MXPA01002350A - Treatment of cervical cancer - Google Patents

Abstract

Vectors for DNA immunization againstcervical cancer comprise a nucleic acid molecule encoding at least one none-toxic T-cell epitope of the E6 and/or E7 antigens of a strain of human papilloma virus (HPV) associated with cervical cancer, such as HPV-16, and a promoter operatively coupled to the nucleic acid molecule for expression of the nucleic acid molecule in a host to which the vector is administered.

Description

TREATMENT OF CERVICAL CANCER FIELD OF THE INVENTION The invention relates to cancer immunotherapy, specifically cervical cancer.

BACKGROUND OF THE INVENTION Cervical cancer is worldwide the second most common cause of cancer-related deaths in women. There are both epidemiological and experimental data that relate the etiology of cervical cancer with infection caused by human papillomavirus (HPV) types 16 and 18. The HPV virus is predominant between 35% and 40% of young women. Although the treatment of the disease in its early stage is relatively successful, recurrent disease occurs in 15% of patients. The results with patients with recurrent disease are relatively scarce. Accordingly, there is a need for a novel therapeutic approach (references 1, 2, 3, several references are mentioned in parentheses to better describe the state of the art to which this invention pertains.) The complete bibliographic information for each citation is at the end of the specification, just before the claims.

Pl 5 • ~ and considers that it is part of this, as a reference). The close association of HPV infection and cervical cancer suggests that a specific immunotherapeutic approach to a viral antigen could be a feasible strategy in the treatment of cervical cancer. The goal of specific immunotherapy is to stimulate the immune response of a patient who has the tumor, to attack and eradicate the tumor lesions. This strategy has been made feasible with the identification of tumor-associated antigens (TAA). The close association between HPV-16 infection and cervical cancer has made this disease a good candidate for the immunotherapeutic intervention (reference 4). In cervical cancer types with HPV DNA positive, the oncogenic proteins E6 and E7 are expressed. Experimental evidence suggests that these two proteins are responsible for the carcinogenic progression of cervical cancer types, since their expression leads to a transformed and immortalized state in cell cultures human epithelial cells (reference 5). Therefore, these two proteins are potential candidates for antigen-specific immunotherapy in cervical cancer induced by HPV, which is evaluated in the present. Although there are many questions related to the nature of immunity against HPV infection Natural and cervical cancer, it is evident that there is an immune component, since immunosuppressed individuals have a higher risk of developing a malignant cervical tumor (reference 6). In addition, it is most likely that this immunity is mediated by the cell branch of the immune response. Vast cellular infiltrates are observed when examining spontaneous regressions of cervical tumors (reference 7). So, it seems that a specific cellular response to the antigen is required to treat patients with cervical cancer. Although the nature of the results of an immunotherapeutic strategy has been identified, the ability to induce this type of response, using current vaccine technology, is limited. The development of the prophylactic vaccine for HPV, has focused on the preparations of recombinant subunits that consist of structural proteins of virion Ll and L2. In eukaryotic cells, the Ll (major capsid protein) organizes itself into particles analogous to the papillomavirus (VLPs) (reference 17). Although LL alone is sufficient for the assembly of VLPs, the coexpression of L2 (minor capsid protein) maintains a higher production of capsid (reference 18). In contrast, the development of the therapeutic vaccine in general has been aimed at the expression of P125 ? • * - wild type E6 and / or E7 protein. The expression vectors employed include vaccinia virus (e.g., as described in U.S. Patent No. 5,744,133 (reference 19, 20), alphavirus (for example, U.S. Patent No. 5,843,723) or others. poxivirus (e.g., U.S. Patent No. 5,676,950) Thus, it was determined that a DNA vector encoding HPV antigens involved in the carcinogenic progression of the disease, is the method Optimal by which an immunotherapeutic strategy could be successfully achieved. However, as previously observed HPV antigens, E6 and E7, are supposedly oncogenic and therefore immunization with a DNA construct that encode each or both proteins, could result in the induction of another malignant tumor (references 5, 10, 11). Therefore, in order to minimize the risks of toxicity, a genetically detoxified E7 molecule was encoded in a DNA construction. This detoxified molecule is modified through the deletion of the retinoblastoma binding region (Rb) (references 8, 9). Another method to achieve antigen-specific immunity without the inherent risks of oncogenic transformation, is the use of a A strategy with the epitope in which only key parts of the molecule are administered to induce a specific immune response (references 12 to 16). Here this approach was used in the design of the DNA-polyepitope construct where several T cell epitopes derived from both E6 and E7 are joined. A comparison of these two approaches is made here in a murine model of cervical cancer associated with HPV.

SUMMARY OF THE INVENTION According to the present invention, novel DNA constructs are provided for the administration of HPV antigens to a host, in order to provide an immune response therein. The invention extends to immunotherapy methods of tumors caused by HPV, in particular cervical cancer. The antigens chosen for the immunotherapy of cervical cancer types with HPV positive DNA, can be those expressed by these types of cancer. One of these HPV antigens is the E7 antigen, which has already been shown to have protective capacity in a vaccinia vector system. There are elements of potential toxicity with the use of a native form of E7 in a vaccine, due to its ability to bind to the Rb protein and thus promote an oncogenic state. A new version was built here P1254 Av E7 detoxified by deletion of the Rb binding site and was incorporated into pC V3 (Figure 2) in order to provide the pCMV-dE7 vector (Figure 1) for immunization. The detoxified E7 coding sequence was prepared from the unmodified coding sequence by replacing approximately 210 bp with DNA formed from paired oligos. The substitute sequence omitted a stretch of 18 nucleotides that code for the E7 region involved in complex formation with proteins of the cellular retinoblastoma (Rb) family, especially amino acids 21 to 26 of the native E7 protein. Immunization with the pCMV-dE7 construct resulted in significant protection against a tumor growth after grafting of live C3 tumor cells expressing the wild-type E7 molecule, showing that the E7 DNA construct can be used successfully to stimulate protective immunity without risks of associated toxicity. 20 Another of these HPV antigens is the E6 antigen. Antigens can be provided in DNA constructs, as full length proteins or in the form of specific T cell epitopes. To assess the focus of T cells, prepared a synthetic minigene containing sequences P125 nucleotides coding for T cell epitopes of both E6 and E7 proteins of HPV-16 (Figure 5A). A DNA construct (pCMV3-HPVT # 1) containing the minigene (Figure 6) was used to immunize mice. Mice immunized with the DNA construct of Figure 6 were 100% protected against tumor growth. The results of these studies indicate that DNA immunization can be used successfully to protect against tumor stimulation with live C3 and thus can be effective in the clinical treatment of cervical cancer. Therefore, in one aspect of the present invention, there is provided a vector comprising a nucleic acid molecule encoding at least one nontoxic T cell epitope of the E6 and / or E7 antigen of a human papilloma virus strain (HPV) ) associated with cervical cancer, eg, HPV-16 and a promoter operably coupled to the nucleic acid molecule for the expression of the nucleic acid molecule in a host to which the vector is administered. The promoter of preference is a cytomegalovirus promoter. The nucleic acid molecule may be contained within the CMV-3 plasmid containing the immediate early cytomegalovirus promoter that includes the activating and intron sequences, together with the polyA tail P1254 of bovine growth hormone and a kanamycin resistance gene. The elements of pCMV-3 are shown in Figure 2. The nucleic acid molecule, in one embodiment, is a coding sequence for E7 antigen, detoxified to prevent oncogenic replication in the host. The detoxification can be carried out in any convenient manner, including eliminating from the native sequence, the nucleic acid encoding the Rb-binding site, including that encoding amino acids 21-26 of the HPV-16. The vector which contains that nucleic acid molecule may have the identifying characteristics of pCMV3-dE7, including the restriction map and the building elements as seen in Figure 1. In another embodiment, the nucleic acid molecule encodes antigen epitopes E7, comprising amino acids 11 to 20, 49 to 57, 82 to 90 and 86 to 93 and epitopes of E6 antigen comprising amino acids 29 to 38 of HPV-16. In particular, in this embodiment, the nucleic acid molecule can be the one having SEQ ID NO: 4 or 5 or it can be the one that codes for an amino acid sequence having SEQ ID NO: 6. The vector containing this molecule of Nucleic acid has the identifying characteristics of pCMV3-HPVT # 1, including the restriction map and construction elements as observed P125 »... , .-. ' : *, .. * & »> * .- ,, Jaste¿-fts ^ £ a »& - ^ 3gaas & 1. & * ti¡Ü? Ü. in Figure 6. In another aspect, the present invention provides an immunogenic composition for in vivo administration to a host, comprising a vector as provided herein, which may include a pharmaceutically acceptable carrier therefor. In another aspect, the present invention provides a method for immunizing a host against cervical cancer caused by human papilloma virus (HPV), which comprises administering to the host an effective amount of the immunogenic composition of the invention. In a further aspect, the present invention provides a method for the treatment of a host having cervical cancer, comprising administering to the host an effective amount of the immunogenic composition of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS Figure IA is a map of a pCMV-dE7 plasmid comprising the plasmid pCMV-3 with detoxified E7 of HPV-16 inserted therein; Figure IB shows the nucleotide sequence (SEQ ID NO: 1) of dE7 amplified by PCR (polymerase chain reaction) from HPV-16 (pSE859.2). The 5 'and 3' PCR primers (SEQ ID Nos: 2, 3) are indicated in this figure P125 by means of pictures; Figure 2 is a map of a pCMV3 vector plasmid containing the CMV immediate early promoter including the activator and intron sequences, bovine growth hormone polyA (BGH PA) and kanamycin resistance (KN (R)); Figure 3 shows the assembly of the plasmid vector pCMV3 of pCMV2K; Figure 4 is a map of plasmid vector pEN-1; Figure 5A shows the nucleotide (SEQ ID NO: 4, full length; SEQ ID NO: 5, coding sequence) and the derived amino sequence (SEQ ID NO: 6) for a synthetic HPV minigene encoding a protein consisting of of five HPV 16 T cell epitopes (from NH2 to COOH terminal) E7: 49 to 57, 11 to 20, 82 to 90, 86 to 93 and E6: 29 to 38. Three spawning alanins were introduced between the epitopes; Figure 5B shows the assembly of synthetic oligonucleotides containing epitopes of E6 and E7 of HPV-16, to form the minigene of Figure 5A. The five individual oligonucleotides are indicated as I to V (SEQ ID Nos: 7, 8, 9, 10, 11). The epitopes of E6 and E7 were indicated above, the specific sequences and numbers correspond to the amino acid sequence of the full length E6 and E7 proteins of HPV-16; Y P125 Figure 6 shows the assembly of pCMV-3HPVT # l by inserting the synthetic minigene shown in Figure 5A in the polylinker, between the SalI and EcoRl sites of the vector pCMV3 of Figure 3.

GENERAL DESCRIPTION OF THE INVENTION The present invention provides an immunotherapeutic approach for cervical cancer caused by human papilloma virus (HPV), based on immunization with DNA. A series of experiments was performed on the C3 cervical cancer model system. Through the use of a DNA delivery platform, several vaccines based on E7 antigen were evaluated in order to check their ability to prevent tumor growth after stimulation of living tumor cells. Although the E7 antigen has been shown in a vaccine vector system that has some protective ability, as noted above, there are concerns related to the potential toxicity of using a native form of E7 in a vaccine, due to its ability to bind to the Rb protein and thus promote an oncogenic state. Therefore, a DNA construct encoding a "detoxified" version (dE7) of E7 of HPV-16 was developed. Immunization with the dE7 DNA construct (pCMV3-dE7, P125 Figure 1) resulted in significant protection against tumor growth after grafting of live C3 tumor cells, which express the native type E7 molecule. This finding indicates that a dE7 DNA construct could be successfully used to stimulate protective immunity, without any of the associated toxicity risks. A specific epitope approach was also evaluated. A DNA construct consisting of T cell epitopes derived from both E6 and E7 proteins of HPV-16 was used to immunize mice (pCMV3-HPVT # 1, Figure 6). Notable results were obtained in the group of mice immunized with this polyepitope construct, since 100% protection against tumor growth was observed. It is quite apparent to one skilled in the art that several of the embodiments of the present invention have many applications in the field of vaccination, diagnosis and treatment of HPV infections. In addition, a non-limiting analysis of these uses is presented below. Preparation and Use of the Vaccine Immunogenic compositions, suitable for use as vaccines, can be prepared from HPV genes, epitopes and vectors, as set forth in P1254 SAan'te tif '? -_ «- fe ^» j_ > _-. A »» - > «-«. * 3-I 13 I presented. The vaccine provides an immune response in a subject that induces a protective or therapeutic antitumor response. Immunogenic compositions, including vaccines, which contain the nucleic acid, can be prepared as injectable preparations, in physiologically acceptable solutions or emulsions for administration of polynucleotides. Nucleic acids in acceptable liquids can be used as direct immunizing agents (e.g., as generally described in U.S. Patent No. 5,589,466). Alternatively, the nucleic acid can be combined with liposomes, such as lecithin liposomes or other liposomes known in the art, such as a nucleic acid liposome (e.g., as described in WO 93/24640, reference 21) or the nucleic acid may be associated with an adjuvant, as described below in greater detail. Liposomes comprising cationic lipids interact spontaneously and rapidly with polyanions, such as DNA and RNA, resulting in liposome / nucleic acid complexes that capture up to 100% of the polynucleotide. In addition, the polycationic complexes fuse with the cell membranes, resulting in an intracellular delivery of the polynucleotide that ignores the degrading enzymes of the liposomal compartment. The published PCT application WO 94/27435 describes compositions P125 A.JE 14 for genetic immunization comprising lipids and cationic polynucleotides. With advantage, agents that collaborate in the cellular uptake of nucleic acid can be used, for example, calcium ions, viral proteins and other transfection facilitating agents. Immunogenic polynucleotide preparations can also be formulated as microcapsules, including biodegradable controlled release particles. Thus, U.S. Patent No. 5,151,264 discloses a particulate carrier phospholipid / glycolipid / pol saccharide that has been termed Bio Vecteurs Supra Moleculars (BVSM). The particulate carriers are intended to transport in one of the layers thereof, a variety of molecules that have biological activity. U.S. Patent No. 5,075,109 describes the encapsulation of the trinitrophenylated antigens hemocyanin of one type of limpet (Diodora) and staphylococcal enterotoxin B in 50:50 poly (DL-lactideco-glycolide). Other polymers for encapsulation are suggested, such as poly (glycolide), poly (DL-lactide-co-glycolide), copolyoxalates, polycaprolactone, poly (lactide-co-caprolactone), poly (stearamides), polyorthoesters and poly (β-hydroxybutyric acid) ) and polyanhydrides. The published PCT application WO 91/06282, P1254 and? - I describe a delivery vehicle comprising a plurality of microspheres and bioadhesive antigens. The microspheres are starch, gelatin, dextran, collagen or albumin. This supply vehicle is intended for particular for the uptake of the vaccine through the nasal mucosa. The supply vehicle may also contain an absorption enhancer. The vectors can be mixed with pharmaceutically acceptable excipients that are compatible. Sayings excipients may include water, saline, dextrose, glycerol, ethanol and combinations thereof. The vaccines and immunogenic compositions may additionally contain auxiliary substances, for example, wetting or emulsifying agents, pH regulating agents or adjuvants, to reinforce their effectiveness. The immunogenic compositions and vaccines provided herein may be administered parenterally, by injection, subcutaneously, intravenously, intradermally or intramuscularly, possibly after pretreatment of the injection site with a local anesthetic. Alternatively, the immunogenic compositions formulated according to the present invention can be formulated and delivered in such a way as to elicit an immune response on the surfaces of the mucous membranes. Thus, the immunogenic composition can be P125 administer to mucosal surfaces, for example, nasally or orally (intragastric). Alternatively, other forms of administration including oral formulations and suppositories may be convenient. For suppositories, binders and carriers may be included, for example, polyalkylene glycols or triglycerides. Oral formulations may include commonly used excipients, for example, pharmaceutical grades of saccharin, cellulose and magnesium carbonate. The immunogenic preparations and vaccines are administered in a manner that is compatible with the dosage formulation and in such an amount as to be therapeutically effective, protective and immunogenic. The amount that is administered will depend on the subject to be treated, including, for example, the ability of the individual's immune system to synthesize the antigens associated with the tumor and the antibodies thereto and, if necessary, to produce a mediated immune response. by cells. The precise amounts of active ingredient that is required to be administered will depend on the physician's judgment. However, dosage ranges can easily be determined by one skilled in the art and can be in the range of approximately 1 μg to 1 mg of the vectors. The regimens for initial administration and adequate booster doses P1254 They are also variable, but they may include an initial administration followed by consecutive administrations. Immunogenicity can be significantly improved if the vectors are coadministered with adjuvants, which are usually used from 0.05 to 0.1 percent in phosphate buffered saline. The adjuvants reinforce the immunogenicity of an antigen but, in themselves, are not necessarily immunogenic. The adjuvants can act by retaining the antigen locally, near the site of administration to produce a depot effect that facilitates a sustained and slow release of the antigen to the cells of the immune system. The adjuvants can also attract cells of the immune system to an antigen deposit and stimulate said cells to elicit immune responses. Immunostimulants or adjuvants have been used for many years to improve host immune responses, for example to vaccines. Thus, it has been identified that codyuvantes reinforce the immune response to antigens. However, some of these adjuvants are toxic and can cause undesirable side effects, which makes them unsuitable for use in humans and in many animals. Indeed, only aluminum hydroxide and aluminum phosphate (al P125 "" "which is generally referred to as alum) are routinely used in vaccines for human and veterinary use.A wide range of extrinsic adjuvants and other immunomodulatory materials can elicit potent immune responses to antigens.These include, saponins complexed with membrane protein antigens to produce complexes in a stimulant (ISCOMS), pluronic polymers with mineral oil, mycobacteria killed in mineral oil, Freund's complete adjuvant, bacterial products, for example, muramyl dipeptide (MDP) and lipopolysaccharide (LPS), as well as monoporyl lipid A, QS 21 and polyphosphazene In particular embodiments of the present invention, the vector can be delivered together with a targeted or targeted molecule that directs the vector to selected cells, including cells of the immune system.The polynucleotide can be delivered to the host by a variety of procedures, for example, Tan g et al. (reference 22) states that the introduction of gold microprojectiles coated with DNA encoding bovine growth hormone (BGH) in the skin of mice resulted in the production of anti-BGH antibodies in the mice, while Furth et al. (reference 23) showed that a jet injector could be used to P1254 transfect skin, muscle, fat and mammary tissues of live animals.

EXAMPLES 5 The above discussion describes the present invention in general terms. The invention will be better understood by reference to the following specific Examples. These Examples are described for illustrative purposes only and are not intended to limit the scope of the invention. Changes in the form and substitution of equivalents are considered circumstances that may be inferred or considered convenient. Although specific terms have been used here, these terms are taken in a descriptive sense and not for limiting purposes. 15 The methods of molecular genetics, protein biochemistry and immunology are used but are explicitly described in this exposition and in these Examples, but they are exhaustively reported in the scientific literature and are within the abilities of the experts in the art.

Example 1. This Example describes the immunization protocol that is employed herein. 25 Female C57B1 / 6 mice were purchased with a weight P125"- - - - - - - - - - - - - - - - - - - - - from 18 to 20 grams from Charles River (St. Constant, Quebec, Canada). The mice were housed in micro-isolators according to guidelines established by the Canadian Council for Animal Protection (CCAC Canadian Council on Animal Care). Ten animals were included in each treatment group. On day 0, the mice were immunized with 100 μg of each respective DNA construct, either by the intramuscular route (i.m.) in the anterior tibial muscle or intradermally (i.d.). Immunizations were repeated, with equivalent doses of each construct, on days 21 and 42 of the study. Serum samples were extracted from the mice on days 20, 40 and 56.

Example 2: This Example describes the protocol for stimulation with tumor cells. Two weeks after the last booster dose with the DNA construct according to the procedure of Example 1, each mouse was injected subcutaneously (s.c.) in the back of the neck, at a dose of 5x105 live C3 tumor cells. The C3 tumor cell line was kindly provided by R. Offringa and C. Melief. The C3 tumor cell line was generated by transfecting B6 mouse embryonic cells with the complete HPV-16 genome, transformed with the ras oncogene. HE P125 «-«? S- requires the expression of the oncogenic proteins E6 and E7 of HPV 16 to maintain the transformed state. After the injections were given, the mice were examined three times a week at the time the study lasted. Tumor measurements were made using vernier calipers. The volume of the tumor mass was calculated by the following formula: volume = ab2 / 2, where a = larger diameter and b = the smaller measurement of the two. The mice that remained without tumor for a period of approximately three months after this initial stimulation with live tumor cells, were then stimulated again on day 141 of the study with the same dose and in the same way as the initial stimulation in the day 57 described above. After the new stimulation, the mice were monitored to observe the appearance of tumor growth as described above.

Example 3 This Example describes the IgG immunoassays specific for E7 and dE7 (EIA). Nunc Maxisorp immunoassay plates were coated overnight with recombinant E7 antigen or recombinant dE7 antigen at a concentration of 10 μg / mL diluted in 50 mM carbonate buffer pH P125 9. 6. The next day the plates were washed in PBS (buffered saline phosphate solution) containing 0.05% Tween 20 (PBS-T) and then blocked with 1% milk solution for one hour at room temperature. After the blocking step, the plates were washed in PBS-T and the serum samples were added to the plates in various dilutions. The samples were incubated on the plates overnight at 4 ° C. The samples in the plates were washed the next day with PBS-T and a peroxidase-labeled sheep anti-mouse IgG conjugate was added to each well, at a dilution of 1 / 25,000. After one hour of incubation at room temperature, the plates were washed in PBS-T and the colorimetric reaction was developed using TMB substrate (ADI). The reaction was read 450 nm in a Dynatech MR 5000 96-well plate reader.

Example 4 This Example describes the construction of pCMV-dE7 Plasmid pCMV3 contains segments from several sources and the building elements are depicted in Figure 2. Briefly, the prokaryotic vector pBluescript SK (Stratagene) is the main structure of pCMV3 and was modified by the substitution of the AmpR gene with a KanR gene and the deletion of fl and the LacZ region. The P1254 modifications were achieved by deletion of the sequences between the restriction sites Ahdl (nucleotide 2041) and Sac I (nucleotide 759) of pBluescript SK, which contains the AmpR, fl origin and LacZ sequences. A Pst I 1.2 kb fragment of the pUC-4K plasmid (Pharmacia) containing the KanR gene was blunt-ended to the Ahd I site of the modified plasmid pBluescript SK in a counter-clockwise direction relative to its transcription. An Ssp I / Pst I 1.5 kb fragment containing the promoter, activator and intron A sequences of the immediate cytomegalovirus immediate gene was ligated to the other end of the KanR gene so that transcription of the CMV promoter proceeded in the direction of the hands of the clock . This procedure yielded the plasmid pCMV2K, as shown in Figure 3. A 0.2 kb fragment containing the bovine growth hormone (BGH) polyadenylation signal sequence was removed from the pEN-3 plasmid by restriction with Xba I, followed by of klenow treatment to produce a blunt end and then restriction with Bam Hl. The 192 bp fragment containing the BGHpA fragment was isolated by means of agarose gel electrophoresis. Plasmid pEN-3 was prepared by cloning bGHpA to pEN-1 (Figure 4). The plasmid pCMV2K was then subjected to restriction with Bgl II, treated with klenow and subjected to P125 * • it 24 J '3? D-t: riction with Bam Hl. The 4.2 kb fragment (see Figure 3) was then isolated and ligated to the 192 bp BGHpA fragment that had already been isolated, to produce the plasmid pCMV3. The dE7 gene was amplified by PCR from plasmid pSE859.2 using primers that introduced a Pst I site at the 5 'end (SEQ ID NO: 2 CTGCAGCAGGCTAGCATGCATGGAGATACACCT) and a Sal I site at the 3' end (SEQ ID NO: 12 GTCGACTTATGGTTTCTGAGAACAGATGGGGCACA). Sequence amplified is shown in Figure IB and contains detoxified E7 protein of HPV-16. The PCR fragment was inserted into pCR2.1 restricted with Pst I and Sal I (Invitrogen) and the insert was sequenced (Figure IB). The Pst I to Sal I fragment was then subcloned to pCMV3 from Pst I to Sal I, to produce Plasmid pCMV-dE7, as shown in Figure IA.

Example 5: This example illustrates the use of pCMV-dE7 to protect animals against subsequent tumor growth to the graft with a tumor cell line expressing wild type HPV-16 E7. The mice were immunized with a DNA construct encoding the detoxified E7 protein (pCMV-dE7, prepared as described in Example 4; Figure 1), a DNA control (pCMV-3; Figure 2) or PBS by P125 - *** • r * «* intramuscularly, according to the protocol of Example 1. After three successive immunizations, live C3 tumor cells expressing the wild-type E7 protein were grafted at a dose of 5 × 10 5 cells, following the protocol of Example 2. The number of tumor-free mice after stimulation with live cells is shown below in Table 1. Approximately one month after stimulation (day 30), all mice in the groups of control (control groups pCMV-3 and PBS) had palpable tumors. However, in contrast, none of the mice in the group immunized with pCMV-dE7 had palpable tumors. On day 60, a mouse from the group immunized with pCMV-dE7, which had previously been negative to the tumor, showed the initial indication of a palpable tumor. On day 90, this mouse was sacrificed due to the large volume of the tumor. However, the remaining mice from the group immunized with pCMV-dE7 remained without tumor. In this way, a significant difference was observed in the state without tumor, between the mice of the group immunized with dE7 and the control groups. Thus, the results indicated that immunization with a DNA construct that codes for a genetically detoxified E7 molecule (dE7) induced an immune response capable of protecting the animal against subsequent tumor cell grafts P125 alive Table 1 Percentage of Mice without Tumor in Various Post-Graft Time Points with Live C3 Tumor Cells Example 6: This Example illustrates that sera derived from mice immunized with pCMV-dE7 construction, are reactive with both detoxified E7 protein and wild type E7. In order to provide serological evidence that mice immunized with the E7 protein genetically detoxified according to Example 5, could generate immunity that was reactive cross-over with the wild-type E7 protein, sera from mice immunized by EIA were analyzed, following the procedure of Example 3. The serum sample that was analyzed was derived from a blood sample taken on day 56, one day before stimulation with live tumor cells and two weeks after the last P125 27 reinforcement immunization. The serum reactive antibody titer, as shown in Table 2 below, was equivalent whether it was analyzed in specific EIA of dE7 or EIA specific of E7. So, at the level of antibodies, the antibodies generated by immunization with dE7 cross-reacted with the E7 protein.

Table 2 Example 7: This Example illustrates that the specific route of immunization has an important effect in the induction of protective immunity. The effect on the induction of antitumor immunity of the intramuscular route (i.m.) was investigated.

P125 #TO*.? ^ F »nf *» 28 with respect to the intr & lfermica route (i.d.) for DNA immunization. Briefly, the two groups of mice were immunized with the same construct (pCMV-dE7, prepared as described in Example 4, Figure 1) at the doses and frequency described above in Example 1. However, a group was immunized via im, while the other group was immunized via id After stimulation with live C3 tumor cells, according to the procedure of Example 2, the group immunized via 10 i.d. presented tumor growth, in direct contrast with the immunized group via i.m. who remained without a tumor In this way, it was determined that DNA vaccination with dE7 DNA construction elicited protective immunity against stimulation with tumor cells only when the immunization was given i.m. The results are shown in Table 3 below.

Table 3 Percentage of Mice without Tumor After Grafting of 20 Live C3 Tumor Cells P125? 29 In example 8: This Example describes the preparation of plasmid pCMV3-HPVT # l. The synthetic minigene that codes for the five T-cell epitopes of the E6 and E7 proteins of HPV-16 (Figure 5A), was constructed by oligonucleotide synthesis using an Applied Biosystems 3AB DNA synthesizer. The synthetic minigene was assembled using five synthetic oligonucleotides (I to V, Figure 5B) that contained a Sal I restriction site at the 5 'end and an Eco Rl site at the 3' end. This assembled gene (Figure 5) was cloned into the plasmid pCMV3 restricted in Sal I / Eco Rl to produce a pCMV3-HPVT # l plasmid as shown in Figure 6. The construction of pCMV3 was described in Example 3 and the elements are shown in Figure 2.

Example 9 This Example illustrates the use of pCMV3-HPVT # 1 to induce protective anti-tumor immunity. C57B1 / 6 mice were immunized with either a DNA construct that codes for several epitopes of the HPV-16 E6 and E7 proteins (pCMV3 -HPVT # 1, was prepared as described in Example 8, Figure 6), or with controls (vector pCMV-3 alone, Figure 2 or PBS), depending on the protocol of Example 1. Prophylactic immunization with the polyepitope DNA construct, according to the protocol of Example 2, induced protective anti-tumor immunity in 100% of the stimulated mice. In contrast, there were no mice without tumor in the control groups. The results are presented in Table 4 below.

Table 4 Percentage of Mice without Tumor After Grafting of Live C3 Tumor Cells Example 10: This Example illustrates the effect of a second restimulation with live C3 tumor cells in mice without tumor, three months after the initial stimulation with live tumor cells. In order to determine whether the protective anti-tumor immunity induced either by the pCMV-dE7 construct, P125 SSfwS ^ j x ^ ry 31 prepared as described in Example 4 (Figure 1), as seen in Example 7 or the pCMV-polyepitope construct, prepared as described in Example 8 (Figure 6), as shown in Example 9, was long-term, the mice that survived the initial stimulation with tumor cells and remained without tumor for a period of three months, were grafted again with live C3 tumor cells. Then the mice were monitored for signs of tumor development. As indicated below in Table 5, an animal in the pCMV-dE7 group developed a tumor after the second dose of stimulation. However, all mice in the group immunized with DNA-polyepitope remained without tumor. Thus, the highest level of protection against tumor cell stimulation appears to be in the group immunized with polyepitope.

Table 5 Percentage of Mice without Tumor After Second Graft 20 of C3 Tumor Cells Live P125 LISTING "OF SEQUENCES X > : 126 > TREATMENT OF CERVICAL CANCER '' ': 14Q »? Cx / CAt» / ooao7: X «1 > l »» »- 09-03 sVSl »X» _ »ß-05-04 to»? a «X"! 0 P- Cß? tln Ver. 3 .1 CÍ13 > OKA < 8XS »Artificial Sequence. «ÍS4» Description of Artificial Sequence: Synthetic mini-gene; oteí? «, ßß * < re tffe * wß * tflß »t¡ßg-kg * fca« »aatacfcttga a_tgMt«? gfctigatttg G? or $ * F > ß * ß. «? M? gM-fcg »aage taag» OT * OT sgBK gtti-Ke MVCgfct? W- txa 5β- l8 * ß »« m on.g ** aBra * e * ß * ßrea-M-fc £ «.» «£« et. «r t-Mßat t ß engoMß g 180 9% i_m« e? «< e .aaatfefrtß wßfc * ja »a * a? aaiB «, isgc & a acatceatlO cecgg« av? a to «0 etffitc ** tpp flßaaßotag? r -U-fctB gtg« aacatctgtt ßta * ga * aae to «** g og * ß 3 DO < m > as aya »SVA < J? J > Artificial Sequence! «ßft3 * Description of Artificial Sequence: Synthetic Mini-gene. < 400 > 2 atg < s * 0c * 3ß ctaa = »tßea C0ßr -. s-tc« ea Cd »< aa.? > a * 8a # »xa» "* Sequence .Artificial * aso »? 23a 3 Description of Synthetic Mini-gen Artificial Sequence < ß # *% (* f «« i * ftfca vm *** & *** a kegzaravtic Offtßb 3S 32S > W 31.3 > Artificial Sequence: 230fc ti I * 'Description of Artificial Sequence Synthetic Mini-gene «40?) > 4 t «$ t * ß» ßßjro ßßsßa saga geac3a.tte.en. .t * ttgtta.o ßtttgßagaa gobca acgc ao tifa * tgoa moaagagaca aotgcagocg oßßtgttaat gggca-Hscrea gMßc gtg? uo sß «« fßß * aß a-.fca55aa.tto gtgtgoooc * ttgcwgt-age cm. i.i .inaE gah * .taat «ta laO Mqtáattgt * gtaßfcagfcg * ff *» tfr «tg * tf * goaoatca oaatattgtt * aßfet gocg ** 0 ß%« y? t-.t -. * ?. gt «aa-gattt.ß aaaQßagaga caaa * agaaga ogatctgcta atjgggcacao 9oo t * 93 * atttgfe ggaßgagsog ißaaeafgaa trgcgcgcoß aatagcavaa g« -.ot fe «e acó« tsgattat attagaaeg * gcgt.cs ase «IlC * s < to?? > lao < a? s & _MA «« 415 * Artificial Science «s * 30» «»% > »Description of Synthetic Artificial Sequence googocigaa ftAtgftaßa. tJttgcaaßoa f 0 Baat-am * ttgtggßcgc gega.ß «« a iao g «jaat at.g aeaooacagc ageagooaet ataoatgat * taatattoaga ^ gtg gca * 2ß« «311» 59 < 313 > Artificial Sequence «¡•« 4 »4383» Description of Synthetic Artificial Sequence, _. «FctSfc *« Hat Avg the Hta tr A * U XI * val «tor rt» Ala Ala Ala t »Mete wu. 1 9 16 iE Aßp &«u? L» tlwr uu ßiy? I «v * l Al« Al * Ove luí aiy II * al cya? Ro xla Ala Ala 31 40 43 1A Tur X to Kt * a? xla *? * ?? U. a Cy «ea 80 35 .2X1! »32 .23.2 * &WA 23.3» »Artificial Sequence (339» - t232 »> Artificial Artificial Sequence Depapment leflütoawßge eaeeBLtßaga ffficcattuoa atattattao efittgoegcß gcotatatgt SO? agici-tgßa aacagagaea atrtgcagtscg ct »2 s310 > 8 c311 > 10 »< 313 > CHA «3% ^» Artificial Sequence «sao * < t23 * > Description of Synthetic Artificial Sequence «400» ß a tgt-t al-.es geaßaofcagg aatugtß? Roa gogrgagacao taßßaatetgt stpaaocateo co gßagßageß etacaoacga cacßacattta ssa sreg-frgt; «6 * ge $ wf ZA» < 310 > 9 < 211 > 93 * M2 »SUCA« 212 * Artificial Sequence * 2A2 »• Description of Synthetic Artificial Sequence« 00 »9 0 * teat_aggeg gßg or * a» ßo «a.'aeAa'U-kVí,« .mat'gsB ^ «fettft &wcgs« &9a'l M «? M * 10 e2A2 OKA. * 2i2 »Artificial Sequence - * «SA» * Description of Artificial Synthetic Juanea Sequence «*? Athetlo < 40? lg a * t * 3-t *** - tsr ee? OT «< ? eßfcjjr ttfpx &gneev * e? ffMj > A »zz kpsBcf. 4ff < ** M- > 1 * «4« 29.2 »BJ A« 212 J »Artificial Sequence »G > ) 23 Description of Synthetic Artificial Sequence 400 # 11 feg. * Fß «?« * «Catresveg * fg ff * ffa e &ttt ** e *? * A S9ga ** 8 * 3 * - agtßflßcgar« or tf áj & ISA M. tt? TAiÉ? Cst aaaßaeatta tßaßtßtta * 100 «Ato» 12 312). OSA 2 2 »Artificial Sequence 22 ** Description of Synthetic Artificial Sequence jfe.iiraiiett.aii oßtttetgaß aaoagafcggg go-vaa 33 ? r. 32 REFERENCES Pisani P. et al., Estimates of the worldwide mortality from major cancers in 1985. Implications for prevention and projections of the future ** s burden, Int. J. Cancer 1993: 55: 891-903.

Piver .S., Handbook of gynecologic oncology. Boston: Little, Brown, 1996 Kurman R.J. et al., Interim guidelines for management of abnormal cervical cytology. The 1992 National Cancer Institute orkshop, IAMA 1994-271: 1866-9.

Bosch FX, et al., Prevalence of human papillomavirus in cervical cancer - a worldwide perspective. International biological study on cervical cancer (IBSCC) Study Group [see comments], J. Natl. Cancer Inst. 1995: 87: 796-802.

Pecoraso G. et al., Differential effects of human papillomavirus - type 6, 16 and 18 DNAs on immortalization and transformation of human cervical epithelial cells. Proc. Natl. Acad Sci. USA, 1989: 86: 563-7. 33 Shamanin V., * "& al., Specific types of human papillomavirus f% te? D in benign preliferations and carcinomas of the skin in immunosuppressed patients, Cancer Res. 1994: 54: 4610-3.

Hilders C.G., et al., Association between HLA-expression and infiltration of immune cells in cervical carcinoma [see comments]. Lab. Invest. 1993: 69: 651-9.

Munger K., et al., Interactions of HPV E6 and E7 oncoproteine with tumor suppressor gene products. Cancer Surv. 1992: 12: 197-217. 9. Dyson N., et al. The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science: 1989: 243: 934-7.

. Munger K., et al. The E6 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes. J. Virol 1989: 63: 4417-21. 11. Crook T., et al., Continued expression of HPV-16 E7 protein is required for maintenance of the Pl25 3. 4 transofmred phenotype of cells, co-transformed by HPV-16 plus EJ-ras. EMBO. J. 1989: 8: 513-9. 12. Ressing M.E., et al., Human CTL epitopes encoded by human papillomavirus type 16 E6 and E7 identified through in vivo and in vitro immunogenicity studies of HLA-A * 0201 -binding peptides. J. Immunol. 1995: 154: 5934-5943. 13. Kast .M. et al., Role of HLA-A motifs in identification of potential CTL epitopes in human papillomavirus type 16 E6 and E7 proteins. J. Immunol, 1994: 152: 3904-3912. 14. Feltka p M.C., et al., Vaccination with cytotoxic T. lymphocyta epitope -containing peptide protects against a tumor induced by human papillomavirus type 16-transformed cells. Eur. J. Immunol. 1993: 23: 2242-2249.

. Alexander M., et al., Generation of tumor specific cytolytic T-lymphocytes of blood cancer patients by in vitro stimulation with a synthetic HPV-16 E7 epitope. Am. J. Obster, Gynecol 1996: 175: 1586-1593. 16. Steller M.A. , et al., Human papillomavirus immunology P125 - * £ '' * s 35 and vaccine prospects. J. Natl. Cancer Inst. Monogr. 1996: 21: 145-148. 17. Schiller J.T., et al., Papillomavirus vaccines: current status and future prospects. Adv. Dermatol. 1996: 11: 355-80; discussion: 355-80; discusstion: 381. 18. Kimbauer R., et al., Efficient self-assembly of human papillomavirus type 16 Ll and L1-L2 into virus-like particles, J. Virol. 1993: 67: 6929-6936. 19. Borysiewicz L.K., et al., A recombinant vaccinia virus encoding human papillomavirus type 16 and 18, E6 and E7 proteins as immunotherapy for cervical cancer [see comments]. Lancet 1996: 347: 1523-1527.

. Irvine K.R., et al., Synthetic oligonucleotide expressed by a recombinant vaccinia virus elicits therapeutic CTL. J. Immunol. 1995: 154: 4651-7. 20 21. WO 93/24640 22 Tang et al. , Nature 1992, 356: 152-154. 23. Furth et al. , Analytical Biochemistry, 1992, 205: 365-368.

P125

Claims (11)

1. < - • 36 CLAIMS; A vector characterized by a nucleic acid molecule, which codes for at least one nontoxic T cell epitope of the E6 and / or E7 antigen of a human papillomavirus (HPV) strain associated with cervical cancer and a promoter operatively coupled to the nucleic acid molecule, for the expression of the nucleic acid molecule in a host to which the vector is administered.
2. 2. The vector according to claim 1, characterized in that the promoter is a cytomegalovirus promoter.
3. 3. The vector according to claim 1 or 2, characterized in that the nucleic acid molecule is contained within the CMV-3 plasmid.
4. 4. The vector according to claim 1, characterized in that the nucleic acid molecule is a sequence encoding the detoxified E7 antigen to prevent oncogenic replication in the host.
5. The vector according to claim 4, characterized in that the detoxification is carried out by eliminating from the native sequence, the nucleic acid encoding amino acids 21 to 26 of HPV-16.
6. 6. The vector according to claim 5, which is pCMV-dE7. P125 37
7. 7. The vector according to claim 1, characterized in that the nucleic acid molecule encodes epitopes of E7 antigen, comprising amino acids 11 to 20, 49 to 57, 82 to 90 and 86 to 93 and epitope of E6 antigen comprising amino acids 29 to 38 HPV-16.
8. The vector according to claim 7, characterized in that the nucleic acid molecule has SEQ ID NO: 4 or 5.
9. 9. The vector according to claim 7, characterized in that the nucleic acid molecule encodes an amino acid sequence that has SEQ ID NO: 6.
10. 10. The vector according to claim 7, which is pCMV3-HPVT # l.
11. 11. An immunogenic composition for in vivo administration to a host, comprising a vector according to any of claims 1 to 10. P1254