . (1993) J. Biol. Chem. 268.19896; Nitsch, et al . (1993) Mol. Cell. Biol. 13-4494), stomach specific (Kovaπk, et al . (1993) J. Biol. Chem.. 268:9917, pituitary specific (Rhodes, et al . (1993) Genes Dev. 7:913, prostate specific antigen promoter (United States Patent No etc
Examples of different phenotypic states would include the neoplastic phenotype versus a normal phenotype in a given cell type Selective replication is achieved by the use of viral replication control elements The term viral replication control element refers to a DNA sequence engineered into the vector of the present invention such that the virus is preferentially enabled to replicate the viral genome in a particular type of target cell.
In order to achieve expression of the adenovirus in tumor cells, one may employ a tumor specific promoter to dn ve expression of an early gene. The term "tumor specific promoters" refers to promoters which are active in tumor cells and inactive in cells which are not transformed Examples of tumor specific promoters include the alpha-fetoprotein promoter, the tyrosinase promoter The use of tumor specific promoters to achieve conditional replication of adenoviral vectors is descπbed in co-pending United States Patent Application 08/433,798 filed May 3, 1995 and International Patent Application No PCT/US96/06199 published as International Publication No WO 96/34969 on November 7, 1996 the entire teaching of which is herein incorporated by reference
For example the alpha-fetoprotein promoter could be used to replace the endogenous E4 promoter and achieve greater selectivity in conditional replication Other factors such as NF-IL6 can substitute for Ela in regulating Ela responsive promoters in the adeno\ιrus in the absence of EIA function (Speιgel,M et al (1992) J. Virol 66.1021-1030) and this can be avoided by substitution of the E4 promoter with a tumor specific promoter Although one may use the pathway responsive promoter to dπve replication of the virus in the presence of a functional pathway, alternatively, the one may use a
pathway responsive promoter to drive expression of a repressor of viral replication to control expression. The term "repressor of viral replication" refers to a protein, if expressed in a given cell, substantially represses viral replication. As will be appreciated by those of skill m the art, the repressor of viral replication will be dependent on the nature of the parent adenoviral vector from which the recombinant vector of the present invention is deπved. For example, in the case of adenoviral vectors or other DNA tumor viruses, the E2F-Rb fusion construct as descπbed in European Patent Application No. 94108445.1 published December 6, 1995 (Publication number. 0 685 493 Al) may be employed. E2F-Rb fusion protein consists of the DNA binding and DPI heterodimeπzations domains of the human E2F transcπption factor protein (amino acids 95-286 of wild type E2F) fused to the Rb growth suppression domain (amino acids 379-928 of the wild type Rb protein) The E2F-Rb fusion protein is a potent repressor of E2F-dependent transcπption and arrests cells in Gl. The DNA binding domain is located at amino acids 128-193 and the dimeπzation domain is located at 194-289 The sequence of the human E2F-1 protein is available from GenBank under accession number M96577 deposited August 10, 1992 The sequences of E2F from other E2F family members of E2F from other species may be employed when constructing a vector for use in other species In the situation where the recombinant virus is based on adenoassociated virus, the rep protein and its deπvates is an effective repressor of viral replication in the absence of adenovirus infection In the situation where the virus is denved from herpes simplex virus, the ICPO-NX, a deleted form of the immediate early protein ICPO (Liun, et al (1998) J Virol 72 7785-7795), protein may be used as an effective repressor of viral replication Similarly, any protein w ith dominant negative activity can be used as a repressor of viral replication
The term "inducible promoter' refers to promoters which facilitate transcnption of the therapeutic transgene preferable (or solely) under certain conditions and/or in response to external chemical or other stimuli Examples of inducible promoters are known in the scientific literature (See. e g Yoshida and Hamada (1997) Biochem Biophys Res Comm 230 426-430, Iida, et al . (1996) J. Virol 70(9):6054-6059, Hwang, et al (1997) J Virol 71(9) 7128-7131 , Lee, et al (1997) Mol Cell. Biol
17(9):5097-5105; and Dreher, et al . (1997) J. Biol. Chem. 272(46); 29364-29371. Examples of radiation inducible promoters include those induced by ionizing radiation such as the Egr-1 promoter (as described in Manome, et al. (1998) Human Gene Therapy 9: 1409-17; Takahashi, et al . (1997) Human Gene Therapy 8:827-833; Joki. et al. Human Gene Therapy (1995) 6:1507-1513; Boothman, et al (1994) volume 138 supplement pages S68-S71; and Ohno, T (1995) Tanpakushitsu Kakusan Koso 40:2624-2630), X-ray inducible promoters such as the XRE promoter (as descrbed in Boothman, et al . (1994) Radiation Research 138(Suppl.l):S68-S71), and UV inducible promoters such as those isolated from Clostridium perfήngens (Gamier and Cole (1988) Mol. Microbiol. 2:607-614.
Addtionally modfications to increase the potency of the vectors of the present invention include but are not limited to alterations within EIB. The vectors of the present invention may be modified to introduce mutations in E4 to increase the cytotoxicity (Muller, et al. (1992) J. Virol. 66:5867-5878) or contain upregulation of viral death proteins such as E4orf4 or E3 1 1.6K proteins. For example, the E4 region of the adenovirus genome has been implicated in viral DNA replication, host protein synthesis shut off and viral assembly. E4orf6 is sufficient for DNA replication and late protein synthesis in immortalized cells. However, E4orf6/7 appears to be required for replication in non-dividing cells. Consequently elimination of E4orf6/7 assists in restricting the replication of the virus in immortalized (i.e. tumor cells) and may be incorporated into the vectors of the present invention.
Furthermore. E4 deletions have been shown to reduce the immongenicity of the vectors (Wang, et al. (1997) Gene Therapy 4:393-400; Dedieu, et al. (1997) J. Virol 71 :4626-37), but can affec the persistence of transgene expression depending on the open reading frames of E4 retained and the promoter used to drive expresion of the transgene (Armentano, et al. (1997) J. Virol 71 :2408-2416). The E4 region also encodes a protein (E4orf6) capable of binding to and inactivating the transciptional activity of p53. Dubner. et al. ( 1996) Science 272:1470-73). Therefore, it may be desirable to modify the E4 region to delete those open reading frames with undesriable properties for the particular virus construct while retina those with desired properties. For example, for the conditionally replication virus described
herein as the ElBdl55K-MLP-p53, the E4 orfό region may be deleted to reduce inactivation of p53 while retaining E4orf3 to allow continued expression of p53 and replication of the virus. In order to preserve replication competency of the virus, one of E4orf6 and E4orf3 must be retained. Examples of other E4 deleted adenoviral vectors are described in Gregory, et al United States Patent No 5,670.488 issued September 23, 1997.
The E3 region of the adenovirus encodes proteins which help adenivrally infected cells avoid clearance by the immune system (Wold, et al (1995) Curr. Top. Microbiol. Immunol. 199:237-274). Upregulation of this region and subfragments thereof has been shown to prevent or decrease the immune response to virally infected cells, leading to longer term gene expression. (Ilan, et al (1997) PNAS 94:2587:2592, Bunder, et al (1997) J. Virol. 71 :7623-28). Therefore, modifications to the E3 region (or sub-components thereof) to overexpress their proteins (e.g. by upregulating the E3 region using a strong constituitive promoter such as CMV) may be desirable to allow for a greater degree of viral replication due to its ability to avoid or delay the immune mediated clearance of infected cells.
The recombinant adenoviral vectors of the present invention may also be modifed to be defective in E1B19K function. The term "defective in E1B19K function" refers to the introduction of modifications to the E1B19K gene so as to eliminate its ability of the E1B19K protein to bind to bcl-2 family members and/or its ability to interact with FADD and CED4. This may include complete elimination of theElB19K coding sequence. Alternatively, one may delete only portions of the E1B19K coding sequence to produce a virus defective in E1B19K function. In the prefeπed practice of the invention, a modification is introduced into the viral genome to delete nucleotides 1682 to 3534 to eliminate both E1B55K and E1B19K function.
The replication competent recombinant adenoviruses may also be optionally modified to possess "targeting modifications" in order to achieve preferential targeting of the virus to a particular cell type. The term "targeting modification" refers to modifications to the viral genome designed to result in preferential mfectivity of a particular cell type. Cell type specificity or cell type targeting may also be achieved in vectors deπved from viruses having characteristically broad mfectivities such as
adenovirus by the modification of the viral envelope proteins. For example, cell targeting has been achieved with adenovirus vectors by selective modification of the viral genome knob and fiber coding sequences to achieve expression of modified knob and fiber domains having specific interaction with unique cell surface receptors. Examples of such modifications are descπbed in Wickham, et al (1997) J. Virol 71(ll):8221-8229 (incorporation of RGD peptides into adenoviral fiber proteins); Arnberg, et al. (1997) Virology 227:239-244 (modification of adenoviral fiber genes to achieve tropism to the eye and genital tract); Hams and Lemoine (1996) TIG 12(10):400-405; Stevenson, et al. (1997) J Virol. 71(6):4782-4790; Michael, et α/.(1995) Gene Therapy 2.660-668 (incorporation of gastnn releasing peptide fragment into adenovirus fiber protein), and Ohno, et al (1997) Nature Biotechnology 15:763- 767 (incorporation of Protein A-IgG binding domain into S dbis virus) Other methods of cell specific targeting have been achieved by the conjugation of antibodies or antibody fragments to the envelope proteins (see, e.g. Michael, et al. (1993) J. Biol. Chem. 268-6866-6869. Watkins, et al (1997) gene therapy 4:1004-1012, Douglas, et al (1996) Nature Biotechnology 14: 1574-1578 Alternatively, particular moieties may be conjugated to the viral surface to achieve targeting (See, e.g. Nilson, et al (1996) gene therapy 3.280-286 (conjugation of EGF to retroviral proteins). These recombinantly modified \ectors may be produced in accordance with the practice of the present invention
In some instances, it may be desirable to include a suicide gene in the adenovirus This provides a "safety valve" to the viral vector delivery system to prevent widespread infection due to the spontaneous generation of replication competent viral vectors The term "suicide gene" refers to a nucleic acid sequence, the expression of which renders the cell susceptible to killing by external factors or causes a toxic condition in the cell A well known example of a suicide gene is the thymidine kinase (TK) gene (see e g Woo, et al United States Patent No. 5,631,236 issued May 20, 1997 and Freeman, et al United States Patent No 5,601,818 issued February 11, 1997) in hich the cells expressing the TK gene product are susceptible to selective killing by the administration of gancyclovir
IX Selectively Replicating
The term "selectively replicating" refers to a recombinant viral vector capable of preferential replication m one cell type versus another cell type (cell-type specific), a cell in one phenotypic state relative to another phenotypic state (cell state specific), or m a given cell type in response to an external stimuli (inducible)
Cell type specific replication may be achieved by operably linking a cell type specific promoter to an early adenoviral gene such as the El, EIA or E2 gene The term "cell type specific promoter" refers to promoters which are differentially activated in as a result of cell cycle progression or in different cell types Examples of cell-type specific promoters include cell cycle regulatory gene promoters, tissue specific promoters or pathway responsive promoters
The term "cell cycle regulatory gene promoters" descπbe promoters for genes which are activated substantially upon entry into S-phase. Examples of such promoters include the E2F regulated promoters (e.g DHFR, DNA polymerase alpha, thymidylate synthase. c-myc and b-myb promoters)
The term "tissue specific promoter" are promoters which are substantially more active in one tissue type versus another tissue type within the same organism Tissue specific promoters are well known in the art and include promoters active preferentially in smooth muscle ( -actin promoter), pancreas specific (Palmiter, et al (1987) Cell 50 435). ver specific Rovet, et al (1992) J Biol Chem 267 20765, Lemaigne, et al ( 1993) J Biol Chem 268 19896, Nitsch, et al (1993) Mol Cell Biol 13-4494), stomach specific (Kovank, et al ( 1993) J Biol Chem . 268-9917, pituitary specific (Rhodes, et al (1993) Genes Dev 7 913, prostate specific (United States Patent 5,698,443, Henderson, et α/.issued December 16, 1997), etc The term "pathway-responsive promoter" refers to DNA sequences that bind a certain protein and cause nearby genes to respond transcπptionally to the binding of the protein in normal cells Such promoters may be generated by incorporating response elements which are sequences to which transcnption factors bind Such responses are generally induc e. though there are several cases where increasing protein levels decrease transcπption Pathway-responsive promoters may be naturally occurnng or
synthetic. Pathway -responsive promoters are typically constructed in reference to the pathway or a functional protein which is targeted. Synthetic pathway-responsive promoters are generally constructed from one or more copies of a sequence that matches a consensus binding motif. Such consensus DNA binding motifs can readily be determined. Such consensus sequences are generally arranged as a direct or head- to-tail repeat separated by a few base pairs.
Examples of pathway-responsive promoters useful in the practice of the present invention include synthetic insulin pathway-responsive promoters containing the consensus insulin binding sequence (Jacob, et al. (1995). J. Biol. Chem. 270:27773- 27779), the cytokine pathway-responsive promoter, the glucocorticoid pathway- responsive promoter (Lange, et α/.(1992) J Biol. Chem. 267: 15673-80), LL1 and IL6 pathway-responsive promoters (Won K.-A and Baumann H. (1990) Mol.Cell.Biol. 10: 3965-3978), T3 pathway-responsive promoters, thyroid hormone pathway- responsive promoters containing the consensus motif: 5' AGGTCA 3.', the TPA pathway-responsive promoters (TREs), TGF-β pathway-responsive promoters (as described in Grotendorst, et α/.(1996) Cell Growth and Differentiation 7: 469-480). Additionally, natural or synthetic E2F pathway responsive promoters may be used. An example of an E2F pathway responsive promoter is described in Parr, et al. (1997, Nature Medicine 3: 1145-1 149) which describes an E2F-1 promoter containing 4 E2F binding sites and is reportedly active in tumor cells with rapid cycling. Examples of other pathway-responsive promoters are well known in the art and can be identified in the Database of Transcπption Regulatory Regions on Eukaryotic Genomes accessible through the internet at http://www.eimb.rssi.ru/TRRD.
In one embodiment of the invention, the vector comprises a synthetic TGF-b pathway-responsive promoter active in the presence of a functional TGF-β pathway such as the promoter containing SRE and PAI pathway responsive promoters. PAI refers to a synthetic TGF-β pathway-responsive promoter comprising sequences responsive to TGF-β signally isolated from the plasminogen activator-I promoter region. The PAI-pathway-responsive promoter may be isolated as a 749 base pair fragment isolatable from the plasmid p8001uc (as described in Zonneveld, et al. (1988) PNAS 85:5525-5529 and available from GenBank under accession number J03836).
SRE refers to a synthetic TGF-β response element compπs g a repeat of 4 of the Smad-4 DNA binding sequences (GTCTAGAC as descπbed in Zawel, et al. (1988) Mol. Cell 1:611-617. The SRE response element may be generated by annealing complimentary o gonucleotides encoding the Smad-4 binding sequences and cloning in plasmid pGL#3 - promoter luciferase vector (commercially available from ProMega).
The use of the term "cell state specific replication" is used herein to refer to an adenovirus has been modified such that it preferentially replicates in response to a phenotypic condition within a cell. Examples of different phenotypic states may include the neoplastic phenotype versus a normal phenotype in a given cell type. Alternatively, the phenotypic state may be a rapidly dividing phenotype versus a quiescent cell.
In order to achieved selective replication of the adenovirus in rapidly dividing cells, the adenovirus may contain modifications to the EIA coding sequence so as to produce EIA gene products which are deficient in binding to one or more p300 protein family members and one or more Rb protein family member protein but retain the transactivatmg function of the EIA CR3 domain and a deletion of the amino acids from approximately 219 to approximately 289 of the EIA 289R protein (or approximately amino acids 173 to approximately amino acid 243 of the El A 243R protein In the preferred practice of the invention the deletion of the binding to the p300 family members is achieved by introducing a deletion corresponding to amino acids 4-25 of the EIA 243R and 289R proteins or amino acids 38-60 of the EIA 243R and 289R proteins In the preferred practice of the invention the deletion of the binding to the pRb family members is achieved amino acids 111-123 of the EIA 243R and 289R proteins Alternatively, deletion of the binding to the pRb family members may be achieved by eliminate of amino acids 124-127 of the EIA 243R and 289R proteins
In order to achiev e expression of the adenovirus in tumor cells, one may employ a tumor specific promoter to dπve expression of an early gene The use of tumor specific promoters to achieve conditional replication of adenoviral vectors is descπbed in co-pending United States Patent Application 08/433,798 filed May 3, 1995 and International Patent Application No PCT/US96/06199 published as
International Publication No. WO 96/34969 on November 7, 1996 the entire teaching of which is herein incorporated by reference.
A phenotypic state which may be present may be a difference in a functional pathway capable of being responded to through the use of pathway responsive promoters. Although one may use the pathway responsive promoter to drive replication of the virus m the presence of a functional pathway as described above, one may alternatively use a pathway responsive promoter to drive expression of a repressor of viral replication to control expression. The term "repressor of viral replication" refers to a protein, if expressed in a given cell, substantially represses viral replication. As will be appreciated by those of skill in the art, the repressor of viral replication will be dependent on the nature of the parent adenoviral vector from which the recombinant vector of the present invention is deπved. For example, in the case of adenoviral vectors or other DNA tumor viruses, the E2F-Rb fusion construct as described in European Patent Application No. 94108445.1 published December 6, 1995 (Publication number. 0 685 493 Al) may be employed. E2F-Rb fusion protein consists of the DNA binding and DPI heterodimeπzations domains of the human E2F transcπption factor protein (amino acids 95-286 of wild type E2F) fused to the Rb growth suppression domain (amino acids 379-928 of the wild type Rb protein). The E2F-Rb fusion protein is a potent repressor of E2F-dependent transcπption and arrests cells in Gl . The DNA binding domain is located at amino acids 128-193 and the dimeπzation domain is located at 194-289. The sequence of the human E2F-1 protein is available from GenBank under accession number M96577 deposited August 10, 1992. The sequences of E2F from other E2F family members of E2F from other species may be employed when constructing a vector for use in other species. In the situation where the recombinant virus is based on adenoassociated virus, the rep protein and its denvates is an effective repressor of viral replication in the absence of adenovirus infection. In the situation where the virus is deπved from herpes simplex virus, the ICPO-NX. a deleted form of the immediate early protein ICPO (Liun, et al. ( 1998) J. Virol. 72:7785-7795), protein may be used as an effective repressor of viral replication. Similarly, any protein with dominant negative activity can be used as a repressor of viral replication.
One may also achieve replication of the vectors of the present invention in respone to external stimuli. For example, an early adenoviral gene may be operably linked to an inducible promoter such that the replication is permitted in reponse to the external stimuli which activates the inducible promoter. The term "inducible promoter" refers to promoters which facilitate transcnption preferably (or exclusively) under certain conditions and/or in response to external chemical or other stimuli. Examples of inducible promoters are known in the scientific literature (See, e.g. Yoshida and Hamada (1997) Biochem. Biophys. Res. Comm. 230:426-430; Iida, et al. (1996) J. Virol. 70(9):6054-6059: Hwang, et al. (1997) J. Virol 71(9):7128-7131; Lee, et al. (1997) Mol. Cell. Biol. 17(9):5097-5105; and Dreher, et al. (1997) J. Biol. Chem. 272(46); 29364-29371. Examples of radiation inducible promoters include those induced by ionizing radiation such as the Egr-1 promoter (as described in Manome, et al. (1998) Human Gene Therapy 9:1409-17; Takahashi, et al. (1997) Human Gene Therapy 8:827-833; Joki, et al Human Gene Therapy (1995) 6: 1507-1513; Boothman, et al (1994) volume 138 supplement pages S68-S71; and Ohno, T (1995) Tanpakushitsu Kakusan Koso 40:2624-2630), X-ray inducible promoters such as the XRE promoter (as descrbed in Boothman, et al. (1994) Radiation Research 138(Suppl.l):S68-S71). and UV inducible promoters such as those isolated from Clostridium perfringens (Gamier and Cole (1988) Mol. Microbiol. 2:607-614. X Pharmaceutical Formulations:
The present invention further provides a pharmaceutically acceptable formulation of the replication competent recombinant adenoviruses in combination with a earner. The vectors of the present invention may be formulated for dose administration in accordance with conventional pharmaceutical practice with the addition of earners, excipients, and/or delivery enhancing agents. Dosage formulations may include intravenous, intratumoral, intramuscular, mtrapeπtoneal, topical, matπx or aerosol delivery formulations.
The term "earner" refers to compounds commonly used on the formulation of pharmaceutical compounds used to enhance stability, sterility and dehverability of the therapeutic compound. When the virus is formulated as a solution or suspension, the delivery system is in an acceptable earner, preferably an aqueous earner. A vanety of
aqueous earners may be used, e.g , water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be steπ zed by conventional, well known steπhzation techniques, or may be steπle filtered The resulting aqueous solutions may be packaged for use as is, or lyophihzed, the lyophihzed preparation being combined with a steπle solution pnor to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffeπng agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloπde, potassium chlonde, calcium chloπde, sorption monolaurate, tnethanolamme oleate, etc
The present invention further provides pharmaceutical formulations of the replication competent recombinant adenoviruses of the present invention with a earner and a delivery enhancing agent(s) The terms delivery enhancers" or "delivery enhancing agents" are used interchangeably herein and includes one or more agents which facilitate uptake of the virus into the target cell. Examples of delivery enhancers are descnbed in co-pending United States Patent Application Senal No 09/112,074 filed July 8, 1998 Examples of such delivery enhancing agents include detergents, alcohols, glycols, surfactants, bile salts, hepaπn antagonists, cyclooxygenase inhibitors, hypertonic salt solutions, and acetates Alcohols include for example the aliphatic alcohols such as ethanol, N-propanol. isopropanol. butyl alcohol, acetyl alcohol Glycols include glyceπne, propyleneglycol, polyethyleneglycol and other low molecular weight glycols such as glycerol and thioglycerol Acetates such as acetic acid, glucomc acid, and sodium acetate are further examples of delivery-enhancing agents Hypertonic salt solutions like 1M NaCl are also examples of de very- enhancing agents Examples of surfactants are sodium dodecyl sulfate (SDS) and lysolecithm, polysorbate 80, nonylphenoxypolyoxyethylene. lysophosphatidylcholme, polyethyleneglycol 400, polysorbate 80, polyoxyethylene ethers, polyglycol ether surfactants and DMSO Bile salts such as taurocholate, sodium tauro-deoxycholate, deoxycholate, chenodesoxycholate, glycocho c acid, glycochenodeoxycho c acid and other astπngents such as silver nitrate may be used Hepann-antagomsts like quaternary amines such as protamine sulfate may also be used Cyclooxygenase
inhibitors such as sodium salicylate, salicylic acid, and non-steroidal antnnflammatory drug (NSAIDS) like lndomethacin, naproxen, diclofenac may be used.
Delivery-enhancing agents includes compounds of the Formula I: O
H H
Xi •N- "(CH2)n N- "(CH2)n- -N- -R
C= :0
X,
wherein n is an integer from 2-8, XI is a cholic acid group or deoxychohc acid group, and X2 and X3 are each independently selected from the group consisting of a cholic acid group, a deoxychohc acid group, and a sacchaπde group. At least one of X2 and X3 is a sacchande group The sacchaπde group may be selected from the group consisting of pentose monosacchande groups, hexose monosacchaπde groups, pentose-pentose disacchaπde groups, hexose-hexose disacchaπde groups, pentose- hexose disacchaπde groups, and hexose-pentose disacchaπde groups.
The term "detergent" includes anionic, cationic, zwittenonic, and noniomc detergents Exemplary detergents include but are not limited to taurocholate, deoxycholate, taurodeoxycholate. cetylpyπdium, benalkonium chloπde, Zwιttergent3- 14 detergent, CHAPS (3-[(3-Cholamιdopropyl) dιmethylammonιol]-l- propanesulfonate hydrate), Big CHAP, Deoxy Big CHAP, Tπton-X-100 detergent, C12E8, Octyl-B-D-Glucopyranoside, PLURONIC- F68 detergent, Tween 20 detergent, and TWEEN 80 detergent (CalBiochem Biochemicals)
Unit dosage formulations of the present invention may be included in a kit of products containing the replication competent recombinant adenovirus of the present invention lyophihzed form and a solution for reconstitution of the lyophihzed product along with instructions for use Recombinant adenoviruses of the present invention may be lyophihzed by conventional procedures and reconstituted
C Calpain Inhibitors:
Another example of a delivery enhancing agent which may be employed in the formulations of present invention include calpain inhibitors. The "calpam inhibitor" (abbreviated "CI") refers to a compound which inhibits the proteolytic action of calpain-I, e.g. μ-calpains The term calpam inhibitors as used herein includes those compounds having calpain I inhibitory activity in addition to or independent of their other biological activities A wide vaπety of compounds have been demonstrated to have activity in inhibiting the proteolytic action of calpains. Examples of calpain inhibitors are useful in the practice of the present invention include N-acetyl-leu-leu- norleucinal also known as "calpam inhibitor 1 " Additional calpain inhibitors are descπbed in the following United States Patents, herein incorporated by reference, United States Patent No 5,716,980 entitled Alcohol or aldehyde deπvatives and their use, United States Patent No. 5,714,471 entitled Peptide and peptide analog protease inhibitors, United States Patent No. 5,693,617 entitled Inhibitors of the 26s proteolytic complex and the 20s proteasome contained therein, United States Patent No 5,691,368 entitled Substituted oxazolidme calpain and/or cathepsin B inhibitors; United States Patent No. 5,679,680 entitled.α -substituted hydrazides having calpain inhibitory activity. United States Patent No 5,663,294 entitled Calpain-mhibiting peptide analogs of the kininogen heavy chain. United States Patent No. 5,661,150 entitled Drug for neuroprotection. United States Patent No 5,658,906 entitled
Cysteine protease and senne protease inhibitors. United States Patent No. 5,654,146 entitled Human ice homolog. United States Patent No 5,639,783 entitled Ketone deπvatives. United States Patent No 5.635.178 entitled Inhibition of complement mediated inflammatory response using monoclonal antibodies specific for a component forming the C56-9 complex which inhibit the platelet or endothe al cell activating function of the C56-9 complex. United States Patent No. 5,629,165 Neural calcium- activated neutral protemase inhibitors. United States Patent No 5,622,981 entitled Use of metabotropic receptor agonists in progressive neurodegenerative diseases, United States Patent No 5.622,967 entitled Quinolone carboxamide Calpain inhibitors. United States Patent No 5,621 ,101 entitled Protein kinase inhibitors for treatment of neurological disorders, United States Patent No 5,554,767 entitled
Alpha-mercaptoacrylic acid deπvatives having calpain inhibitory activity; United States Patent No. 5,550,108 entitled Inhibition of complement mediated inflammatory response; United States Patent No. 5,541,290 entitled Optically pure calpain inhibitor compounds; United States Patent No. 5,506,243 entitled Sulfonamide deπvatives, United States Patent No 5,498,728 entitled Deπvatives of L-tryptophanal and their use as medicmals; United States Patent No. 5,498,616 entitled Cysteme protease and seπne protease inhibitors, United States Patent No. 5,461,146 entitled Selected protein kinase inhibitors for the treatment of neurological disorders; United States Patent No. 5,444,042 entitled Method of treatment of neurodegeneration with calpain inhibitors, United States Patent No. 5,424,325 entitled am oketone denvatives; United States Patent No 5,422,359 entitled . -ammoketone denvatives; United States Patent No 5,416.1 17 entitled Cyclopropenone denvatives, United States Patent No 5,395,958 entitled Cyclopropene denvatives; United States Patent No. 5,340,922 entitled Neural calcium-activated neutral proteinase inhibitors, United States Patent No 5,336,783 entitled Calpain inhibitor cystamidin A and its production; United
States Patent No 5,328.909 entitled Cyclopropenone deπvatives; and United States Patent No. 5,135,916 entitled Inhibition of complement mediated inflammatory response. The uses of calpam inhibitors in gene therapy protocols is further descπbed in Atencio, et al , co-pending United States Patent Applications Senal Nos. 09/172.685 and 60/104.321 filed October 15, 1998
XI Therapeutic Applications
The present invention provides a method of eliminating neoplastic cells by the administration of a recombinant adenoviral vector of the present invention to said neoplastic cell The term "neoplastic cell" is a cell displaying an aberrant growth phenotype charactenzed by independence of normal cellular growth controls. As neoplastic cells are not necessaπly replicating at any given time point, the term neoplastic cells compπse cells which may be actively replicating or in a temporary non- rep cative resting state (Gl or GO) Localized populations of neoplastic cells are refeπed to as neoplasms Neoplasms may be malignant or benign Malignant neoplasms are also referred to as cancers The term cancer is used interchangeably
herein with the term tumor Neoplastic transformation refers the conversion of a normal cell into a neoplastic cell, often a tumor cell
The present invention provides a method of ablating neoplastic cells in a mammalian organism vivo by the administration of a pharmaceutically acceptable formulation of the replication competent recombinant adenovirus descπbed above The term "ablating" means the substantial reduction of the population of viable neoplastic cells so as to alleviate the physiological maladictions of the presence of the neoplastic cells The term "substantial" means a reduction m the population of viable neoplastic cells in the mammalian organism by greater than approximately 20% of the pretreatment population The term "viable" means having the uncontrolled growth and cell cycle regulatory charactenstics of a neoplastic cell The term "viable neoplastic cell" is used hereing to distinguish said cells from neoplastic cells which are no longer capable of replication For example, a tumor mass may remain following treatment, however the population of cells compπsing the tumor mass may be dead These dead cells have been ablated and lack the ability to replicate, even though some tumor mass may remain The term "mammalian organism" includes, but is not limited to, humans, pigs, horses, cattle, dogs, cats
Preferably one employs an adenoviral vector endogenous to the mammalian type being treated However, in some instances it may be advantageous to use vectors denved from different species which possess favorable pathogenic features For example, since humans may not have been exposed to equine or ovine adenoviruses, they may have a reduced humoral immunological response to such vectors By minimizing the immune response, rapid systemic clearance of the vector is avoided resulting in a greater duration of action of the vector and greater efficacy of administration over a course of therapy pnor to any immunological response can be mounted by the organism For example, it is reported (WO 97/06826 published Apnl 10, 1997) that ovine adenoviral vectors may be used in human gene therapy to minimize the immune response characteπstic of human adenoviral vectors
In the preferred practice of the invention as exemplified herein, a recombinant adenovirus containing a deletion of the E1B55K gene function and expressing the p53 gene from a constituitive promoter (e g CMV) is formulated with a pharmaceutically
acceptable earner for administration by intravenous, mtrapentoneal, or intratumor injection. The appropnate dose and method of administration of the vector to be administered to the mammalian organism in need of treatment will be determined by the skilled artisan taking into account the extent of metastatis of the pπmary tumor, the delivery enhancer(s) included in the formulation, the extent to which the immunological response is suppressed, etc. Each of these latter factors will decrease the dosage of the vector provided to the mammalian organism in need of treatment. In the preferred practice of the invention, a dosage of approximately 1 x 105 to 1 x 1013 particles (preferably 1 x 106 to 1 x 10" particles, most preferably x 107 to 1 x 1010 particles) will be administered to the mammalian organism in one or more doses in a treatment regimen. The typical course of treatment will be the daily administration of a pharmaceutically acceptable formulation of the vector of the present invention over a penod of three to ten days, preferably five to eight days
In a further preferred practice of the invention, the pharmaceutically acceptable earner contains a delivery enhancing agent In a further preferred practice of the invention, the delivery enhancing agent is a calpain inhibitor. In the most preferred practice of the invention as exemplified herein, the recombinant adenoviral vector d]55K-CMV-p53 is formulated in a earner solution further compnsing the calpain inhibitor n-acetyl-leu-leu-norcinal (calpain inhibitor 1) at a concentration of from approximately 1 to 50 micromolar In such instances, the daily dosage may be reduced as compared to a formulation absent such delivery enhancing agents by a factor of one to two logs.
While the present invention provides a method of use of the replication competent recombinant adenoviruses alone, the replication competent recombinant adenoviruses of the present invention and formulations thereof may be employed in combination with conventional chemotherapeutic agents or treatment regimens. Examples of such chemotherapeutic agents include inhibitors of puπne synthesis (e.g., pentostatin, 6-mercaptopuπne, όthioguanine, methotrexate) or pynmidine synthesis (e.g Pala, azarbine), the conversion of πbonucleotides to deoxynbonucleotides (e.g hydroxyurea), inhibitors of dTMP synthesis (5-fluorouracιl), DNA damaging agents (e g. radiation, bleomycines, etoposide, teniposide, dactmomycine, daunorubicin,
doxorubicin, mitoxantrone, alkylating agents, mitomycin, cisplatin, procarbazme) as well as inhibitors of microtubule function (e.g vmca alkaloids and colchicme) and farnesyl protein transferase (FPT) inhibitors. Chemotherapeutic treatment regimens refers pπmanly to non-chemical procedures designed to ablate neoplastic cells such as radiation therapy.
It has been observed that the immune system is capable of recognizing and eliminating recombinant adenoviral vectors. As this would effectively reduce the quantity of adenovirus reaching the target cell, it may be advantageous to instances to administer the compounds of the present invention combination with immunosuppressive agents such as etoposide. In the preferred practice of the invention, the immunosuppressive agent is administered in advance, preferably for about a week in advance of the introduction of the recombinant viral vector of the present invention to eliminate the humoral immune response to the viral particles. In the prefeπed practice of the invention, a pharmaceutically acceptable formulation of the vector of the present invention is administered intratumorally following the administration of a immunosuppressive agent for a peπod of from about one day to about two weeks in advance of administration of the vector. The vector is preferably an adenoviral vector and further contains a deletion of the E1B-55K protein and contains an expression cassette expressing the p53 tumor suppressor gene from the CMV promoter In the prefeπed practice of the invention, the immunosuppressive agent is etoposide and is administered daily for a penod of from about 1 to 7 days (preferably 3-7 days) pnor to administration of the vector. In such instances, the daily dosage in the course of treatment is reduced in companson to those dosages provided absent such immunosuppressive agents The present invention also provides a method of ablating neoplastic cells in a population of normal cells contaminated by said neoplastic cells ex vivo by the administration of a replication competent recombinant adenovirus of the present invention to said population An example of the application of such a method is cuπently employed in ex vivo applications such as the purging of autologous stem cell products commonly known as bone marrow purging The term "stem cell product" refers to a population of hematopoietic, progenitor and stem cells capable of
reconstituting the long term hematpoietic functionvof a patient who has received myoablative therapy. Stem cell products are conventionally obtained by apheresis of mobilized or non-mobilized peπpheral blood. Apheresis is conventionally achieved through the use of known procedures using commercially available apheresis apparatus such as the COBE Spectra Apheresis System, commercially available from COBE International, 1185 Oak Street, Lakewood, CO. It is preferred that treatment conditions be optimized to achieve a "3-log purge" (i.e. removal of approximately 99 9% of the tumor cells from the stem cell produce) and most preferably a "5-log purge" (removal of approximatley 99.999% of tumor cells from the stem cell product) In the prefeπed practice of the invention, a stem cell product of 100 ml volume would be treated at a concentration of from about lxlO6 to lxlO10 particles/ml of the recombinant adenovirus of the present invention for a peπod of approximately 4 hours at 37°C
The present invention provides a recombinant viral vectors capable of recruiting immature dendπtic cells to a tumor site and exposing the dendπtic cells to a localized high concentration of tumor antigens characteπstic of the tumor present in the patient. The vectors of the present invention are specifically engineered to induce killing of tumor cells The lysed tumor cell (or the apoptotic bodies produced by an apoptosed tumor cell) provides a πch localized concentration of tumor specific proteins By introducing a gene encoding a dendπtic cell chemoattractant. immature dendπtic cells capable engulfing tumor antigens are recrutited to the site of the lysed tumor cells thereby engulfing tumor antigents and presenting these antigens to the immune system The term "dendπtic cell chemoattractants" refers to chemotactic chemokines capable of attracting and/or directing the migration of dendπtic cells to a particular location. It has been demonstrated that certain chemokines. fMLP (representative of formyl peptides of bacteπal oπgm), C5a and the C-C chemokines monocyte chemotactic protein (MCP)- 3. macrophage inflammatory protein (MIP)-l alpha/LD78, and RANTES, have been involved in the recruitment and chemotactic migration of dendntic cells. Sozzani, et al ( 1995 ) J Immunol 1995 155(7) 3292-5 Xu. et al suggest that all C-C chemokines, including MCP-1 , MCP-2, MCP-3, MIPl alpha, MIP-1 beta, and RANTES, induced migration of DC-enπched cells cultured with or without FL-4 Xu, et al . (1996) J.
Leukoc. Biol. 60(3):365-71. Greaves, et al (1997) J. Exp. Med. 186(6):837-44, indicate that MIP-3-alpha specifically interacts with the CC chemokine receptor 6 expressed on dendπtic cells capable of directing migration of dendπtic cells. In the preferred practice of the invention, the dendπtic cell chemoattractant is MIP-3-alpha. The dendπtic cell chemoattractant may be expressed lntracellular form where it is released upon cell lysis or in secreted form by the use of a signal peptide. Upon expression of the dendritic cell chemoattractant, the dendritic cells then engulf the tumor antigens or apoptotic bodies, mature and migrate through existing pathways to the lymph and present the tumor antigens to the T-cells. The resulting T-cells are then capable of recognizing and killing tumor cells The dendπtic cell chemoattractant may be expressed from a viral or cellular promoter active in the target tumor cell. The chemoattractant may also be expressed in conjunction with another gene, such as the p53 gene, from the same promoter through the use of IRES elements. See for example, Lindley, et al (1994) Gene 138: 165-170, or He, et al. (1996) Gene 172.121-125.
XII Diagnostic Applications
In addition to therapeutic applications descnbed above, the vectors of the present invention are also useful for diagnostic purposes For example, the vectors of the present invention may incorporate a reporter gene in conjuction with the p53 gene The term "reporter gene" refers to a gene whose product is capable of producing a detectable signal alone or in combination with additional elements. Examples of reporter genes includes the beta-galactosidase gene, the luciferase gene, the green fluorescent protein gene, nucleotide sequences encoding proteins detectable by imaging systems such as X-rays or magnetic field imaging systems (MRI) Alternatively, such vectors may also be employed to express a cell surface protein capable of recognition by a binding molecule such as a fluorescently labelled antibody Examples of in vivo applications include imaging applications such as X-ray, CT scans or Magnetic Resonance Imaging (MRI)
XII Method of Making The Compositions:
The present invention further provides a method of producing the replication competent recombinant adenovirus compnsing the modifications to packaging domains descπbed above, said method compnsing the steps of: a. infecting a producer cell with a replication competent recombinant adenovirus b. cultuπng said infected producer cell under conditions so as to permit replication of the viral genome in the producer cell, c. harvesting the producer cells, and d punfying the replication competent recombinant adenovirus
The term "infecting" means exposing the replication competent recombinant adenovirus to the producer cell under conditions so as to facilitate the infection of the producer cell with the replication competent recombinant adenovirus In cells which have been infected by multiple copies of a given virus, the activities necessary for viral replication and viπon packaging are cooperative Thus, it is prefeπed that conditions be adjusted such that there is a significant probability that the producer cells are multiply infected with the virus An example of a condition which enhances the production of virus in the producer cell is an increased virus concentration in the infection phase However, it is possible that the total number of viral infections per producer cell can be overdone, resulting in toxic effects to the cell Consequently, one should stnve to maintain the infections in the virus concentration in the range of 106 to 1010, preferably about 109, viπons per ml Chemical agents may also be employed to increase the infectivity of the producer cell line For example, the present invention provides a method to increase the infectivity of producer cell lines for viral infectivity by the inclusion of a calpain inhibitor Examples of calpain inhibitors useful in the practice of the present invention include calpain inhibitor 1 (also known as N-acetyl- leucyl-leucyl-norleucinal. commercially available from Boehnnger Mannheim) Calpa inhibitor 1 has been observed to increase the infectivity of producer cell lines to recombinant adenovirus
The term "producer cell" means a cell capable of facilitating the replication of the viral genome of the recombinant adenovirus to be produced and capable of complementing the packaging defects of the recombinant adenovirus. A vanety of mammalian cell lines are publicly available for the culture of recombinant adenoviruses. For example, the 293 cell line (Graham and Smiley (1977) J. Gen.
Virol. 36:59-72) has been engineered to complement the deficiencies in Elfunction and is a preferred cell line for the production of the current vectors. In a similar manner, cell lines may be developed incorporating viral sequences stably integrated into the viral genome For example, Cunningham and Davidson ((1997, Virol 197:116-124) demonstrate that overlapping cosmids may be used to complement deleted viral functions for herpes viral vectors A similar approach may be employed to complement adenoviral 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, multi-drug resistance, thy mi dine kinase, beta-galactosidase, dihydrofolate reductase (DHFR), and chloramphenicol acetyl transferase Examples of other producer cells parent cell lnes which may be employed include HeLa cells, PERC.6 cells (as descnbed in publication WO/97/00326. application serial No PCT/NL96/00244 and the A549-E1 cell line (as descnbed in International Patent Application No PCT/US97/810039 published February 23, 1998 as International Publication No WO98/US3473
The term "cultuπng under conditions to permit replication of the viral genome" means maintaining the conditions for the infected producer cell so as to permit the virus to propagate in the producer cell It is desirable to control conditions so as to maximize the number of v iral particles produced by each cell Consequently it will be necessary to monitor and control reaction conditions such as temperature, dissolved oxygen, pH, etc Commercially available bioreactors such as the CelliGen Plus Bioreactor (commercially available from New Brunswick Scientific, Inc 44 Talmadge Road, Edison, NJ) have provisions for mo tonng and maintaining such parameters Optimization of infection and culture conditions will vary somevv hat, however, conditions for the efficient replication and production of virus may be achieved by
those of skill in the art taking into considerations the known properties of the producer cell line, properties of the virus, type of bioreactor, etc. When 293 cells are employed as the producer cell line, oxygen concentration is preferably maintained from approximately 50% to approximately 120% dissolved oxygen, preferably 100% dissolved oxygen. When the concentration of viral particles (as determined by conventional methods such as HPLC using a Resource Q column) begins to plateau, the reactor is harvested
The term "harvesting" means the collection of the cells containing the recombinant adenovirus from the media. This may be achieved by conventional methods such as diffential centπfugation or chromatographic means. At this stage, the harvested cells may be stored or further processed by lysis and punfication to isolate the recombinant virus For storage, the harvested cells should be buffered at or about physiological pH and frozen at -70C.
The term "lysis" refers to the rupture of the producer cells. Lysis may be achieved by a vanety of means well known in the art When it is desired to isolate the viral particles from the producer cells, the cells are lysed, using a vanety of means well known in the art. For example, mammalian cells may be lysed under low pressure (100-200 psi differential pressure) conditions or conventional freeze thaw methods. Exogenous free DNA/RNA is removed by degradation with DNAse/RNAse The term "puπfying" means the isolation of a substamally pure population of recombinant virus particles from the lysed producer cells. Conventional punfication techniques such as chromatographic or differential density gradient centπfugation methods may be employed In the prefened practice of the invention, the virus is puπfied by column chromatography in substantial accordance with the process of Huyghe et al (1995) Human Gene Therapx 6 1403-1416 as descπbed in co-pendmg United States Patent application Seπal No 08/400,793 filed March 7, 1995
Additional methods and procedures to optimize production of the recombinant adenoviruses of the present invention are descπbed in co-pending United States Patent Application Seπal No 09/073,076, filed May 4, 1998
The puπfied virus is then admixed with appropπate earners or delivery enhancing agents. The solution is stenhzed for individual packaging and vialed for storage.
Alternatively, the virus may be lyphophi zed for storage and reconsituted in a solution containing delivery enhancing agents, buffers, preservatives, cryoprotectants and/or earners. The lyophihzed virus and the reconsitution solution may be packaged together as a tat for consumption by the end user along with instructions for appropπate handling and administration.
EXAMPLES
As will be apparent to those skilled in the art to which the invention pertains, the present invention may be embodied in forms other than those specifically disclosed below, without departing from the spiπt or essential characteπstics of the invention. The particular embodiments of the invention descπbed below, are, therefore to be considered as illustrative and not restπcti ve The scope of the present invention is as set forth in the appended claims rather than being limited to the examples provided below
Example 1 Construction of d!55K/CMVp53 (cFAIC)
The ElB55K-CMV-p53 (cFAIC) adenovirus was prepared by using o gonucleotide site directed mutatgenesis technique of Deng and Nickoloff (1992)
Anal Biochem 200. 81-88 AH of the reagents, bacteπal strains, and vectors used for mutagenesis were provided in the Transformer Site-Directed mutagenesis tat (commercially availble from Clontech, Palo Alto, CA) The Ad5 region containing the sequence to be mutated was inserted into Clontech plasmid pEGFP-1 and named pXB- EIB Two pπmers were designed to anneal to. (1) Ad5 sequence 2236 to 2260 and modify G2247 to thymidine and T2248 to cytosine and (2) Ad5 sequence 3255 to 3284 and modify T3272 to cytosine A third pnmer was designed to eliminate the Hindlll site within the onginal vector sequence, to provide a method for selection
For the mutagenesis reaction, the mutagenic ohgonucleotides were first phosphorylated at the 5' end, and then annealed to denatured pXB-ElB template DNA The annealed
pπmer/template reactions were incubated with T4 DNA polymerase, T4 DNA ligase and deoxyribonucleotides (dATP, dCTP, dGTP and dTTP) to synthesize a complementary mutant strand. The complementary strand synthesis reaction was then transfomed into the bactenal strain, BMH 71-18 mutS. This bacteπal strain is defective for mis-match repair, preventing undesired repair of the mutant strand. The transformants were digested with HindEII to cut any parental plasmid strands, and retransformed into DH5α for amplification. Potential EIB mutants were then screened by restπction enzyme analysis to confirm the desired mutations.
This procedure w as used to introduce restπction enzyme nuclease cleavage sites in the E1B55K coding region The first site was introduced by modifying positions 2247 and 2248 of the wild type Ad5 genome wherein a guanme2247 was replaced with a thymidine and thymidine2248 replaced with cytosine (respectively) to introduce a EcoRl cleavage site. This results in a modification of the EIB coding sequence at position 77 from valine to seπne. A second restnction site was introduced at position 3272 wherein thymidine3272 was replaced with cytosine site (silent mutation) to introduce an Xhol site. The new restnction enzyme sites were used m a restπciton enzyme digest with EcoRI and Xhol .
A cassette containing the p53 coding sequence, under control of the adenovirus Major Late Promoter and tπpartite leader sequence, was removed by EcoRl and partial Xhol digestion from the plasmid, pAd-MLP-p53 This plasmid is based on the pBR322 denvative pML2 (pBR322 deleted for base pairs 1 140-2490) and contains an adenovirus type 5 sequences extending from base pair 1 to 5788 except that it is deleted foi adenovirus type 5 base pans 357-3327. At ths site the Ad5 357-3327 deletion, a transciption unit is inserted which is compnsed of the adenovirus type 2 major late promoter, the adenovirus type 2 tπpartite leader DNA and human p53 cDNA This EcoRI Xhol fragment is inserted into the EcoRl and Xhol sites introduced into the ElB55k coding region The polyA sequence that follows wtAd5 pIX was amplified by PCR (Ad5 sequence 4001-4368). The pπmers used for amplification included sequences to introduce an EcoRl site at the 5' end of the Ad5 sequence, and a SacII sue at the 3' end of the Ad5 sequence. This fragment was then inserted into the EcoRl-SacII sites immediately following the E1B19K coding
sequence The SacII site was included in the MLP-p53 cassette inserted previously, and was upstream of the MLP promoter sequence The resulting EIB mutation results in a sequence encoding the first 76 ammo acids of the E1B55K protein followed by 11 missense amino acids resulting in a non-functional deleted EIB protein The CMV promoter was obtained from PCR amplification of the corresponding region of
SCH58500 Duπng PCR, a BamHl site was introduced onto the 3' end of the CMV sequence The MLP sequence of d!55K-MLP-p53 was then replaced by digesting with SacII and BamHl, and inserting the CMV promoter PCR fragment (with the same cohesive ends) back into the vector Construction of the EIB mutant/ CMV-p53 adenovirus was earned out by using homologous recombination in the adenovirus El-region containing 293 cell line by the method of Chartier, et al (1996) J Virol 70 4805-4810 This method requires two fragments of DNA, one a transfer plasmid containing the E1B55K deleted CMV- p53 cassette and the other Ad5 viral DNA containing the wtAd5 genome ("Ad5 large fragment"), from Ad5 bp918 to the 3' ITR (bp 35935) The transfer plasmid used, pXCl-CMV53, contains wtAd5 sequences from 22-2246 For recombination to produce adenovirus cFAIC, the viral large fragment and pXCl-CMV53 were cotransfected into 293 cells by calcium phosphate mediated transfection After 5 hours the precipitate was nnsed from the cells and normal media replaced At 15 days after the initial transfection, viral "comets' w ere isolated, plaque puπfied two times, and subsequently viral DNA was screened using restπction enzyme analysis and DNA sequencing Viral stocks were punfied by double cesium chlonde gradients and quantitated by column chromatography as descπbed in Huyghe, et al (1995) Human Gene Therapy 6 1403-1416 Example 2 Ev aluation of In \ ltro Efficicacv of d!55K7CMVp53 (FAIC)
In order to demonstrate that the dJ55K7CMVp53 (FAIC) virus induced apoptosis in tumor cells SK-BR3 cells Six well plates were seeded with 6 x 10s SK-
BR3 cells per well Infection was performed for a one hour pulse in a volume of 250 microliters at two different concentrations, 1 8x10s particles/ml and 1 8xl09 particles/ml of the following vectors 1 Mock Non-infected cells
2. rAdcon: a recombinant adenovirus lacking El and protein IX function without a p53 coding sequence (Wills, et al.) 3 ElBdJ55K. A replication competent recombinant adenovirus containing the E1B-55K deletion descnbed in Example 1 above with no exogenous transgene cassette.
4. rAd-p53: ACN53 (Wills, et al.)
5. dl55K/CMVp53. The replication competent recombinant adenovirus FAIC prepared in substantial accordance with the teaching of Example 1 above.
6 Ad5WT: Wild type adenovirus type 5. The cells were harvested by scraping at 48 hours. The cells were resuspended in lysis buffer (lOmM Tπs-HCl, 5mMEDTA, lOOmM NaCl, 0.05% SDS, 0.5mg/mL Proteinase K, 0.25mg/mL Pronase E) and incubated for 4 hours at 37C. The sample salt concentration was then raised to 1M by addition of 5M NaCl solution. The samples were then frozen and stored at -20°C overnight The supernatant was clanfied by centπfugation and the DNA recovered by phenol/chloroform extraction and ethanol precipation The recovered DNA was then applied to an agarose gel and stained with EtBr The results are presented in the Figure 2 of the attached drawings. The characteristic smear of fragmented DNA is seen in those lanes representative of apoptosis As can be seen from the data presented, the FAIC vector induces substantial apoptosis in the SK-BR3 tumor cells
Example 3 Demonstration of Apoptosis in Tumor Cell Lines d!55K/CMV-p53
(FAIC)
The ability to induce apoptosis was demonstrated in a second tumor cell line, the NCI H538 cells seeded in six well plates with 1.5 x 106 NCI H358 cells per well. The expeπment was performed in substantial accordance with the teaching of Example 2 above, except that no wild-type was included and only a single concentration (1.8 x 109partιcles/ml) was evaluated The results are presented in Figure 3 of the attached drawings The characteristic smear of fragmented DNA is seen in those lanes representative of apoptosis Example 4 Demonstration of Replication of d!55K-MLP-p53 (FAIC)
In order to demonstrate replication of the_d!55K/CMVp53 (FAIC) vector in tumor cells. SK-BR3 cells were infected in substantial accordance with the teaching of Example 2 above However, prior to loading on the agarose gel. the recovered DNA
was subjected to a digestion with the Hindlll restriction endonuclease for approximately 1 hour. The DNA was then applied to an agarose gel and stained with EtBr. The results are presented in the upper panel of Figure 3 of the attached drawings. The results demonstrate viral replication of the_dl55K/CMVp53 (FAIC) construct in tumor cells. The foregoing experiment was repeated in NCI H358 lung non-small cell carcinoma line (p53nu", pRb+) tumor cells in substantial accordance with the teaching of Example 3 above and the data presented in the lower panel of Figure 3 of the attached drawings. The results demonstrate viral replication of the dl55K/CMVp53 (FAIC) construct in NCI H358 tumor cells. Example 5 Demonstration of Therapeutic Efficacy In Vivo
On day 0, 36 athymic nude-nu mice (Harlan-Sprague-Dawley, Indianapolis IN) were injected in each flank with approximately 5xl06 PC-3 cells (prostate carcinoma, p53 null) in 200 micro ters of Dulbecco's Modified Eagle Medium (DMEM were injected subQ into flanks of nude mice. The tumors were allowed to grow for 11 days at which time they had reached a palpable size of approximately 100 mm3 Animals were randomized by tumor size into six groups and each of 5 virus or control PBS soluton was intratumorally injected for 5 consecutive days at a dose of 1x10'° particles per injection; days 11-15 post cell injection of the PC-3 cells . Tumor volume was evaluated on days 1 , 4, 7, 1 1 , 15, 19, 23, and 27 The results of the evaluations are presented in Figure 5 of the accompanying drawings and presented in the Table 1 above.