WO1995014102A1

WO1995014102A1 - Recombinant viruses coding for thymidine kinase in gene therapy

Info

Publication number: WO1995014102A1
Application number: PCT/FR1994/001285
Authority: WO
Inventors: Jean-François DEDIEU; Aude Le Roux; Michel Perricaudet
Original assignee: Rhone-Poulenc Rorer S.A.
Priority date: 1993-11-18
Filing date: 1994-11-07
Publication date: 1995-05-26
Also published as: FR2712603B1; AU8147294A; FR2712603A1; IL111681A0; ZA949104B

Abstract

The invention concerns recombinant adenoviruses comprising a DNA sequence coding for thymidine kinase under the control of heterologous expression signals preparation thereof, and their use in the treatment and prevention of cancers.

Description

RECOMBINANT ADENOVIRUSES ENCODING THYMIDINE KINASE IN GENE THERAPY

The present invention relates to recombinant vectors of viral origin and their use for the treatment of cancers. More particularly, it relates to recombinant adenoviruses comprising a DNA sequence coding for thymidine kinase under the control of heterologous expression signals. The invention also relates to the preparation of these vectors, the pharmaceutical compositions containing them and their use in gene therapy.

Gene therapy consists of correcting a deficiency or an anomaly (mutation, aberrant expression, etc.) by introducing genetic information into the affected cell or organ. This genetic information can be introduced either in vitro into a cell extracted from the organ, the modified cell then being reintroduced into the organism, or directly in vivo into the appropriate tissue. Different techniques have been described for the introduction of this genetic information, among which various transfection techniques involving complexes of DNA and DEAE-dextran (Pagano et al., J. Virol. 1 (1967) 891), d DNA and nuclear proteins (Kaneda et al., Science 243 (1989) 375), DNA and lipids (Felgner et al., PNAS 84 (1987) 7413), the use of liposomes (Fraley et al. , J. Biol. Chem. 255 (1980) 10431), etc. More recently, the use of viruses as vectors for gene transfer has emerged as a promising alternative to these physical transfection techniques. In this regard, different viruses have been tested for their ability to infect certain cell populations. In particular, retroviruses (RSV, HMS, MMS, etc.), the HSV virus, adeno-associated viruses, and adenoviruses.

Many applications of gene therapy are being studied, such as genetic diseases (myopathy, cystic fibrosis, SCID, etc.), pathologies of the central nervous system (alzheimer, Parkinson, etc.), cardiovascular diseases (hemophilia, atherosclerosis ), AIDS or cancers.

The present invention relates to a recombinant adenovirus capable of inducing, in the presence of therapeutic agents, the selective death of infected cells. The adenoviruses of the invention can be used for the selective destruction of cancer cells infected with viruses (HIV, hepatitis), etc. More particularly, it relates to recombinant adenoviruses comprising a DNA sequence coding for the thymidine kinase under control.

The herpes simplex virus thymidine kinase is capable of phosphorylating nucleoside analogs such as acyclovir and ganciclovir. These molecules modified can be incorporated into a DNA chain during elongation, which results in the arrest of DNA synthesis, causing the mon of the cell (Nishiyama et al., J. Gen. Virol. 45 (1979) 227; FL Moolten, Cancer Res. 46 (1986) 5276). This strategy thus makes it possible to specifically eliminate the cells expressing the suicide gene. In addition, since DNA synthesis is the target of toxicity, only the cells being divided are affected.

The therapeutic use of the thymidine kinase gene has already been envisaged in the prior art. Thus, application WO 93/02556 describes the use of cells taken from the tumor, genetically modified ex vivo by introduction of the thymidine kinase gene, then re-administered to the patient. However, this approach requires surgical steps, and moreover, the stability of the toxic gene in the transformed cell ex vivo has not been demonstrated. Similarly, application WO 93/04167 describes the treatment of certain tumors by implantation, in the vicinity of tumors, of cells producing retro viruses containing the TK gene. The grafted producer cells are said to produce retroviruses capable of infecting tumor cells and sensitizing them to ganciclovir. However, this technique involves the implantation of retrovirus-producing cells, which (1) requires a heavy and delicate surgical step, (2) poses immunogenicity problems linked to the presence of these cells, (3) poses problems of side effects linked to possible secretion products of these implanted producer cells, and (4) makes it very difficult to control the amount of viral particles produced by these cells after implantation.

The present invention provides an advantageous solution to these problems. It indeed provides viral vectors usable directly in gene therapy, to direct the expression of thymidine kinase in target cells. The present invention therefore makes it possible to avoid the use of producer cells, the numerous drawbacks of which have been mentioned above. It also makes it possible to control the quantity of virus administered and therefore of thymidine kinase produced, and thus offers better control of the treatment. Furthermore, the adenoviruses of the invention have the advantage of not integrating into the genome of the cells they infect, of remaining therein in a very stable manner which considerably limits the risks of dissemination to neighboring cells, and to have a very broad host spectrum, which offers many therapeutic applications. In addition, recombinant adenoviruses can be obtained with high titers, which makes it possible to work at high multiplicities of infection. In addition, the use of heterologous expression signals makes it possible to optimize for each therapeutic application, and therefore, for each cell type concerned, the levels and conditions of expression of thymidine kinase.

A first object of the invention therefore resides in a defective recombinant adenovirus comprising a DNA sequence coding for thymidine kinase under the control of heterologous expression signals.

The invention also relates to the use of such a defective recombinant adenovirus for the preparation of a pharmaceutical composition intended for the treatment or prevention of cancers and / or viral diseases.

The defective adenoviruses according to the invention are adenoviruses incapable of replicating autonomously in the target cell. Generally, the genome of the defective adenoviruses used in the context of the present invention is therefore devoid of at least the sequences necessary for the replication of said virus in the infected cell. These regions can be either eliminated (in whole or in part), or made non-functional, or substituted by other sequences and in particular by the inserted gene. Preferably, the defective virus nevertheless retains the sequences of its genome which are necessary for the packaging of the viral particles.

There are different serotypes of adenovirus, the structure and properties of which vary somewhat. However, these viruses are not pathogenic for humans, especially non-immunocompromised people. Among these serotypes, it is preferred to use, within the framework of the present invention, human adenoviruses of type 2 or 5 (Ad 2 or Ad 5) or adenoviruses of animal origin (see application FR 93 05954). Among the adenoviruses of animal origin which can be used in the context of the present invention, mention may be made of adenoviruses of canine, bovine, murine origin (example: Mavl, Beard et al., Virology 75 (1990) 81), ovine, porcine , avian or even simian (example: SAN). Preferably, the adenovirus of animal origin is a canine adenovirus, more preferably a CAN2 adenovirus [Manhattan strain or A26 / 61 (ATCC NR-800) for example].

Preferably, in the context of the invention, adenoviruses of human or canine or mixed origin are used. As indicated above, the adenoviruses of the invention carry a DNA sequence coding for thymidine kinase. It is preferably the thymidine kinase gene of the herpes simplex virus (HSV-tk). Even more preferably, the thymidine kinase gene from the human herpes simplex virus (hTK HSV-1) is used in the context of the present invention. The sequence of this gene has been described in the literature (see in particular McKnight et al, Nucleic Acid. Res. 8 (1980) 5931). This sequence is placed under the control of heterologous expression signals allowing its expression in infected cells. Within the meaning of the present invention, by heterologous expression signals is meant signals different from those naturally responsible for the expression of a TK gene. They may in particular be sequences responsible for the expression of other proteins, or synthetic sequences. In particular, they may be promoter sequences of eukaryotic or viral genes. For example, they may be promoter sequences originating from the genome of the cell which it is desired to infect. Likewise, they may be promoter sequences originating from the genome of a virus, including the radenovirus used. In this regard, there may be mentioned, for example, the promoters E1A, MLP, CMV, RSV, etc. In addition, these expression sequences can be modified by adding activation, regulation sequences, etc. Furthermore, when the DNA sequence coding for thymidine kinase does not contain expression sequences, it can be inserted into the genome of the defective virus downstream of such a sequence. In a first particular particular embodiment of the invention, the RSV promoter is preferably used, which allows long-lasting and significant expression of thymidine kinase in cells of the nervous system, in particular central.

In another particular embodiment of the invention, expression signals active specifically in tumor cells are used. In this way, the gene used is only expressed and produces its effect when the virus has actually infected a tumor cell. More preferably, these are expression signals induced by or active in the presence of viruses responsible for or associated with tumors. Even more preferably, in the context of the present invention, an expression signal inducible by the Epstein-Barr virus (EBN) or by the papilloma virus is used.

Epstein-Barr virus (EBN) is associated with two types of human cancer: Burkitt's lymphoma and nasopharyngeal cancer. The use of a recombinant adenovirus comprising a toxic gene under the control of a promoter inducible by EBN advantageously makes it possible to specifically express this toxic gene in tumor cells of the nasopharynx. In biopsies of nasopharyngeal cancer, a single nuclear antigen is regularly present, EBΝA1, which is involved in the maintenance of the viral genome in cells infected with EBV in latent phase, and which transactivates the viral promoter BCR2. A particular object of the invention therefore lies in the use, for the specific expression of a gene in cancer cells of the nasopharynx, of a sequence responding to EBN A1 (EBNA1-RE: EBNA1 "responsive element"). In particular, the invention relates to an adenovirus comprising, as expression signal, a chimeric promoter comprising a sequence responding to EBNA1 fused upstream of another viral promoter, the promoter of the terminal protein 1 gene (TPI). The examples described in the present application clearly show that this chimeric promoter is inducible by EBN A1.

Papilloma viruses (notably HPN 16 and 18) are responsible for 90% of cervical cancers in women and have been identified in pre-cancerous epithelial lesions (Riou et al., Lancet 335 (1990) 117). The E6 gene product leads to the formation of tumors by greatly reducing the amount of wild-type p53, an anti-oncogene, in HPV-positive cells (Wrede et al., Mol. Carcinog. 4 (1991) 171). The use of a recombinant adenovirus comprising a toxic gene under the control of a promoter inducible by HPN (for example the E6 protein) advantageously makes it possible to express this toxic gene specifically in the corresponding tumor cells.

In addition, these promoter sequences can be modified by adding activation, regulation, etc. sequences.

They may also be expression signals which are inactive in normal cells and active in tumor cells. In particular, in the context of the present invention, the α-fetoprotein promoter (Alpert E., in Hepatocellular carcinoma, Okuda & Peters (eds), New York, 1976, 353) or 1TGF-II ( Sussenbach et al., Growth Regulation 2 (1992) 1), which are active in adults only in hepatocarcinomas.

The defective recombinant adenoviruses according to the invention can be prepared by any technique known to those skilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham, EMBO J. 3 (1984) 2917). In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid carrying inter alia the heterologous DNA sequence. Homologous recombination occurs after co-transfection of said adenovirus and plasmid in an appropriate cell line. The cell line used must preferably (i) be transformable by said elements, and (ii), contain the sequences capable of complementing the part of the genome of the defective adenovirus, preferably in integrated form to avoid the risks of recombination. As an example of a line, mention may be made of the human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which contains in particular, integrated into its genome, the left part of the genome of a Ad5 adenovirus (12%). Strategies for the construction of vectors derived from adenoviruses have also been described in applications Nos. FR 93 05954 and FR 93 08596.

Then, the adenoviruses which have multiplied are recovered and purified according to conventional techniques of molecular biology, as illustrated in the examples.

The present invention also relates to a pharmaceutical composition comprising one or more defective recombinant adenoviruses as described above. These pharmaceutical compositions can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc. administration. Preferably, the pharmaceutical compositions of the invention contain a pharmaceutically acceptable vehicle for an injectable formulation, in particular for a direct injection into a tumor to be treated. They may in particular be sterile, isotonic solutions, or dry compositions, in particular lyophilized, which, by addition as appropriate of sterilized water or physiological saline, allow the constitution of injectable solutes. Direct injection into the tumor to be treated is advantageous because it allows the therapeutic effect to be concentrated in the affected tissues.

The doses of defective recombinant adenovirus used for injection can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the pathology concerned, of the gene to be expressed, or even of the duration of the treatment sought. . In general, the recombinant adenoviruses according to the invention are formulated and administered in the form of doses of between 10 ⁴ and 10 ¹⁴ pfu / ml, and preferably 10 ⁶ to 10 ¹⁰ pfu / ml. The term pfu ("plaque forming unit") corresponds to the infectious power of a virus solution, and is determined by infection of an appropriate cell culture, and measures, generally after 48 hours, the number of plaques of infected cells. The techniques for determining the pfu titer of a viral solution are well documented in the literature.

The present invention thus provides a very effective means for the treatment or prevention of cancers. It is particularly suitable for the treatment of nasopharyngeal cancer, brain tumors (neuroblastomas, glioblastomas, etc.) and hepatocarcinomas.

Concerning brain tumors, the adenoviral vectors according to the invention have significant advantages, linked in particular to their very high efficiency of infection of nerve cells, making it possible to carry out infections from low volumes of viral suspension. In addition, infection with the adenoviruses of the invention is very localized at the injection site, which avoids the risks of dissemination to neighboring cells.

In addition, this treatment can concern both humans and any animal such as sheep, cattle, domestic animals (dogs, cats, etc.), horses, fish, etc.

The present invention will be more fully described with the aid of the following examples, which should be considered as illustrative and not limiting.

Legend of figures

Figure 1: Representation of the vector pONT-tk Figure 2: Representation of the vector pRSV-tk

General molecular biology techniques

Methods conventionally used in molecular biology such as preparative extractions of plasmid DNA, centrifugation of plasmid DNA in cesium chloride gradient, electrophoresis on agarose or acrylamide gels, purification of DNA fragments by electroelution, the extraction of proteins with phenol or phenol-chloroform, the precipitation of DNA in a saline medium with ethanol or isopropanol, the transformation in Escherichia coli, etc. are well known in the art. skilled in the art and are abundantly described in the literature [Maniatis T. et al., "Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982; Ausubel F.M. et al. (eds), "Current Protocols in Molecular Biology", John Wiley & Sons, New York, 1987].

The pBR322, pUC and phage plasmids of the M13 series are of commercial origin (Bethesda Research Laboratories). For the ligations, the DNA fragments can be separated according to their size by electrophoresis in agarose or acrylamide gels, extracted with phenol or with a phenol / chloroform mixture, precipitated with ethanol and then incubated in the presence of DNA. phage T4 ligase (Biolabs) according to the supplier's recommendations.

The filling of the protruding 5 ′ ends can be carried out by the Klenow fragment of DNA Polymerase I of E. coli (Biolabs) according to the supplier's specifications. The destruction of the prominent 3 'ends is carried out in the presence of the DNA Polymerase of phage T4 (Biolabs) used according to the recommendations of the maker. The destruction of the protruding 5 ′ ends is carried out by gentle treatment with nuclease SI.

Mutagenesis directed in vitro by synthetic oligodeoxynucleotides can be carried out according to the method developed by Taylor et al. [Nucleic Acids Res. 13 (1985) 8749-8764] using the kit distributed by Amersham.

Enzymatic amplification of DNA fragments by the so-called PCR technique

[Polymerase-catalyzed C_hain Reaction, Saiki R.K. et al., Science 22Q (1985) 1350-

1354; Mullis K.B. and Faloona F.A., Meth. Enzym. J.5 5 (1987) 335-350] can be performed using a "DNA thermal cycler" (Perkin Elmer Cetus) according to the manufacturer's specifications.

Verification of the nucleotide sequences can be carried out by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467] using the kit distributed by Amersham.

Examples

Example 1. Construction of the Ad-ONT-tk vector carrying the tk gene under the control of a chimeric promoter EBNA1-RE / TP1 (FIG. 1).

This example describes the construction of a recombinant adenovirus comprising the thymidine kinase gene of the herpes simplex virus (tk) under the control of a promoter specifically active in cells infected with the EBN virus (chimeric promoter EBΝA1-RE TP1).

1.1. Construction of the plasmid p7tkl

This example describes the construction of the plasmid p7tkl containing the open reading phase of the tk gene of 1131 base pairs (ATG 114-116 and stop codon TGA 1242-1244), inserted into a cloning multisite. The BglII-ΝcoI fragment containing the thymidine kinase (tk) gene of the herpes simplex virus type 1 was isolated from the plasmid pHSV-106 (marketed by Gibco BRL), repaired by the action of the klenow fragment and then inserted into the site Smal of the plasmid pGEM7zf (+) (marketed by Promega). The Smal and BglII sites are destroyed during this step, the Νcol site is preserved. The plasmid obtained was designated p7tkl. 1.2. Construction of the plasmid pONTl

This example describes the construction of a plasmid containing a chimeric promoter consisting of a sequence necessary for transactivation by the EBNA1 antigen and of the TPI promoter of the EBN virus. The EcoRI (7315) -SmaI (8191) fragment of the EBN virus was isolated from the strain B95-8. The complete sequence of the EBN virus has been described by Baer et al. (Nature 310 (1984) 207). This fragment contains the sequences necessary for transactivation by nuclear antigen 1 (EBNA1) (D. Reisman & B. Sugden, 1986, Molecular and Cellular Biology, vol. 6 pp. 3838-3846). This fragment was then fused to the Nrul (166,241) -PstI (166,559) fragment of EBN B95-8 (the PstI site was digested with T4 polymerase), containing the TPI promoter. The chimeric promoter thus obtained was then inserted into the multisite for cloning the plasmid pBluescript II SK.

The plasmid obtained was designated pOΝTl.

1.3. Construction of the plasmid pOΝTtk

The plasmid pOΝTtk comprises the gene for the thymidine kinase of the herpes simplex virus (tk) cloned in the plasmid p7tkl, under the control of the chimeric promoter EBΝA1-RE / TP1 cloned in the plasmid pOΝTl.

To construct this plasmid, the BamHI-XhoI fragment from pOΝTl which contains the chimeric promoter transactivated by EBΝA-1 and EBNA-2, and the fragment

X ol-ClaI of p7tkl which contains the open reading phase of tk were cloned at the BamHI (478) and ClaI (4550) sites of the plasmid ρAd.RSVβgal. The plasmid pAd.RSNβGal contains, in the 5 '-> 3' orientation,

- the PvuII fragment corresponding to the left end of the Ad5 adenovirus comprising: the ITR sequence, the origin of replication, the packaging signals and the El A amplifier;

- the gene coding for β-galactosidase under the control of the RSV promoter (from Rous sarcoma virus),

- A second fragment of the genome of the Ad5 adenovirus, which allows homologous recombination between the plasmid pAd.RSVβGal and the adenovirus dl324. The plasmid pAd.RSVβGal has been described by Stratford-Perricaudet et al. (J. Clin. Invest. 90 (1992) 626).

All cloning sites are preserved. The plasmid obtained was designated pONTtk (Figure 1). 1.4. Construction of the Ad-ONT-tk Recombinant Adenovirus The pONTtk vector was linearized and cotransfected with a deficient adenoviral vector, in helper cells (line 293) providing in trans the functions coded by the El regions (El A and E11B) d adenovirus. More specifically, the adenovirus Ad-ONT-tk was obtained by homologous in vivo recombination between the mutant adenovirus Ad-dll324 (Thimmappaya et al., Cell 31 (1982) 543) and the vector pONTtk, according to the following protocol: the plasmid pONTtk, linearized by Xmnl, and the adenovirus Ad-dll324, linearized by the enzyme ClaI, were co-transfected in line 293 in the presence of calcium phosphate, to allow homologous recombination. The recombinant adenoviruses thus generated were selected by plaque purification. After isolation, the DNA of the recombinant adenovirus was amplified in the cell line 293, which leads to a culture supernatant containing the unpurified recombinant defective adenovirus having a titre of approximately 10 ¹⁰ pfu / ml. The viral particles are generally purified by centrifugation on a cesium chloride gradient according to known techniques (see in particular Graham et al., Virology 52 (1973) 456). The Ad-ONT-tk adenovirus can be stored at -80 ° C in 20% glycerol.

Example 2. Construction of the Ad-RSV-TK vector

This example describes the construction of a recombinant adenovirus comprising the thymidine kinase gene of the herpes simplex virus (tk) under the control of the RSV promoter. This adenovirus was constructed by homologous recombination between the defective adenovirus Ad-dll324 and the plasmid pRSVtk carrying the tk gene under the control of the RSV promoter.

2.1. Construction of the plasmid pRSVtk

This plasmid was constructed from the plasmid pONTtk (Example 1.3.), By substitution of the promoter transactivable by EBNA1 by the promoter RSV. For this, the RSV promoter was isolated in the form of a BamHI-SalI fragment from the plasmid pAd.RSV.βgal (Stratford-Perricaudet et al., J. Clin. Invest. 90 (1992) 626), then cloned at the BamHI (478) and Sall (1700) sites of the plasmid pONTtk. The resulting plasmid was designated pRSVtk (Figure 2). 2.2. Construction of the Ad-RSV-tk adenovirus

This adenovirus was constructed according to the protocol described in Example 1.4. The adenovirus Ad-RSV-tk thus obtained can be stored at -80 ° C in 20% glycerol.

Example 3. Use of the Adenovirus Ad-RSN-tk for the Destruction of Tumor Nerve Cells

The Ad-RSN-tk adenovirus was used to infect a nerve tumor cell line (C6 cells: rat glioma accessible to ATCC under the number ATCC CCL 107). The infection was carried out at a titer of approximately 10 pfu / cell. 24 hours after infection, the cells are treated in the presence of 10 ^~ 5 M of gancyclovir and the mortality rate is determined 48 hours after. The results obtained show that 100% of the cells infected with the adenovirus Ad-RSN-tk die after 48 hours, while the cells not infected or infected under the same conditions with the adenovirus Ad-RSN.βgal survive. These results clearly show that the adenovirus Ad-RSN-tk made the glioma cells sensitive to gancyclovir.

Claims

1. Defective recombinant adenovirus comprising a DNA sequence coding for thymidine kinase under the control of heterologous expression signals.

2. Adenovirus according to claim 1 characterized in that it lacks the regions of its genome which are necessary for its replication in the target cell.

3. Adenovirus according to claim 2 characterized in that it is a human adenovirus type Ad 5 or canine type CAV-2.

4. Adenovirus according to one of claims 1 to 3 characterized in that the heterologous DNA sequence codes for the thymidine kinase of the herpes simplex virus.

5. Adenovirus according to claim 4 characterized in that the heterologous DNA sequence codes for the thymidine kinase of the human herpes simplex virus.

6. Adenovirus according to claim 5 characterized in that the expression signals are chosen from viral promoters.

7. Adenovirus according to claim 6 characterized in that the expression signals are chosen from promoters El A, MLP, CMV and RSV.

8. Adenovirus according to claim 1 characterized in that it comprises an expression signal active specifically in tumor cells.

9. Adenovirus according to claim 8 characterized in that it comprises an expression signal inducible by the Epstein-Barr virus (EBV), or by the papilloma virus.

10. Adenovirus according to claim 9 characterized in that the expression signal comprises a sequence responding to the EBNAl antigen.

11. Adenovirus according to claim 10 characterized in that the expression signal consists of a chimeric promoter comprising a sequence responding to EBNA1 fused upstream of another viral promoter, preferably the promoter of the protein 1 terminal gene (TPI).

12. Defective recombinant adenovirus comprising a heterologous DNA sequence coding for thymidine kinase under the control of the RSV promoter.

13. Defective recombinant adenovirus comprising a heterologous DNA sequence coding for thymidine kinase under the control of the promoter EBNAl - RE / TPI.

14. Use of an adenovirus according to one of claims 1 to 13 for the preparation of a pharmaceutical composition intended for the treatment and / or prevention of cancers.

15. Use according to claim 14 for the preparation of a pharmaceutical composition intended for the treatment and / or prevention of cancers of the nasopharynx, brain tumors (neuroblastomas, glioblastomas) or hepatocarcinomas.

16. Use according to claim 14 or 15 for the preparation of a pharmaceutical composition for direct administration in the tumor to be treated.

17. Pharmaceutical composition comprising one or more defective recombinant adenoviruses according to one of claims 1 to 13.

18. Pharmaceutical composition according to claim 17 characterized in that it is in injectable form.

19. Pharmaceutical composition according to claim 17 characterized in that it comprises between 10 ⁴ and 10 ¹⁴ pfu / ml, and preferably 10 ⁶ to 10 ¹⁰ pfu / ml defective recombinant adenoviruses.