Pharmaceutical composition for treating papillomavirus tumours and infection

CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412 Pharmaceutical composition against tumors and butterfly virus infections The present invention relates to a pharmaceutical composition intended for the treatment or prevention of lesions associated with papillomaviruses and more particularly with papillomaviruses human (HPV) types 16, 18, 31, 33 and 45.

Papillomaviruses are DNA viruses with a circular genome of about 7,900 base pairs surrounded by a protein capsid.

A number of types of bovine papillomavirus (BPV), human (HPV) have been identified and their genome sequenced (Pfister, 1987, in The papovaviridae: The Pcrpillomaviruses (Salzman and Howley edition) Plenum Press, New York, p 1-38 ).

It includes an early region and a late region.

The late region contains two reading frames L1 and L2 which encode the major components of the capsid.

The early region contains at least the E1, E2, E4, E5, E6 and E7 reading frames.

The E 1 and E2 expression products regulate viral replication and the expression of viral genes, while those of the E5, E6 and E7 regions are involved in the processes of oncotyenic transformation of infected cells.

Indeed, it has been shown experimentally that the E5 protein of BPV-1 can transform cells in vitro (Schlegel et al., 1986, Science 233, 464-467).

The E6 proteins of BPV-1 and E7 of HPV-16 are involved in the induction and maintenance of oncogenic transformation.

The transforming power of E7 has been demonstrated for HPV-16 and HPV-18 (Kanda et al., 1988, J.

Virol. 62, 610-613 Vousden et al., 1988, Oncogene Res. 3, 1-9; Bedell et al., 1987, J.

Virol.

61, 3635-3640).

No function was found at E4.

In humans, HPVs are associated with conditions ranging from benign skin infection to warts and malignant tumors.

These viruses are highly specific for the epidermal epithelium of the genital, oral and respiratory tract.

Epidemiological data strongly suggest the role of certain strains CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412 -2 in cancer of the cervix and lower pathways, in particular HPV16 and 18 and in a lesser degree HPV-31, 33 and 45.

Cervical cancer is the second leading cause of female cancer in the world.

According to the WHO, 460,000 new cases are listed each year, of which at least 200,000 cases are clearly associated with E-TPV.

A whole series of studies demonstrate the transforming role of these viruses, their specific integration into the genome of neoplastic cells, their gene activity in cancer cells and the importance of the expression of the early E6 and E7 genes in the maintenance of the phenotype. malignant HPV positive neoplastic cells (Monsenego, J.

Impact Medecin, March 11, 1994).

The pathologies associated with HPV viruses pose a therapeutic problem because of their persistent and recurrent nature.

Many approaches have already been used in the treatment of these diseases such as surgery, chemotherapy, antiviral agents and immunotherapy.

In this regard, European patent EP 0 462 187 describes a vaccination approach using the early genes of papillomaviruses to establish immunity against tumors resulting from the integration of the HPV genome into cellular DNA and in which the proteins capsid are no longer expressed.

Application WO 93/02184 teaches a therapeutic approach based on the use of capsid antigens as immunogenic agents.

These documents do not suggest the possibility of combining the preventive effect provided by the early polypeptides and the curative effect conferred by the late polypeptides of papillomaviruses to generate compositions suitable for all serious pathologies due to HPV.

Furthermore, in recent years, it has been proposed to use polypeptides having immunostimulatory activity in order to activate T cells with a more or less beneficial result depending on the pathologies targeted (see for example WO 96/11279 ), The present invention relates more precisely to a preparation based on a mixture of antigens originating from the early and late regions of a papillomavirus or a vector expressing them simultaneously, with the aim of establishing lasting immunity against infected cells.

The vaccine candidates proposed in the context of the present invention can be used as a preventive measure CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412 -3 (immunoprophylaxis) to limit the development or the spread of the viral infection to neighboring tissues or as a curative (immunotherapy) to prevent or reduce tumor development in infected patients.

The use of the capsid antigens will induce the production of antibodies against the antigenic epitopes localized on the surface of the viral particles preventing the infection from establishing itself durably. The use of early proteins will make it possible to induce immunity against infected cells after integration of the viral DNA.

The present invention also provides a preparation combining a polypeptide originating from a papillomavirus and an immunostimulatory molecule.

One of the advantages of such a composition is that it combines the specific immunity induced by the viral antigens and the nonspecific immunity induced by the immunostimulatory molecule and intended to enhance the specific response.

The aim of the present invention is to make pharmaceutical compositions available to the public allowing the treatment of HPV infections and more particularly serious pathologies such as cancer of the cervix of the uterus, with improved efficacy compared to the compositions. of the prior art.

This is why the present invention relates to a pharmaceutical composition intended for the treatment or prevention of a papillomavirus infection or tumor which comprises, as therapeutic agents:

(1) at least one polypeptide originating from the early region of a papillomavirus and at least one polypeptide originating from the late region of a papillomavirus, (2) at least one polypeptide originating from the early region of a papillomavirus, at least a polypeptide originating from the late region of a papillomavirus and at least a polypeptide having immunostimulatory activity, or (3) at least one polypeptide originating from an early or late region of a papillomavirus and at least one polypeptide having immunostimulatory activity.

In general, the term polypeptide refers to all or part CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412 -4 of the native polypeptide, to a chimeric polypeptide resulting from the fusion of sequences of different origins or to a variant characterized by at least one mutation (deletion, insertion and / or substitution) of an amino acid.

More particularly, a polypeptide in use in the context of the present invention has in particular an amino acid sequence of which the degree of similarity with the sequence of the native protein is greater than 75%, advantageously greater than 85% and, of preferably greater than 95%.

The degree of similarity can be easily calculated using an appropriate computer program or by aligning the sequences so as to obtain the maximum degree of homology and by counting the number of positions in which the amino acids of the two sequences are found. identical to the total number of positions.

The sequence of the HPV-16 and HPV-18 genomes is disclosed in Genebank under accession numbers K02718 and X05015 respectively.

As recalled above, the genome of viruses of the papillomavirus family, in particular BPV and HPV, codes for at least 8 polypeptides, two late polypeptides LI and L2 making up the viral capsid and 6 early polypeptides (E1, E2, E4, E5, E6 and E7) involved in the regulation, maintenance of the viral genome and the transformation of infected cells.

Although all of the early proteins of a papillomavirus can be used in the context of the present invention, it is advantageously chosen to use a polypeptide derived from the E6 protein, from the E7 protein or from the E6 and E7 proteins.

It may be advantageous to have recourse to a non-oncogenic variant mutated at the level of the regions involved in the process of transformation of the infected cells.

Such variants are described in the literature (Munger et al., 1989, EMBO J. 8, 4099-4105; Crook et al., 1991, Cell 67, 547556; Heck et al., 1992, Proc.

Natl.

Acad.

Sci. USA 89, 4442-4446; Phelps et al., 1992, J.

Virol. 66, 2418-2427).

A preferred polypeptide originating from the late region of a papillomavirus is derived from the L1 protein, the L2 protein or the L1 and L2 proteins.

According to particularly advantageous embodiments (2) and (3), a composition according to the invention also comprises a polypeptide having a CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412 -5imunostimulatory activity.

By "immunostimulatory" is meant the ability to stimulate a humoral immune response by activating B lymphocytes in order to enhance the production of antibodies directed against papillomavirus antigens or to stimulate a cell-mediated immune response by activating T lymphocytes to to elicit a significant cytotoxic response against tumor cells or cells infected with a papillomavirus. As an indication, the immunostimulation can be evaluated in an animal model (by comparison of the rejection rate in an animal to which a tumor expressing the target antigen has been grafted, this in the presence and in the absence of the immunostimulator). More generally, the means for demonstrating immunostimulation are indicated in Roitt (in Inrmtinology, 4th edition, Moby Ltd).

It is possible to use a native immunostimulatory molecule such as found in a mammal and, in particular in man, a part thereof, a chimeric molecule resulting from the fusion of sequences of various origins or a mutant, on the condition, however, of maintain immunostimulatory function.

Among all the molecules that can be envisaged, it will be preferable to use a polypeptide consisting of or derivative of interleukin-2, interleukin-7, interleukin-12 and coadhesion molecules B7.1 and B7. 2, interleukin-2 and the B7.1 molecule being particularly preferred in the context of the present invention. A preferred composition according to the invention comprises (1) a polypeptide originating from the E6 region, a polypeptide originating from the E7 region, a polypeptide originating from the LI region and a polypeptide originating from the L2 region of a papillomavirus, (2 ) a polypeptide originating from the E6 region, a polypeptide originating from the E7 region of a papillomavirus and a polypeptide derived from interleukin-2, (3) a polypeptide originating from the E6 region, a polypeptide originating from the E7 region of a papillomavirus and a polypeptide derived from the B7.1 molecule, (4) a polypeptide originating from the E6 region, a polypeptide originating from the CA 02234263 1998-03-30 WO 98/04705 PCT1FR97 / 01412 -6 E7 region of a papillomavirus, a polypeptide derived from niolecule B7.1 and a polypeptide derived from interleukin-2, (5) a polypeptide originating from the E6 region, a polypeptide originating from the E7 region , a polypeptide originating from the LI region, a polypeptide originating in the L2 region of a papillomavirus and a polypeptide derived from interleukin-2, (6) a polypeptide originating in the E6 region, a polypeptide originating in the E7 region, a polypeptide originating in the LI region, a polypeptide originating from the L2 region of a papillomavirus and a polypeptide derived from the B7 molecule. 1, or (7) a polypeptide originating from the E6 region, a polypeptide originating from the E7 region, a polypeptide originating from the L1 region, a polypeptide originating from the L2 region of a papillomavirus, a polypeptide derived from the B7 molecule. 1 and a polypeptide derived from interleukin-2.

Given the observations recalled above on the frequency of infection by certain types of HPV in cases of cervical cancer, a composition according to the invention comprises a polypeptide originating from a high-risk papillomavirus, of the type HPV-16, HPV-18, HPV-31, 1-1PV-33 and / or HPV45 and in particular the HPV-16 virus.

Of course, in the case where the composition includes several papillomavirus antigens, these can be of a common or different origin.

In general, a polypeptide originating from a papillomavirus or having immunostimulatory activity can be produced by conventional methods of chemical synthesis or by recombinant DNA techniques (see for example Maniatis et al., 1989, Laboralory Mainral, Cold Sprinçl, Harbor, Laboratory Press, Cold Spcing Harbor, NY).

More particularly, a method of preparation comprises the act of cultivating a cell transformed with a DNA fragment encoding the polypeptide in question to generate a producer cell and the act of harvesting said polypeptide from the culture.

The producer cell can be of any origin and without limitation, a bacterium, a yeast or a mammalian cell, insofar as CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412 -7fragment d The DNA under consideration is either integrated into its genony or integrated into an appropriate expression vector capable of replicating.

Of course, the DNA fragment is placed under the control of transcription and translation signals allowing its expression in the producer cell.

Expression vectors and control signals are known to those skilled in the art.

The present invention also relates to a pharmaceutical composition intended for the treatment or prevention of a papillomavirus infection or tumor which comprises, as therapeutic agent (s) one or more recombinant vector (s) in the (s) which are inserted DNA fragments coding for:

(1) at least one polypeptide originating from the early region of a papillomavirus and at least one polypeptide originating from the late region of a papillomavirus, (2) at least one polypeptide originating from the early region of a papillomavirus, at least a polypeptide originating from the late region of a papillomavirus and at least one polypeptide having immunostimulatory activity, or (3) at least one polypeptide originating from an early or late region of a papillomavirus and at least one polypeptide having immunostimulatory activity;

said DNA fragments being placed under the control of the elements necessary for their expression in a cell or a host organism.

According to this moreover preferred alternative, the therapeutic agent is a vector into which are inserted the DNA fragments encoding the polypeptides originating from a papillomavirus or immunostimulators as defined above.

This type of composition has the advantage of inexpensive production and high stability under various environmental conditions.

In particular, the storage conditions are less restrictive.

The DNA fragments encoding a polypeptide originating from a papillomavirus can be obtained by cloning, by PCR (Polymerase Chain Reaction) or by chemical synthesis according to conventional techniques CA 02234263 1998-03-30 WO 98/04705 PCT / Fit97 / 01412 - Commonly in use from papillomavirus positive cells obtained from patients or collections.

The gene encoding a polypeptide having immunostimulatory activity can also be isolated according to standard techniques from the genome of a cell (genomic type) or from the messenger RNAs of a cell in which it is expressed (complementary DNA type).

Furthermore, the gene in question can code for (i) a soluble molecule, either intracellular or secreted into the external medium or (ii) a molecule anchored in the membrane and therefore present on the surface of the cells which express it.

A preferred recombinant vector in the context of the invention is a viral vector into the genome of which the aforementioned DNA fragments have been inserted so as to allow their transfer and expression in a host cell or organism.

A viral vector which can be used in the context of the present invention can be derived in particular from a poxvirus, from an adenovirus, from a retrovirus, from a herpes virus or from a virus associated with adenovirus.

Advantageously, it will be a non-integrative vector and of attenuated virulence.

Such vectors as well as their preparation techniques are known to those skilled in the art.

In the case where an adenoviral vector is used, a non-replicating vector will preferably be used by deletion of regions essential for replication and, in particular, of the majority of the E1 region in order to prevent its propagation to the region. within the host organism or environment.

It goes without saying that it is possible to modify or delete other regions of the adenoviral genome, insofar as the defective essential hains are complemented in trans.

A preferred adenoviral vector according to the invention will retain the sequences essential for packaging, namely the 5 ′ and 3 ′ ITRs (Inverted Terminal Repeat) and the packaging region.

The various adenoviral vectors as well as their preparation techniques are conventional and are described in Graham and Prevect (199 1, in Metfzods in Molecttlar Biology, vol 7, p 109-128; Ed:

EJ

) 0 Murey, The Human Press Inc.) and international application WO 94/28152. If CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412 -9 is a retrovirus, we keep the LTRs (Loiig Terininal Repeat) and the encapsidation sequences (see for example Naviaux and Verma , 1992, Current Opinion in Biotechnology 3, 540-547).

According to an advantageous embodiment, a recombinant viral vector according to the invention is derived from a poxvirus and, in particular, from an avian poxvirus, such as canary poxvirus, from a fowlpox or from a vaccinia virus, the latter being preferred.

Among all the vaccinia viruses that can be envisaged in the context of the present invention, the Copenhagen, Wyeth and Modified Ankara strains are preferably chosen (MVA for Modified Vaccinia Virus Ankara).

The general conditions for obtaining a vaccinia virus capable of expressing a heterologous gene are taught in European patent EP 83 286 and application EP 206 920.

As for the MVA virus, it is more particularly described in (Mayr et al., 1975, Infection 3, 6-14; Sutter and Moss, 1992, Proc.

Natl.

Acad.

Sci. USA 89, 10847-10851).

Of course, in the context of the present invention, the DNA fragments are placed under the control of the elements necessary for their expression.

These include the appropriate elements of transcription regulation as well as signals for the initiation and termination of translation.

The promoter is of particular importance. In general, we will use a promoter functional in the organism or the host cell that we want to treat and adapted to the vector used.

In addition, it can be modified so as to contain regulatory sequences, for example an element which activates transcription or sequences responding to certain cellular signals. In this regard, it may be advantageous to use a tissue-specific promoter since the lesions associated with papillomaviruses are localized in the genital tract or a promoter responding to specific tumor signals (for example activated in the presence of growth factors generally overexpressed by tumor cells) in order to limit expression to tumor cells only.

Among the promoters that can be envisaged in the context of the invention, CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412 - 10 mention the SV40 (Siinian Virus 40), HMG (Hydroxy-Metliyl-Glutarylcoenzyme A) promoters. ), TK (Thymidine Kinase), CMV (cytomegalovirus), RSV (Rous Sarcoma Virus), MLP (Major Late Protnoter) of adenoviruses adapted to the adenoviral vector and the LTR of Mo-MLV (Moloney Murine Leukemia Virus) more specific retroviral vectors.

When the vector is derived from a poxvirus, the promoter will preferably be the promoter of a gene of the poxvirus used, for example the promoter of the gene encoding the 7.5K, H5R, TK or KIL protein of the vaccinia virus.

The latter are described in the literature and can be cloned from the viral genome by standard techniques.

Furthermore, the elements necessary for expression may also include sequences improving expression or maintenance in the host cell (intron, signal sequence, transcription terminator sequence, translation initiation site, sequences modifying the presentation. polypeptide to host immune system cells ....).

However, in the case of a vector derived from a poxvirus, the use of introns will be avoided.

A composition according to the invention can be obtained either with several recombinant vectors each expressing a given polypeptide or with a single vector expressing the DNA fragments corresponding to the chosen polypeptides placed under the control of independent or common elements.

According to this last option, we can have recourse to sequences making it possible to initiate translation internally (IRES) or to in-phase fusions of the various genes.

The general conditions for obtaining a recombinant vector in use in the present invention are widely described in the state of the art.

As regards a poxviral vector, reference may be made to European patent EP 83 286, the content of which is incorporated here by reference.

These conditions are applicable to other viruses acceptable as vectors which possess a non-essential genomic region into which the expression blocks can be incorporated.

Of course, they can be inserted in the same locus or a different locus.

For example, when using a vaccinia virus from CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412 - I1 strain Copenhagen, the preferred insertion site is Iocus TK and / or KI L locus.

The insertion at the level of the viral TK gene has the effect of inactivating the latter and thus facilitating the selection of the recombinants.

In the context of a vaccinia virus of the MVA strain, the insertion of the papillomavirus and immunostimulator genes can be carried out within one of the excisions 1 to VI and, preferably, the excision zone II or III (Meyeret al., 1991, J.

Gen.

Virol. 72, 1031-1038; Sutter et al., 1994, Vaccine 12, 1032-1040).

In accordance with the aims pursued by the present invention, a recombinant vector may further comprise an expression block for a selection marker gene in order to facilitate the steps of isolation and purification of the recombinant virus.

Mention may in particular be made of the Neo gene conferring resistance to the antibiotic G418, the pac gene for resistance to puromycin, the TK gene of the herpes simplex virus type 1 (HSV-1) which confers sensitivity to certain analogues. nucleosides such as ganciclovir or acyclovir, the bacterial LacZ genes encoding β-galactosidase and gus A encoding β-glucuronidase.

These last two enzymatic markers make it possible to identify the recombinant viruses by staining in the presence of the substrates X-Gal (5-bromo-4-chloro-3-indolyl- (3-D-galactopyranoside) and XglcA (5-bromo-6-chloro -3-indolyl-pD-glucoronide) respectively.

A pharmaceutical composition according to the invention for the treatment or prevention of a papillomavirus infection or tumor, comprises one or more recombinant vector (s) derived (s) from a vaccinia virus of the Copenhagen strain or MVA in which (s) are inserted:

(1) a DNA fragment encoding the E6 protein of a papillomavirus, a DNA fragment encoding the E7 protein of a papillomavirus and a DNA fragment encoding the B7 molecule. 1, (2) a DNA fragment encoding the E6 protein of a papillomavirus, a DNA fragment encoding the E7 protein of a papillomavirus and a DNA fragment encoding interleukin-2, (3 ) a DNA fragment encoding the E6 protein of a papillomavirus, a CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412 - 12 DNA fragment encoding the E7 protein of a papillornavirus, a DNA fragment encoding the B7.1 molecule and a DNA fragment encoding interleukin-2, (4) a DNA fragment encoding the E6 protein of a papillomavirus, a DNA fragment encoding the E7 protein of a papillomavirus, a DNA fragment encoding the LI protein of a papillomavirus and a fragment DNA encoding the L2 protein of a papillomavirus, (5) a DNA fragment encoding the E6 protein of a papillomavirus, a DNA fragment encoding the E7 protein of a papillomavirus, a fragment DNA encoding the LI protein of a papillomavirus, a DNA fragment encoding the L2 protein of a papillomavirus and a DNA fragment encoding the B7.1 molecule, (6) a DNA fragment encoding the E6 protein of a papillomavirus, a fragment of DNA encoding the E7 protein of a papillomavirus, a DNA fragment encoding the LI protein of a papillomavirus, a DNA fragment encoding the L2 protein of a papillomavirus, and a DNA fragment encoding the L2 protein of a papillomavirus. interleukin-2, or (7) a DNA fragment encoding the E6 protein of a papillomavirus, a DNA fragment encoding the E7 protein of a papillomavirus, a DNA fragment encoding the LI protein of a papillomavirus, a DNA fragment encoding the L2 protein of a papillomavirus, a DNA fragment encoding the B7.1 molecule and a DNA fragment encoding interleukin-2.

On the other hand, a pharmaceutical composition according to the invention more particularly intended for immunoprophylaxis purposes, comprises one or more recombinant vector (s) derived (s) from a vaccinia virus of the Copenhagen strain or MVA in which are inserted:

(1) a DNA fragment encoding the L1 protein of a papillomavirus, a DNA fragment encoding the L2 protein of a papillomavirus and a DNA fragment encoding the B7 molecule. 1, (2) a DNA fragment encoding the L 1 protein of a papillomavirus, a CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412 - 13 fra (DNA component encoding the protein L2 of a butterfly virus and a DNA fragment encoding interleukin-2, or (3) a DNA fragment encoding the L1 protein of a papillomavirus, a DNA fragment encoding the L2 protein of a papillomavirus, a DNA fragment encoding interleukin-2 and a DNA fragment encoding the B7 molecule. 1.

A composition according to the invention can be prepared according to the methods known in the field of vaccines and the applicable doses can vary over a wide range.

They depend in particular on the polypeptides and the virus employed, the pathology to be treated, the patient's condition and other parameters which can be evaluated by the clinician.

In general, however, the dose of virus per kilogram will be 104-1011, preferably 106-1010, and preferably 107-109 plaque-forming units (pfu) when the therapeutic agent is a viral vector and 0. 05 to 500 mg, advantageously from 0.5 to 200 mg and preferably from 1 to 100 mg when the therapeutic agent is of polypeptide origin.

A composition according to the invention can be administered by any conventional route of administration, in particular by the intravenous, intramuscular, subcutaneous or subepithelial route or else by scarification.

In the case of an accessible tumor, it is also possible to use direct injection into or near the tumor site or topical application. As a vaccine, a composition according to the invention can be administered according to current practices in the field, for example in a single dose or repeated one or more times after a certain time interval.

On the other hand, as part of a curative treatment, it can be administered frequently for a period sufficient for the treatment to be effective.

When the therapeutic agent is a viral vector, this virus is preferably in live form. As this is a poxviral vector, it will be preferable to use an attenuated strain such as the MVA strain or the Copenhagen thymidine kinase negative strain.

Finally, a recombinant viral vector can be attenuated by an appropriate chemical treatment known to those skilled in the art.

However, it is also possible to envisage injecting a killed recombinant vector.

CA 02234263 2006-05-30 -14 According to a preferred embodiment, a pharmaceutical composition according to the invention comprises a therapeutically effective amount of a recombinant vector in combination with an acceptable carrier from a pharmaceutical point of view.

The support is chosen so as to allow its administration by injection into humans or animals.

It can also comprise a vehicle, a diluent and / or an adjuvant and be provided in liquid or lyophilized form.

The present invention also relates to a pharmaceutical composition according to the invention, as a medicament for the treatment or prevention of cervical cancer, low-grade cervical dysplasia and papillomavirus infection.

The present invention also relates to a method of treatment or prevention of the pathologies mentioned above, according to which an individual in need of such treatment is administered a pharmaceutically effective amount of a mixture of polypeptide or a recombinant vector in use in the present invention.

The present invention also relates to a pharmaceutical composition intended for the treatment or prevention of a papillomavirus infection or tumor which comprises, as therapeutic agents, a polypeptide originating from the early E6 region, a polypeptide originating from the early E7 region, a polypeptide originating from the late L1 region, a polypeptide originating from the late L2 region of a papillomavirus and at least one polypeptide having immunostimulatory activity selected from the group consisting of interleukin-2, interleukin-7 and coadhesion molecules B7.1 and B7.2 , it being understood that the polypeptide having an immunostimulatory activity is a native polypeptide and the polypeptides originating in the early region and the original polypeptides CA 02234263 2008-03-31 - 14 (a) naires of the late region are native polypeptides or character variants. sés by at least one mutation of an amino acid.

The present invention also relates to a pharmaceutical composition intended for the treatment or prevention of a papillomavirus infection or tumor which comprises, as therapeutic agent (s) one or more recombinant vector (s) derived from a poxvirus in which are inserted DNA fragments encoding:

(1) a polypeptide originating in the early E6 region, a polypeptide originating in the early E7 region, a polypeptide originating in the L1 region, a polypeptide originating in the L2 region of a papillomavirus, or (2) at least one originating polypeptide from the early region of a papillomavirus, at least one polypeptide originating from the late region of a papillomavirus and at least one polypeptide having immunostimulatory activity selected from the group consisting of interleukin-2, interleukin-7 and co-adhesion molecules B7.1 and B7.2, the DNA fragments being placed under the control of independent elements necessary for their expression in a cell or a host organism, the composition comprising a support pharmaceutically acceptable.

The present invention also relates to a pharmaceutical composition for the treatment or prevention of a papillomavirus infection or tumor which, as therapeutic agents, consists of one or more polypeptide (s) originating from an early region. a papillomavirus and one or more polypeptide (s) having immunostimulatory activity selected CA 02234263 2006-05-30 - 14 (b) from the group consisting of interleukin-2, interleukin-7 and co-adhesion molecules B7.1 and B7.2.

Finally, the present invention also relates to a pharmaceutical composition intended for the treatment or prevention of a papillomavirus infection or tumor which, as therapeutic agent (s), consists of one or more recombinant vector (s) into which are inserted DNA fragments encoding one or more polypeptide (s) originating from an early region of a papillomavirus and one or more polypeptide (s) having immunostimulatory activity selected from the group consisting of interleukin-2, interleukin-7 and coadhesion molecules B7.1 and B7.2, said DNA fragments being placed under the control of the elements necessary for their expression in a cell or a host organism.

The present invention is illustrated with reference to the following Figures.

Figure 1 is a schematic representation of the vector pTGS021 allowing the transfer to the TK locus of vaccinia Copenhagen of the late genes L1 and L2 of I-IPV-16 placed under the control of the p7.5K promoter, the two cassettes being in opposite orientation one in relation to the other, Fioure 2 is a schematic representation of the vector pTG5065 allowing the transfer to the KIL locus of the Copenhagen vaccinia of the early E6 and E7 genes of HPV-16 placed under the control of the pH5R promoter (the two cassettes being in opposite orientation relative to each other. to the other), the human IL-2 gene (IL2h) placed under the control of the p7.5K promoter and the LacZ marker gene (btaGAL) placed under the control of the pK 1 L promoter.

Figure 3 illustrates schematically the strategy which can be employed to introduce the late genes LI and L2 of I II'V-16 within the exclusion zone II of a vaccinia virus MVA, the recombination arms left and right being indicated (BRG2 and BRD2) respectively.

CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412 -15E \ EIYiPLES The present invention is more fully described, without being limited, with the aid of the following examples.

The constructions described below are carried out according to the general techniques of genetic engineering and molecular cloning, detailed in Maniatis et al., (1989, supra) or according to the manufacturer's recommendations when a commercial kit is used.

The mutagenesis directed in vitro by synthetic oligonucleotides is carried out using the kit distributed by Amersham.

PCR amplification techniques are known to those skilled in the art (see for example PCR Protocols-A guide to methods and applications, 1990, edited by Innis, Gelfand, Sninsky and White, Academic Press Inc). Regarding the repair of restriction sites, the technique used consists of filling the protruding 5 'ends with the large fragment of DNA polymerase I from E. coli (Klenow).

The cloning steps, the recombinant M 13 bacteriophages are multiplied on the E. coli strain NM522 (Stratagene) in a minimum agar medium (7.5% agar) or in a rich liquid LBM medium.

The recombinant plasmids carrying the gene for resistance to ampicillin are replicated in the strains E. coli C600 (Stratagene), BJ 5183 (Hanahan, 1983, J.

Mol.

Biol. 166, 557-580) and NM522 on agar or liquid medium supplemented with 100 ~, ~. / Ml of antibiotic.

The BJ5183 strain is preferably used when the cloning is carried out by homologous recombination (Bubeck et al., 1993, Nucleic Acid Res.

21, 3601-3602).

The construction of the recombinant vaccinia viruses is carried out according to conventional technology in the field disclosed in the documents already cited and in Mackett et al. (1982, Proc.

Natl.

Acad.

Sci. USA 79, 7415-7419) and Mackett et al. (1984, J.

Virol. 49, 857-864).

EXAMPLE 1 Construction of the Copenhagen vaccinia virus: ue VVTG5021 & 5065 expressing the E6 zenes. E7. L1 and L2 of H_PV-16 and human izene IL-2 CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412 - 16 A.

Isoleniejlt of the LI and L2 genes of HPVI6 The fragments encoding the L 1 and L2 proteins are isolated by PCR from genomic DNA of Caski cells (ATCC 1550) according to the general techniques of the art.

The amplification fragment carrying the LI sequences is subcloned into the vector M13TG130 (Kieny et al., 1983, Gene 26, 91-99), to give the construction M13TG8171.

The sequence of the cloned LI gene reveals several mutations with respect to the sequence contained in Genebank (accession K02718): C in place of an A in position 248, C in place of an A in position 253, G in place of an A in position 537, G in place of a C in position 682, G in place of an A in position 874, insertion of an ACT triplet in position 1393, deletion of a GAT triplet in position 1390. Insertion of the PCR fragment carrying the L2 sequences into the vector M13TG6131 leads to M13TG9126.

There are 5 point mutations relative to the sequence disclosed in Genebank: C instead of a T at position 378, A instead of a G at position 691, A instead of a G at position 702, G in place of an A in position 990 and C in place of an A in position 1092. As an indication, the vector M 13TG6131 is derived from MI 3TG 131 (Kieny et al., 1983, stipra) by mutation of the internal BgIII site located outside of multiple cloning sites.

B. Constructing dtr vector pTG5021 for the transfer of the LI and L2 genes into the 7K loctts of the vaccinia virus dtr genome The gene encoding the LI protein is modified by creating a BgIII site upstream of the initiator ATG .

The mutated gene is excised from the preceding vector (M13TG8185) by BgIII-SacI digestion and inserted between the Barr1HI and SacI sites of pTG186-poly.

The resulting construct is called pTG4019.

The pTG186-poly transfer vector is described in detail in French patent 2,583,429.

It has 10 restriction sites for inserting the gene to be transferred and the p7.5K promoter for controlling its expression.

The gene encoding the L2 protein is isolated from MI3TG9126 by digestion CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412 - 17 BgtII-HitidIII then cloned between the BamHI and Hijidlll sites in 3 'of the 7.5K promoter .

MI 3TG9127 is obtained in which a SalI site is introduced by localized mutagenesis upstream of the promoter.

The expression block "p7.5K-L2 gene" is isolated from the mutated vector M13TG9129 and cloned into the SacI site of the transfer vector pTG4019 such that the two cassettes p7.5K-L1 and p7.5K-L2 are in reverse orientation.

The construction thus obtained pTG5021 is shown in Figure 1.

The expression blocks are transferred into the genome of the vaccinia virus Copenhagen strain by homologous recombination.

The recombinant virus designated VVTG5021 is isolated by selection with 5-bromodeoxyuridine (5BUDR) C.

Isolation of E6 and E7 gets from HPVI6 The E6 and E7 genes are isolated from the Caski cell line as described in examples 2 and 3 of European patent EP 0 462 187.

Two constructs were derived from the M13E7 / E6 clone containing the E6 and E7 genes of HPV-16 in order to facilitate subsequent cloning steps.

The first designated M 13TG8188 results from the introduction by site-directed mutagenesis of PstI and BaniHI sites respectively upstream and downstream of the E7 gene and the second, M 13TG8189 comprises a PstI site upstream of the E6 gene. The introduction of point mutations upstream of an initiator ATG and downstream of a stop codon are within the abilities of those skilled in the art.

The association of the E7 protein of HPV-16 with the product of the retinoblastoma gene has been demonstrated by various authors (see for example Munger et al., 1989, EMBO J. 8, 4099-4105) and correlated with its transforming power. .

For obvious safety reasons, a non-oncogenic mutant is generated deleted from the sequences encoding amino acids 21 to 26 of the native E7 protein involved in the transformation function by site-directed mutagenesis of the vector M 13TG8188 using the oligonucleotide oTG5 I 18 (SEQ ID NO: 1).

MI 3TG9104 is obtained.

The mutated E7 gene is hereinafter referred to as E7 *.

Likewise, it was demonstrated that the E6 protein of HPV-16 could interact with the expression product of the tumor suppressor gene p53 (Crook et al., CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412-181991, Cell 67, 547-556).

The domain involved in this interaction has been clearly defined and lies between residues 1 I 1 to 115 of the native protein.

The vector M 13TG9125 is generated by mutagenesis of M 13TG8189 using the oligonucleotide oTG5377 (SEQ ID NO: 2).

The mutated E6 gene is hereinafter referred to as E6 *.

D.

Construction of the transfer vector pTG5065 carrying the E6 and E7 genes of HPY 16 and the IL-2 gene integrated hztmaine arr locris KIL.

The 5.2 kb EcoRI K fragment from the left end of the Copenhagen vaccinia virus genome (Guillard et al., 1985, J.

Virol. 53, 316-318) is cloned into the plasmid pUC8 (Gibco BRL) linearized with EcoRI.

The vector thus obtained is then subjected to a controlled digestion with Bg1II followed by ligation in order to delete a fragment of 855 bp encoding the host restriction gene K 1 L (Guillard et al., 1986, Proc.

Natl.

Acad.

Sci., USA 83, 5573-5577).

The 3.4 kb BgII fragment is isolated from this intermediate construct, designated pUCBKhr, then cloned into the BamHI site of the plasmid pUC7 (Gibco BRL).

PBAC 1 is generated in which an XhoI adapter is introduced to the left of the unique Bg1II site.

After digestion with XhoI and BglII, a SalI-BglII fragment carrying the vaccinia promoter p7.5K is inserted.

The two EcoRI sites are removed by EcoRI digestion followed by Klenow treatment and religation.

The vector pTG2147 is obtained by introduction into the unique Bg1II site of a multiple cloning site isolated from the vector p poly II (Lathe et al., 187, Gene 57, 193-201) isolated in the form of a Bg1II-I3amHI fragment .

It therefore comprises the recombination arms allowing insertion at the K 1 L locus flanking the p7.5K promoter followed by restriction sites.

The E6 * and E7 * genes are cloned downstream of the vaccinia promoter pH5R contained in the vector M13TG9132, to give M13TG9138 and M13TG9139 respectively. As an indication, the vector M13TG9132 comes from the insertion of the promoter of the H5R gene isolated by PCR from the viral genome into the phage M13TG6131.

Furthermore, the cDNA encoding human interleukin-2 is isolated from CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412 - 19 plasmid pTG36 (French patent 2,583,770), introduced into an intermediate vector and emerged by SaII-BamHI digestion to be inserted into the transfer vector pTG2147 targeting the K 1 L_ locus The insertion takes place at the PstI and Xba1 sites located downstream of the p7.5K promoter, using adapters cloning.

We obtain pTG5056.

The pH5R-E6 * expression block is isolated from the vector M13TG9139 in the form of a BgIII BamH1 fragment which is introduced into the BamH1 site of the vector pTG5056.

The vector pTG5060 is generated.

The pH5R-E7 * expression block is purified from M 13TG9138 and inserted into the BamHI site of the vector pTG5060, to give pTG 5062.

It is indicated that the insertion at the KIL locus leads to recombinant viruses having a reduced capacity of replication or even destroyed in certain cell types.

For these reasons, a positive selection marker is introduced to facilitate the identification of the recombinant viruses.

The vector pTG5065 results from the cloning into the BamHI site of pTG5062 of a “pK1L-LacZ gene” expression cassette isolated from the plasmid pIV75 (Chen et al., 1993, Virology 196, 682-693) by Bg1II-BamHI cleavage.

As visualized in FIG. 2, it allows the transfer of the E6 *, E7 * and IL-2 blocks within the K1L locus of the vaccinia virus The recombinant vaccinia virus, called VVTG5065, is generated by homologous recombination after transfection of the vector pTG5065 in chicken embryonic cells infected with wild-type Copenhagen vaccinia and identified by staining under X-Gal agar.

Vaccinia viruses VVTG5021 and VVTG5065 are used in double infection assays.

The double recombinants are selected according to two martlueurs: formation of blue plaques in the presence of X-Gal and deletion of the TK marker.

These, called VVTG5021 & 5065, express the p7.5K-IL-2, pH5R-E6 * and pH5R-E7 * blocks integrated at the K1L locus and the p7.5K-LI and p7.5K-L2 blocks integrated at the TK locus.

Expression of HPV genes can be verified by Western blot analysis using appropriate monoclonal or polyclonal antibodies or by reverse PCR.

IL-2 production can be evaluated by ELISA test or CTL test as described in the literature. As an indication, the level of expression in vitro is of the order of 60 ng / ml / 106 infected cells at 1 pfu / cell and 24 h.

CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412 -70EXAMPLE 2: Construction of a vaccinia Copenhagen ~ = vinis, ue expressing the E6 genes. Human E7, eleuene IL-2 The cDNA encoding human interleukin-2 is isolated from plasmid pTG36 by PstI digestion and inserted into the PstI site of plasmid pTG 186, giving rise to pTG 188.

The virus obtained by homologous recombination is called VVTG 188.

The E6 * gene is isolated from M13TG9125 in the form of a PstlBamHI moiety and inserted downstream of the 7.5K promoter between the PstI and XbaI sites of pTG2147 in the presence of a BanrHI-.XbaI adapter, giving rise to pTG5057.

The E7 * gene is placed under the control of the vaccinia H5R promoter producing MI3TG9138 and the cassette bordered by the BanrHI-Bg1I1 sites is subcloned into the BaniHI site of pTG50S7.

We obtain pTG5059, into the BamHI site of which the LacZ selection marker is inserted under the control of the promoter of the vaccinia gene KIL isolated by BgII-BamH1 digestion of the vector pIV75.

The resulting construct is designated pTG5061 and the virus generated by homologous recombination with the vaccinia genome VVTG5061.

A recombinant vaccinia virus expressing the E6 * and E7 * genes integrated into the K1L locus and the human IL-2 gene integrated into the TK locus is obtained by coinfection of chicken embryonic cells with the viruses VVTG5061 and VVTGI8S. As an indication, the latter produces approximately an amount of IL-2 greater than 400 ng per 106 infected cells at 1 pfu per cell for 24 h.

The doubly recombinant virus is designated VVTG5061 & 188.

EXAMPLE 3 Construction of a Copenhagen vaccinia virus expressing the E6_ E7 genes and the ~ ene encoding the human B7.1 adhesion cofactor _ The cDNA encoding B7.1 is isolated from an mRNA preparation obtained from the Daudi cell line (ATCC CCL-213) by reverse PCR using the primers oTG6353 and oTG6352 (SEQ ID NO: 3 and 4).

It is stated that the gene sequence is disclosed in Genebank under the accession number M27533.

The amplified fragment is cloned between the Bglll and EcoRI sites of M 13TG6131 CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412-21 leading to M13TG9149 then between the BanrHI and EcoRI sites of pTG186.

The construct is called pTG5090 and the virus obtained by homologous reconibination VVTG5090.

A vaccinia virus expressing the E6 * and E7 * genes of HPV-16 integrated at the K 1 L locus and the human B7.1 gene integrated at the TK locus can be obtained by co-infection of chick embryonic cells with the VVTG5061 viruses. and VVTG5090. the recombinant virus is designated VVTG5061 & 5090.

EXAMPLE 4: Construction of a vaccinia virus strain MVA expressing the Eb E7 zenes and the B7 1 aene integrated within the excision zone III! 0 The MVA virus is derived from the vaccinia virus strain Ankara.

It is not able to generate infectious particles on mammalian cells but grows properly on embryonic chicken fibroblasts.

Its adaptation to these cells caused the excision of 6 regions not essential for its development and its infectious cycle on this type of cells (disappearance of about 15% of the viral genome; Meyer et al., 1991, J.

Gen.

Virol. 72, 10311038). The integration of exogenous genetic material can be achieved at any of these areas of excision.

In the context of the present invention, the excisions II and III localized at the level of the restriction fragments HiiidII N and A respectively (Altenburger et al., 1989, Arch.

Virol. 105, 1527).

First, the vector pTG6019 is constructed allowing insertion into the excision zone III of the MVA virus.

The homologous recombination arms on either side of the excision zone III are isolated by PCR from the viral genome (see American patent US Pat. No. 5,185,146) and the primers oTG7637 and oTG7638 (SEQ ID NO: 5 and 6) for the left arm and oTG7635 and oTG7636 (SEQ ID NO: 7 and 8) for the right arm.

The amplified fragments are cloned at the EcoRI site of the vector pTG1E, to give pTG6019.

The genetic material to be transferred is inserted between the two recombination arms.

The pTGIE vector is similar to pTGIH (French patent 2,583,429) except for the presence of an EcoRI adapter in place of multiple cloning sites.

Firstly, an expression cassette for the gus marker gene CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412 -22A is inserted.

The 7.5K promoter is first of all cloned into the BanrHI site of pTG60I9.

PTG6021 is obtained into the BaniHI site of which the giis A gene generated in the form of a Bg1II-BaniHI fragment is inserted.

This can be obtained from the sequence disclosed in the literature.

The resulting construct is called pTG6022.

The presence of the marker will make it possible to distinguish wild-type viruses from recombinant viruses by detection of the GUS enzymatic activity by the XglcA substrate.

Red staining reveals {3-glucuronidase activity.

However, for clinical application, it may be useful to be able to remove this bacterial marker from the final product after selection for recombinant viruses.

To do this, advantage is taken of the ability of the vaccine to delete the sequences between two homologous sites. This is why a second p7.5K promoter is inserted downstream of the gus A gene in a sense orientation with respect to that which directs the expression of the latter.

The vector pTG6022 is modified by insertion between the BamIHI and SacI sites of a p7.5K fragment provided with cohesive ends, to give pTG6025.

This is completed by the insertion of the expression cassettes of the mutated E6 * and E7 * genes.

We start by introducing a new promoter sequence p7.5K this time in antisense orientation with respect to the previous ones.

This construction called pTG6039 therefore comprises the labile GUS cassette "p7.5K-> g7tsA-p7.5K--" followed by p7.5K in the opposite orientation.

In parallel, the E6 * and E7 * genes are isolated respectively from the vectors M13TG9104 and M 13TG9125 by BamHI and Pst1 digestion and assembled in orientation opposite to each other at the PstI site of M I3TG6131. The whole is produced in the form of a PstI fragment which is inserted into pTG6039 linearized by this same enzyme.

The vector pTG6056 is obtained which contains the following sequences p7.5K -> gzts A p7.5K --- E7 * -E6 * <- p7.5K ".

The B7.1 immunostimulatory gene is integrated into this latter construction.

To do this, the vector pTG6056 is cleaved by HrjiclllI and Kpri1 before being ligated in the presence of the oligonucleotides oTG 1045 I and oTG 10450 (SEQ ID NO: 9 and 10), to generate pTG6070.

These will allow the cloning of the expression cassette “pH5R-B7.1” by hornologous recombination.

CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412 - 23 To this end, the sequences B7. i isolated from M 13TG9149 are cloned downstream of the vaccinia promoter pH5R, to form MI3TG9184.

The Bg1II-LcoRI fragment carrying the cassette is integrated into the vector pTG6070 linearized with XhoI by homologous recombination in E. coli.

The vector pTG6079 is obtained which in total comprises the gits A marker gene in a "labile" form and the cassettes for expressing the early genes of HPV-16 as well as a cassette for the costimulation gene B7. 1.

The virus generated by homologous recombination with the MVA genome is designated MVATG6079.

The HPV-16 late gene expression blocks can be inserted within excision zone II using the transfer vector pTG60I8.

This is constructed by insertion at the EcoRI site of pTGIE of the left and right recombination arms bordering excision II, generated by PCR using the primers oTG7665 and oTG7584 (SEQ ID NO: 11 and 12) and oTG7639 and oTG7640 (SEQ ID N0: 13 and 14).

As previously, an expression cassette for a positive marker is integrated between the two arms to facilitate the detection of the recombinant viruses, the latter possibly being eliminated after the selection step (Spehner et al., 1990, J.

Virol. 64, 527-533).

The vector pTG6020 results from the cloning of the LacZ gene placed downstream of the p7.5K promoter in the vector pTG6018 linearized with BamHI.

PTG6020 is modified by inserting a p7.5K sequence between its BamHI and SacI sites located downstream of the LacZ gene.

We obtain pTG6024.

The LI and L2 cassettes can then be introduced according to a strategy as shown in Figure 3.

EXAMPLE 5 Construction of a MVA strain vaccinia virus expressing the E6 genes. E7 and IL-2 ç ~ ene integrated within the ITT excision zone. The same strategy as above is used to introduce the IL-2 gene into the vector pTG6056.

After cloning of the recombinant oligonucleotides oTG 10503 and oTG I 0502 (SEQ ID NO: 15 and 16) to produce pTG6076, the latter is cleaved by XhoI and the Bg1II-EcoRI fragment prepared from M 13TG9185 (pH5R-IL-2) inserted by homologous recombination.

The vector CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412 - ~ 4pTG6090 thus obtained comprises the marker gene gus A under a "labile" form, the cassettes for expressing the early genes of 1-IPV-16 as well as a human IL-2 gene cassette. The recombinant virus obtained by homologous recombination with the MVA genome is designated MVATG6090.

The late genes can be integrated into the excision zone II by using pTG6018, as indicated above.

EXAMPLE 6: Validation of the vector VVTG5021 & 5065 in animal model A.

Toxicity levels Nude mice received 10 pfu of VVTG5021 & 5065 or 107 pfu of wild-type vaccinia virus intravenously, intramuscularly, subcutaneously or intracranially.

Groups of 5 mice are made up according to the type of virus injected and the route of administration and the number of animals exhibiting lesions linked to the vaccine is evaluated 26 days after the injection.

The mice which received the recombinant virus show no lesions whatever the route of administration used, whereas the majority of the animals treated with wild-type vaccinia present lesions with a frequency and severity depending on the route of administration.

In addition, deaths are recorded in the case of intravenous and intracranial injection.

These data show that VVTG5021 & 5065 is attenuated compared to wild virus and that it does not cause lesions even after intracranial injection.

B. 1 x Imminoprophylaxis experiments with GT / '7G5021 & 5065 C57BL6 mice were vaccinated three times subcutaneously with 10' pfu of VVTG5021 & 5065.

Three days after the last immunization, these animals are challenged with 103 E7W 1 cells implanted subcutaneously. As an indication, the E7W 1 cells originate from a murine lymphoma line transfected with a vector expressing the oncogenic E7 gene of HPV-16.

The percentage of survival of the animals as a function of time is compared with that CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412 -25 obtained with control mice treated with 107 pfu of a non-recombinant vaccinia virus VVTG186 (derived from the vector TG186 described above).

The monitoring of mortality shows a difference between the two groups.

In the control group, 100% of the animals died on D36 while 40% of the animals vaccinated with V VTG5021 & 5065 are still alive on D36 and more than 30% on D51.

Thus, the recombinant viruses expressing HPV antigens and an immunostimulatory gene exhibit antitumor activity against tumor cells expressing the E7 oncogene of HPV-16.

VS'. L = xperiods of immunotherapy C57 BL6 mice are inoculated with 10 'E7 W 1 cells implanted subcutaneously (OJ). 10 ′ pfu of recombinant viruses are then administered also subcutaneously on D3 then D6 and finally D9 and the percentage of survival of the animals is determined relative to the control animals having received a non-recombinant virus.

While 100% of the control animals are dead, a notable increase in the survival of the animals injected with a mixture of VVTG5061 5021 and VVTG188 is observed.

Similar results are obtained after administration of VVTG5065 5021 and VVTG 188.

These immunotherapy experiments were reproduced using a different tumor model, BMK16 myc cells replacing E7w1 cells.

BMK 16myc cells are kidney cells from newborn mice transfected with the I-IP V-16 genome and the murine c myc gene.

The animals are treated with 10 'MVA TG6090 virus expressing the E6 *, E7 * genes of HPV-16 and human IL-2.

Compared to the controls, the treated mice exhibit a delay in tumor growth up to D 15.

CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412 -26 SEQUENCE LIST (1) GENERAL INFORMATION:

(i) DEPOSITOR:

(A) NAME: TRANSGENE SA (B) STREET: 11 rue de Moisheim (C) CITY: Strasbourg (E) COUNTRY: France (F) POSTAL CODE: 67082 (G) TELEPHONE: (33) 88 27 91 00 (M) FAX: (33) 88 27 91 11 (ii) TITLE OF THE INVENTION: Pharmaceutical composition against tumors and papillomavirus infections (iii) NUMBER OF SEQUENCES: 16 (iv) COMPUTER READABLE FORM:

(A) MEDIA TYPE: Tape (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Release # 1.0, Version # 1.25 (EPO) ( 2) INFORMATION FOR SEQ ID NO: 1:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETIC: NO (iii) ANTI-SENSE: NO (vi) ORIGIN:

(A) ORGANISM: Human papillomavirus (B) STRAIN: HPV-16 (C) ISOLATED INDIVIDUAL: synthetic oligonucleotide oTG5118 (E7 delete 21 to 26) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:

CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412 -27TCTGAGCTGT CATTTAATTG AGTTGTCTCT GGTTGC 36 (2) INFORMATION FOR SEQ ID NO: 2:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETIC: NO (iii) ANTI-SENSE: NO (vi) ORIGIN:

(A) ORGANISM: Human papillomavirus (B) STRAIN: HPV-16 (C) ISOLATED INDIVIDUAL: synthetic oligonucleotide oTG5377 (E6 delete 111 to 115) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:

TGTCCAGATG TCTTTGCAGT GGCTTTTGAC AG 32 (2) INFORMATION FOR SEQ ID NO: 3:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO (iii) ANTI-SENSE : NO (vi) ORIGIN:

(A) ORGANISM: Homo sapiens (C) ISOLATED INDIVIDUAL: synthetic oligonucleotide oTG6353 (PCR gene B7_1) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3:

TCAGCCCCTG AATTCTGCGG ACACTGTTAT ACAGG 35 (2) INFORMATION FOR SEQ ID NO: 4:

CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412 -28 (i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO (iii) ANTI-SENSE : YES (vi) ORIGIN:

(A) ORGANISM: Homo sapiens (C) ISOLATED INDIVIDUAL: synthetic oligonucleotide oTG6352 (PCR gene B7.1) (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 4:

TTGACCCTAA AGATCTGAAG CCATGGGCCA CAC 33 (2) INFORMATION FOR SEQ ID NO: 5:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETIC: NO (iii) ANTI-SENSE: NO (vi) ORIGIN:

(A) ORGANISM: Vaccinia virus (B) STRAIN: Modified Ankara (C) ISOLATED INDIVIDUAL: synthetic oligonucleotide oTG7637 (PCR zone III) (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 5:

GGGGGGGAAT TCAGTAAACT TGACTAAATC TT 32 (2) INFORMATION FOR SEQ ID NO: 6:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412 -29 (D) CONFIGURATION: linear (ii ) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETIC: NO (iii) ANTI-SENSE: YES (vi) ORIGIN:

(A) ORGANISM: Vaccinia virus (B) STRAIN: Modified Ankara (C) ISOLATED INDIVIDUAL: synthetic oligonucleotide oTG7638 (PCR zone III) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6:

GGGGGGGGAT CCGAGCTCAC CAGCCACCGA AAGAGCAAT 39 (2) INFORMATION FOR SEQ ID NO: 7:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETIC: NO (iii) ANTI-SENSE: NO (vi) ORIGIN:

(A) ORGANISM: Vaccinia virus (B) STRAIN: Modified Ankara (C) ISOLATED INDIVIDUAL: synthetic oligonucleotide oTG7635 (PCR zone III) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7:

GGGGGGGGAT CCGGAAAGTT TTATAGGTAG TT 32 (2) INFORMATION FOR SEQ ID NO: 8:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) CA 02234263 1998-03-30 WO 98 / 04705 PCT / FR97 / 01412 e -30 (iii) HYPOTHETIC: NO (iii) ANTI-SENSE: YES (vi) ORIGIN:

(A) ORGANISM: Vaccinia virus (B) STRAIN: Modified Ankara (C) ISOLATED INDIVIDUAL: synthetic oligonucleotide oTG7636 (PCR zone III) (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 8:

GGGGGGGAAT TCTTTGTATT TACGTGAACG 30 (2) INFORMATION FOR SEQ ID NO: 9:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 78 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETIC: NO (iii) ANTI-SENSE: NO (vi) ORIGIN:

(C) ISOLATED INDIVIDUAL: synthetic oligonucleotide oTG10451 (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 9:

CTTATACAGG GCGTACACTT TCCCTTCTCA ATCTCTCTCG AGTTGTATTT ATTTTCATTT 60 TTTAAGTATA GAATAAAA 78 (2) INFORMATION FOR SEQ ID NO: 10:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETIC: NO (iii) ANTI-SENS: YES CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412 -31 (vi) ORIGIN:

(C) ISOLATED INDIVIDUAL: synthetic oligonucleotide oTG10450 (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 10:

AGCTTTTTAT TCTATACTTA AAAAATGAAA ATAAATACAA CTCGAGAGAG ATTGAGAAGG 60 GAAAGTGTAC GCCCTGTATA AGGTAC 86 (2) INFORMATION FOR SEQ ID NO: 11:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETIC: NO (iii) ANTI-SENSE: NO (vi) ORIGIN:

(A) ORGANISM: Vaccinia virus (B) STRAIN: Modified Ankara (C) ISOLATED INDIVIDUAL: synthetic oligonucleotide oTG7665 (PCR zone II) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11:

ATGGTACCGA ATTCCATCTA CCAATTCATC CAACAACAT 39 (2) INFORMATION FOR SEQ ID NO: 12:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETIC: NO (iii) ANTI-SENSE: YES (vi) ORIGIN:

(A) ORGANISM: Vaccinia virus (B) STRAIN: Modified Ankara (C) ISOLATED INDIVIDUAL: synthetic oligonucleotide oTG7584 CA 02234263 1998-03-30 WO 98/04705 PCTIFR97 / 01412 -32 (PCR zone II) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12:

GGCTGCAGGA TCCGAGCTCA TCATGACGTC CTCTGCAATG G 41 (2) INFORMATION FOR SEQ ID NO: 13:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETIC: NO (iii) ANTI-SENSE: NO (vi) ORIGIN:

(A) ORGANISM: Vaccinia virus (B) STRAIN: Modified Ankara (C) ISOLATED INDIVIDUAL: synthetic oligonucleotide oTG7639 (PCR zone II) (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 13:

= GGGGGGGGAT CCTGTGAATC ATCCATTCCA CT - 32 (2) INFORMATION FOR SEQ ID NO: 14:

(i) CHARACTERISTICS OF THE-SEQUENCE:

(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETIC: NO (iii) ANTI-SENSE: YES (vi) ORIGIN:

(A) ORGANISM: Vaccinia virus (B) STRAIN: Modified Ankara (C) ISOLATED INDIVIDUAL: synthetic oligonucleotide oTG7640 (PCR zone II) CA 02234263 1998-03-30 WO 98/04705 PCT / FR97 / 01412 - ~~ (xi ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14:

GGGGGGGAAT TCGTTACTAA ATTGCAAGGA AAT 33 (2) INFORMATION FOR SEQ ID NO: 15:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 69 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETIC: NO (iii) ANTI-SENSE: NO (vi) ORIGIN:

(C) ISOLATED INDIVIDUAL: synthetic oligonucleotide oTG10503 (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 15:

CTCAAGTCAG TGTTGAGATG ATGCTTTGAC AACTCGAGTT TATTTTCATT TTTTAAGTAT 60 AGAATAAAA 69 (2) INFORMATION FOR SEQ ID NO: 16:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETIC: NO (iii) ANTI-SENSE: YES (vi) ORIGIN:

(C) ISOLATED INDIVIDUAL: synthetic oligonucleotide oTG10502 (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 16:

AGCTTTTTAT TCTATACTTA AAAAATGAAA ATAAACTCGA GTTGTCAAAG CATCATCTCA 60 ACACTGACTT GAGGTAC 77