NZ265306A - Recombinant adenovirus containing heterologous dna whose expression in an abnormally dividing cell inhibits cell division - Google Patents

Abstract

Defective recombinant adenovirus contains a heterologous DNA sequence (I), the expression of which in a target cell contributes to the inhibition of cell division.

Description

New Zealand No. 265306 International No.

TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION Priority dates: 22.04.1993; Complete Specification Filed: 15.04.1994 Classification:^) C12N15/86,11,12; C12N7/01; A61K39/235 Publication date: 24 October 1997 Journal No.: 1421 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention: Defective recombinant adenoviruses for gene therapy of tumours Name, address and nationality of applicant(s) as in international application form: RHONE-POULENC SA, 20, avenue Raymond Aron F-92160 Antony, France 2653 WO 94/24297 1 PCT/FR94/00421 DEFECTXVB RECOMBINANT ADENOVIRUSES FOR THE GENE THERAPY OF TUMOURS The present invention relates to recombinant vectors of viral origin and to their use for the 5 treatment of cancers. More particularly, it relates to recombinant adenoviruses containing a heterologous DMA sequence whose expression in an abnormally dividing cell makes it possible to at least partially inhibit the division of the said cell. The invention also 10 relates to the preparation of these vectors and to the pharmaceutical compositions containing them.

Cell growth is regulated in an extremely subtle manner by two types of signals. Some favour the multiplication of the cells, whereas others, in 15 contrast, cause them to enter into a quiescent state or cause them to differentiate, depending on the needs of the body. All cancers are characterized by a disruption of the mechanisms controlling cell division, resulting in an abnormal proliferation. Most often, the 20 development of a cancer therefore involves the activation of genes which favour the multiplication of the cells (genes designated proto-oncogenes, which are activated into oncogenes) and the disappearance or the inactivation of genes which inhibit cell proliferation. 25 The present invention offers the possibility of treating cancers by gene therapy, by the administration, to tumour cells, of one or more of these genes whose expression makes it possible to at 2653 2 least partially inhibit cell proliferation.

Gene therapy consists in correcting a deficiency or an abnormality (mutation, aberrant expression and the like) by introduction of a genetic 5 information into the cell or the affected 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 body, or directly in vivo, into the appropriate tissue. In this second case, 10 various techniques exist, amongst which are various transfection techniques involving complexes of DNA and DEAE-dextran (Pagano et al., J. Virol. 1 (1967) 891), of DNA and nuclear proteins (Kaneda et al., Science 243 (1989) 375), of DNA and lipids (Feigner et al., PNAS 84 15 (1987) 7413), the use of liposomes (Fraley et al., J. Biol. Chem. 255 (1980) 10431), and the like. More recently, the use of viruses as vectors for the transfer of genes appeared as a promising alternative to these physical transfection techniques. In this 20 respect, various viruses have been tested for their capacity to infect certain cell populations, in particular retroviruses (RSV, HMS, MMS and the like), HSV virus, adeno-associated viruses, and adenoviruses.

The possibility of using gene therapy to 25 treat cancers has already been mentioned in the application W091/15580. This application describes the construction of retroviruses containing a gene encoding a ribozyme, whose expression in cell culture can make 265306 3 it possible to destroy an mRNA of an oncogene.

The present invention results- from the demonstration that adenoviruses constitute particularly effective vectors for the transfer and expression of therapeutic genes in the tumours. In particular, adenoviruses have the advantage of not becoming integrated into the genome of the cells which they infect, of being maintained therein in a very stable manner, which makes it possible to obtain a lasting therapeutic effect and to have a very broad host range, which permits application to the treatment of cancers affecting any type of cells. Moreover, the invention is also based on the demonstration that adenovirus-type viruses are capable of transferring and expressing genes capable of at least partially inhibiting cell division directly in tumours.

The present invention accordingly provides a defective recombinant adenovirus containing a heterologous DNA sequence whose expression in a target cell makes it possible to at least partially inhibit cell division, the sequence comprising defective recombinant adenovirus containing a heterologous DNA sequence whose expression in a target cell makes it possible to at least partially inhibit cell division, the sequence comprising - at least one gene chosen from among tumour suppressor genes, antisense genes whose expression in the target cell makes it possible to control the transcription or the translation of genes favouring cell proliferation and genes - 3a - 265306 whose expression product induces apoptosis of the Infected cell; and a promoter sequence permitting the expression, in the infected cell, of the gene, the promoter sequence having a different origin from that of the gene.

The subject of the invention is also the use of such a defective recombinant adenovirus for the preparation of a pharmaceutical composition intended for the treatment or the prevention of cancers.

For the purposes of the present invention, the term "defective adenovirus" designates an adenovirus incapable of autonomously replicating in the target cell. Generally, the genome of the defective 265306 adenoviruses used within the framework of the present invention therefore lacks at least sequences necessary for the replication of the said virus in the infected cell. These regions can be either removed (completely 5 or partially), or rendered non-functional, or substituted by other sequences and especially by the inserted gene. Preferably, the defective virus conserves, nevertheless, the sequences of its genome which are necessary for the encapsulation of the viral 10 particles.

There are various serotypes of adenoviruses, whose structure and properties vary somewhat. However, these viruses are not pathogenic for man, and especially for non-immunosuppressed subjects. Among 15 these serotypes, the use of type 2 or 5 adenoviruses (Ad 2 or Ad 5) is preferred within the framework of the present invention. In the case of the Ad 5 adenoviruses, the sequences necessary for the replication are the E1A and E1B regions. 20 For the purposes of the present invention, the heterologous DNA sequence whose expression makes it possible to at least partially inhibit cell division preferably comprises at least one gene chosen from tumour suppressor genes (or antioncogene) or any active 25 derivative of the said genes; antisense genes, whose expression in the target cell makes it possible to inhibit the expression of genes promoting cell division; or genes whose expression product induces 2653 apoptosis of the infected cell.

Among the tumour suppressor genes which can be used within the framework of the present invention, the following genes may be mentioned more particularly: 5 - p53 gene; The p53 gene encodes a nuclear protein of 53 kDa. The mutated, by deletion and/or mutation, form of this gene is involved in the development of most human cancers (Baker et al.. Science 244 (1989) 217). 10 Its mutated forms are also capable of cooperating with the ras oncogenes to transform murine fibroblasts. The wild-type gene encoding native p53 inhibits, on the other hand, the formation of transformation foci in rodent fibroblasts transfected with various 15 combinations of oncogenes. Recent data emphasize that the protein p53 could itself be a transcription factor and stimulate the expression of other tumour suppressor genes.

- Rb gene The Rb gene determines the synthesis of a nuclear phosphoprotein of about 927 amino acids (Friend et al.. Nature 323 (1986) 643) whose function is to repress the division of the cells by causing them to enter the quiescence phase. Inactivated forms of the Rb 25 gene have been implicated in various tumours, and especially in retinoblastomas or in mesenchymatous cancers such as osteosarcomas. The reintroduction of this gene into the tumour cells where it was 265306 6 inactivated produces a return to the normal state and a loss of the tumorigenicity (Huang et al.. Science 242 (1988) 1563). Recently, it has been demonstrated that the normal Rb protein, but not its mutated forms, 5 represses the expression of the c-fos proto-oncogene, a gene which is essential for cell proliferation. - rap 1A gene The rap 1A gene (also designated k-revl) encodes a protein of 21 kDa associated with the inner face of the cytoplasmic membrane. This protein is capable, at high levels, of reverting transformed cells expressing the mutated ras oncogenes (Kitayama et al.. Cell 56 (1989) 77).

- DCC gene The DCC gene encodes a protein homologous to the cell-adhesion proteins of the N-CAM family. This gene is very frequently deleted in colon carcinomas (Fearon et al.. Science 247 (1990) 49). - k-rev2 and k-rev3 genes The k-rev2 gene encodes a secreted protein of 60 amino acids, and the k-rev3 gene encodes a truncated version of a protein of the extracellular matrix. These two genes are capable of reverting NIH 3T3 cells transformed by the Ki-ras oncogene.

Other genes can be used within the framework of the present invention for their anti-tumour effect, and especially other tumour suppressor genes described in the literature, or amy other gene whose expression 265306 7 product can induce cell apoptosis.

As indicated above, the heterologous DNA sequence may contain the native tumour suppressor gene or an active derivative of the said gene. Such a derivative can be obtained by mutation, deletion, substituion and/or addition of one or more base pairs in the sequence of the gene, according to conventional molecular biology techniques. The activity of the derivative thus obtained can then be confirmed in vitro from tests known to persons skilled in the art, such as those described in the examples.

For the purposes of the present invention, the heterologous DNA sequence may also comprise an antisense gene, whose expression in the target cell makes it possible to control the expression of genes or the transcription of cellular mRNAs encoding proteins which favour cell proliferation. Such genes can for example be transcribed, in the target cell, into RNAs complementary to cellular mRNAs and thus block their translation into protein.

Among the antisense genes which can be used within the framework of the invention, there may be mentioned more particularly any antisense sequence which makes it possible to reduce the levels of production of the ras, myc, fos, c-erb B oncogenes and the like.

Generally, the heterologous DNA sequence also comprises promoter sequences permitting the expression 2653 8 of the gene(s) capable of at least partially inhibiting cell division in the target cell. These-could be promoter sequences which are naturally responsible for the expression of the said gene when these sequences 5 are capable of functioning in the infected cell. They may also be promoter sequences of different origin (responsible for the expression of other proteins, or even synthetic). They may especially be promoter sequences of eukaryotic or viral genes. For example, 10 they may be promoter sequences derived from the genome of the cell which it is desired to infect. Likewise, they may be promoter sequences derived from the genome of a virus, including the adenovirus used. In this respect, there may be mentioned for example the 15 promoters of the E1A, MLP, CMV and RSV genes and the like. In addition, these promoter sequences can be modified by addition of activating and regulatory sequences and the like. Moreover, when the heterologous DNA sequence does not contain expression sequences, it 20 can be inserted into the genome of the defective virus downstream of such a sequence. It is also possible to use inducible promoters.

Moreover, in another embodiment of the invention, the heterologous DNA sequence comprises, in 25 addition to the tumour suppressor gene or the antisense gene, a gene encoding a tumour-specific antigen and/or a gene encoding a lymphokine. The combination of these genes makes it possible, indeed, (i) to stop the cell 2653 9 division in a tumour and thus, to cause the said tumour to regress, and (ii) to increase the immune response of the body against the said tumour.

The tumour-specific antigens are antigenic 5 units which appear at the surface of tumour cells, but which do not exist at the surface of the same, non-tumour, cells. Such antigens are generally used for the diagnosis of cancers. More recently, they were described for the preparation of anti-tumour vaccines 10 (EP 259 212). However, they have never been combined with other therapeutic genes as within the framework, of the present invention.

Among the genes encoding lymphokines, there may be mentioned more particularly genes encoding 15 interleukins (IL-1 to IL-13), interferons, tumour necrosis factors, colony-stimulating factors (G-CSF, M-CSF, GM-CSF, and the like), TGF/5, and the like. Moreover, the lymphokine-encoding gene e nerally comprises, upstream of the coding sequence, an 20 expression sequence and a signal sequence directing the synthesized polypeptide in the secretion pathways of the target cell. This signal sequence may be the natural signal sequence of the lymphokine, but it may also be any other functional signal sequence, or an 25 artificial signal sequence. Such constructs make it possible in particular to increase the lymphokine levels in a very localized manner, and thus, in the presence of a tumour-specific antigen, to amplify the 2653 immune response against a particular type of tumour, which gives a particularly advantageous-effect. Such recombinant adenoviruses are particularly utilizable for the preparation of anti-tumour vaccines.

The defective recombinant adenoviruses according to the invention can be prepared by any technique known to persons 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. The homologous recombination occurs after cotransfaction of the said adenoviruses and plasmid into an appropriate cell line. The cell line used should preferably (i) be transformable by the said elements, and (ii) contain the sequences capable of complementing the defective adenovirus genome part, preferably in integrated form in order to avoid risks of recombination. As an example of a cell line, there may be mentioned the human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which contains especially, integrated in its genome, the left-hand part of the genome of an Ad5 adenovirus (12 %) .

Then, the adenoviruses which have multiplied are recovered and purified according to conventional molecular biology techniques, as illustrated in the examples. . 265306 11 The present invention also relates to a pharmaceutical composition containing one or more defective recombinant adenoviruses as described above. Preferably, the pharmaceutical compositions of the 5 invention contain a vehicle which is pharmaceutically acceptable for a formulation directly injectable into the tumours to be treated. This may be in particular isotonic sterile solutions, or dry, especially freeze-dried, compositions which, upon addition, depending on 10 the case, of sterilized water or physiological saline, permit the preparation of injectable solutions. Direct injection into the tumour to be treated is advantageous since it makes it possible to concentrate the therapeutic effect at the level of the affected 15 tissues.

The doses of defective recombinant adenovirus used for the injection can be adapted according to various parameters, especially according to the mode of administration used, the pathology concerned, the gene 20 to be expressed, or alternatively the duration of treatment desired. Generally, the recombinant adenoviruses according to the invention are formulated and administered in the form of doses of between 104 and 1014 pfu/ml, preferably 10s to 1010 pfu/ml. The term pfu 25 (plague forming unit) corresponds to the infectivity of a virus solution, and is determined by infection of an appropriate cell culture, and measurement, generally after 48 hours, of the number of plagues of infected 265306 12 cells. The techniques for determining the pfu titre of a viral solution are well documented in the literature.

The present invention thus offers a very-effective means for the treatment or prevention of cancers. In addition, this treatment may apply both to man and any animal such as ovines, bovines, domestic animals (dogs, cats and the like), horses, fish and the like.

The present invention will be more completely described with the aid of the following examples which should be considered as illustrative and non-limiting.

Legend to the fiarureB Figure 1 : Representation of the vector mp53wtI-.CMV Figure 2 : Representation of the vector mp53pIX.CMV General molecular bioloov techniques The methods conventionally used in molecular biology, such as preparative extractions of plasmid DNA, centrifugation of plasmid DNA in cesium chloride gradient, agarose or acrylamide gel electrophoresis, purification of DNA fragments by electroelution, phenol or phenol-chloroform extraction of proteins, ethanol or isopropanol precipitation of DNA in saline medium, transformation in Escherichia coli and the like, are well known to persons skilled in the art and are widely described in the literature [Maniatis T. et al., "Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982; Ausubel F.M. et al. (eds), "Current Protocols in 265306 13 Molecular Biology", John Wiley & Sons, New York, 1987] .

The pBR322- and pUC- type plasmids and the phages 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 agarose or acrylamide gel electrophoresis, extracted with phenol or with a phenol/chloroform mixture, precipitated with ethanol and then incubated in the presence of phage T4 DNA ligase (Biolabs) according to the recommendations of the supplier.

The filling of the protruding 5' ends can be performed with the Klenow fragment of E. coli DNA polymerase I (Biolabs) according to the specifications of the supplier. The destruction of the protruding 3' ends is performed in the presence of phage T4 DNA polymerase (Biolabs) used according to the recommendations of the manufacturer. The destruction of the protruding 5' ends is performed by a controlled treatment with SI nuclease.

Site-directed mutagenesis in vitro by synthetic oligodeoxynucleotides can be perforviad according to the method developed by Taylor et al.

[Nucleic Acids Res. 13 (1985) 8749-8764] using the kit distributed by Amersham.

The enzymatic amplification of the DNA fragments by the so-called PCR technique [Polymerase-catalyzed Chain Reaction, Saiki R.K. et 2653 14 al., Science 230 (1985) 1350-1354; Mullis K.B. and Faloona F.A., Meth. Enzym. 155 (1987) 335-350] can be performed using a DNA thermal cycler (Perkin Elmer Cetus) according to the specifications of the manufacturer.

The verification of the nucleotide sequences cam. be performed by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467] using the kit distributed by Amersham.

Examples El. Construction of the vector mp53wtI-CMV carrying the p53 gene under the control of the cytomegalovirus promoter (Figure 1).

The eukaryotic expression vector mp53wtX-CMV was constructed from the plasmid pUC19, by insertion: - of a promoter region of viral origin which corresponds to the cytomegalovirus (CMV) early promoter. This region is surrounded in the vector by unique restriction sites EcoRI-SphI at the CMV/pUC junction and BamHI at the CMV/p53 junction. The presence of unique sites flanking the promoter region makes it possible to replace the CMV region by any other promoter. A second series of vectors is thus obtained in which the p53 gene is placed under the control of an inducible promoter: the metallothionein promoter, which is inducible by heavy metals (cadnium and zinc). - of a sequence of 1173 bp corresponding to the cDNA encoding the mouse p53 protein in its wild-type form (Zakut-Houri et al.. Nature 36 (1983) 594). In this construct, the suppressor gene is in the form of cDNA, that is to say lacking introns. This makes it 5 possible especially to reduce the size of the vector. Moreover, it was verified that the expression levels obtained are comparable in the presence or absence of introns. - of the polyadenylation signal of the late 10 genes of the SV40 virus, which corresponds to a very efficient polyadenylation signal. Two unique Sail and Hindlll restriction sites are situated downstream of the polyadenylation signal. These sites permitted the insertion of the pIX regions of the adenovirus (Cf E3). 15 E2. Activity in vitro of the vector mp53wtI-CMV The functionality of the vector mp53wtI-CMV was confirmed in vitro, by transient expression in HeLa cells. For that, the vector was introduced into the cells by transfection and, 40 hours later, the protein 20 p53 was assayed by immunofluorescence and immunoprecipitation. The results obtained show that more than 50 % of the transfected cells induce high levels of protein p53.

E3. Construction of the vector znp53pXX. CMV 25 The plasmids used to generate, by homologous recombination, the recombinant adenoviruses expressing the p53 gene were constructed as follows: The feukaryotic expression vector mp53pIX.CMV 2653 265306 16 was constructed by insertion of the pIX sequence derived from the adenovirus genome between the Sail and EcoRI sites of mpl3wtl-.CMV. The pIX sequence was isolated from the recombinant plasmid pLTR-jSgal pIX 5 (Stratford-Perricaudet et al., J. Clin. Invest. 90 (1992) 626) by digestion by means of the enzymes EcoRV and Hindlll.

The expression vector mp53pIX.CMV thus obtained (Figure 2) has a unique Hindlll site 10 downstream of the pIX insert, which permits a linearization of the construct (Cf E4.). E4. Construction of a defective recombinant adenovirus carrying the p53 gene under the control of the CMV promoter.

The vector mp53pIX.CMV is linearized and cotransfected with a deficient adenoviral vector, into helper cells (line 293) supplying in trana the functions encoded by the adenovirus El regions (E1A and E11B).

The adenovirus Ad.p53 is obtained by homologous recombination in vivo between the mutant adenovirus Ad.dl324 (Thimmappaya et al., Cell 31 (1982) 543) and the vector mp53pIX.CMV, according to the following procedure: after linearization by the enzyme 25 Hindlll, the plasmid mp5"*pIX.CMV and the adenovirus dl324 are cotransfected into the line 293 in the presence of calcium phosphate, to allow homologous recombination. The recombinant adenoviruses thus 265306 17 generated are selected by purification on plates. After isolation, the recombinant adenovirus DNA is amplified in the 293 cell line, giving a culture supernatant containing the unpurified recombinant defective 5 adenovirus having a titre of about 1010 pfu/ml.

The viral particles are generally purified by cesium chloride gradient centrifugation according to known techniques (see especially Graham et al..

Virology 52 (1973) 456) . The adenovirus Adp53 can be 10 preserved at -80°C in 20 % glycerol.

The capacity of the adenovirus Ad-p53 to infect cells in culture and to express a biologically active form of wild-type p53 in the culture medium has been demonstrated by infecting cells of the human 293 15 line. The presence of p53 in the culture supernatant was then revealed using a p53-specific monoclonal antibody.

These studies make it possible to show that the adenovirus does indeed express a biologically 20 active form of p53. 265306

Claims (18)

18 CLAIMS

1. Defective recombinant adenovirus containing a heterologous DNA sequence whose expression in a target cell makes it possible to at least partially inhibit cell division, the sequence comprising - at least one gene chosen from among tumour suppressor genes, antisense genes whose expression in the target cell makes it possible to control the transcription or the translation of genes favouring cell proliferation and genes whose expression product induces apoptosis of the infected cell; and - a promoter sequence permitting the expression, in the infected cell, of the gene, the promoter sequence having a different origin from that of the gene.

2. Defective recombinant adenovirus according to claim 1 containing, a sequence encoding the wild-type p53 protein under the control of a heterologous promoter.

3. Defective recombinant adenovirus according to claim 2, containing a sequence encoding the wild-type p53 protein tinder the control of the early cytomegalovirus promoter.

4. Adenovirus according to any one of claims 1 to 3, characterized in that it lacks the regions of its genome which are necessary for its replication in the 26530 19 target cell.

5. Adenovirus according to claim 4, characterized in that it is a type Ad 5 adenovirus.

6. Adenovirus according to claim 1, characterized in that the antisense gene makes it possible to reduce the levels of translation of the ras, myc, fos and/or c-erb oncogenes.

7. Adenovirus according to any one of claims 1 to 6, characterized in that the heterologous DNA sequence comprises, in addition, a gene encoding a tumour-specific antigen and/or a gene encoding a lymphokine.

8. A defective recombinant adenovirus according to claim 1 substantially as herein, described with reference to the examples and the accompanying drawings.

9. Use of an adenovirus according to any one of claims 1 to 8, for the preparation of a pharmaceutical composition intended for the treatment and/or the prevention of cancers.

10. Use of an adenovirus according to claim 2, for the preparation of a pharmaceutical composition intended for the treatment and/or prevention of cancers which are associated with the presence of a mutated form of p53.

11. Use according to claim 9 or 10, for the preparation of a pharmaceutical composition for direct administration into the tumour to be treated.

12. Use according to claim 9, for the preparation of an anti-tumour vaccine. 20 265306

13- Pharmaceutical composition comprising one or more defective recombinant adenoviruses according to any one of claims 1 to 8•

14. Pharmaceutical composition according to claim 13, characterized in that it is in injectable form.

15. Pharmaceutical composition according to claim 13, characterized in that it comprises between 104 and 1014 pfu/ml of defective recombinant adenoviruses.

16. Pharmaceutical composition according to claim 13, characterized in that it comprises 10® to 1010 pfu/ml of defective recombinant adenoviruses.

17. Use of a defective recombinant adenovirus according to any one of claims 1 to 8, for the preparation of a pharmaceutical composition intended for the treatment and/or the prevention of cancers.

18. Use of a defective recombinant adenovirus according to any one of claims 1 to 8 containing a heterologous DNA sequence encoding the wild-type p53 protein for the preparation of a pharmaceutical composition intended to induce cell apoptosis. N.Z. PATENT C'r'-r 1 2 0 AUG 1997 RECEIVED " ... i