RU2229307C1 - Recombinant protein composition, method for preparing such composition, pharmaceutical kit of reagents for immunotherapy and prophylactic vaccination of tumor diseases in anus-genital sphere, method for immunotherapy and prophylactic vaccination based on thereof - Google Patents

Abstract

FIELD: medicine, pharmacy. SUBSTANCE: invention relates to composition of recombinant proteins, method for preparing the composition, pharmaceutical kit of reagents used in immunotherapy and prophylactic vaccination of tumor diseases in anus-genital sphere and method for immunotherapy and prophylactic vaccination. Invention involves hybrid proteins consisting of amino acid sequences of oncoprotein E7 of human papilloma virus of 16 and 18 types that are bound covalently with amino acid sequence of heat shock protein with molecular mass 70 kDa (Hsp70) from M. tuberculosis - E716-Hsp70 and E718-Hsp70, respectively. EFFECT: enhanced activity of composition. 6 cl, 5 dwg, 7 ex

Description

The invention relates to the field of medicine, bioorganic chemistry and genetic engineering, specifically it relates to a composition of recombinant proteins, a pharmaceutical kit based on it for the prevention and treatment of chronic palillomovirus infection leading to genital invasive cancer, as well as the treatment of neoplasia and invasive cancer associated with viruses human palillomas. The invention also relates to a developed method of immunotherapy and prophylactic vaccination and a method for producing a composition of recombinant proteins.

About 500,000 women in the world are diagnosed with cervical cancer, which ranks second in the world as the cause of death from cancer among women. Despite screening programs in the United States that detect cervical cancer at an early stage, the incidence of invasive cervical cancer is not decreasing. Over the past 15 years, epidemiological and virological studies have unequivocally confirmed the connection of this pathology with human papillomavirus (HPV) (Sexually transmitted diseases. "Vaccines, prevention and control", 2000, Edited by D.I. Bernstein, Academic Press). The relationship between neoplasia and invasive cancer of the uterus, external genitalia, anus and human papilloma viruses has also been established (Lorine J. et all "Obstet Gynecol." 79: 328-337, 1992).

Papillomavirus is a double-stranded DNA helix virus consisting of 7800-7900 base pairs, a non-enveloped virion and a 20-sided icosahedral capsid of 72 capsomeres. The genome consists of three main areas. One encodes late genes, the other encodes early; the third region is non-coding. It consists of 400 base pairs and contains binding sites for various transcription factors that control the expression of late and early genes. The region of late genes has two reading frames and encodes the L1 protein, which is the main HPV calendine protein, and the L2 protein, which is the minor capsid protein. The region of early genes includes six reading frames and encodes proteins E1, E2, E3, E4, E5, E6 and E7, respectively. Proteins E6 and E7 are cancer proteins and play a crucial role in viral replication, and they are also necessary for immortalization and transformation of cells. Proteins E1, E2 and E4 also play an important role in replication. Protein E4 is necessary for the maturation of the virus. The role of E5 protein has not yet been fully clarified.

Current evidence suggests that HPV capsid proteins are capable of inducing high titers of specific antibodies (humoral immunity) and are promising candidates for the development of prophylactic vaccines (Lowy DR, Schffler JT Papillomaviruses: Prophylactic vaccine prospects. Biochim. Biophys. Acta, 1999 , 1423: M 1-8; Russian Patent No. 2173170, PCT Application 86/05816) or diagnostic kits for determining papilloma viruses (EP No. 386734, 375555).

However, when using these proteins for therapeutic immunization with an established, chronic papillomavirus infection, it was not possible to obtain any significant effect. For therapeutic immunization, another strategy is needed, namely the induction of antigen-specific T-cell immunity. The main requirement for therapeutic vaccines is that they should stimulate the cellular component of specific immunity and block the development of neoplastic processes. The cellular response should be directed to cellular and viral proteins involved in malignancy of the epithelium. Despite intensive studies, little is known about cell targets during tumor transformation, so most experimental vaccines are aimed at the products of the HPV E6 and E7 genes, whose role in oncogenesis has been unequivocally proven.

There are a large number of recombinant proteins that can be used as vaccines and which contain, along with E6 or E7 proteins, other HPV proteins (E1, E2, E4, L1, L2, etc.), (see, for example, PCT application 99 / 48518 et al.). However, such constructs have poor prospects for the treatment of neoplasia and cancer due to the fact that a cancer cell is capable of mutating or losing many less important proteins for its existence as a target for an immunological attack during chemo- or immunotherapy. The inclusion of such proteins in the composition of hybrid constructs is also ineffective because they cannot boost sufficiently specific cellular immunity to E6 and / or E7 due to the insufficient immunogenicity of HPV proteins. More promising constructs should be recognized as those that contain only E6 and / or E7 HPV proteins as antigenic determinants and which are closer to the hybrid protein constructs of the present invention.

The earliest such work is the patent application PCT 92/10513, which describes the design based on the peptide sequence of the immunogenic part of the HPV E7 protein E7 16, directly or indirectly linked to the amino acid sequence corresponding to the E7 epitope of HPV 16 or HPV 18. Using this the design was able to overcome the oncogenicity of the E7 and E6 proteins and cause stimulation of humoral immunity. The design requires an adjuvant.

International application No. 96/19496 describes the design of a hybrid protein based on HPV E6 or E7 protein (types 16, 18, 6, and 11), which cause a humoral and / or cellular immune response and are not dangerous for recipient cells, as they are not capable cause their transformation. For this, HPV E7 or E6 proteins bind covalently to a protein or peptide fragment, which, for example, facilitate the purification of a recombinant protein (e.g. 6His, GST fragment or glutadione s-transferase), or increase its immunogenicity, for which diphtheria or cholera toxin or their non-toxic derivatives. (However, there is no description of such fusion proteins with diphtheria or cholera toxin in the application materials). The construct provided by this invention also needs an adjuvant.

In the international application 97/06821 for the first time the possibility of using heat shock proteins to excite the cellular component of immunity is shown. Example 10 describes a construct that is a complex formed in vitro from a heat shock protein and a hybrid protein from E7 HPV and a peptide site that is capable of complexing with a heat shock protein. To induce an immune response, the construct needs an adjuvant.

A digression should be made and a number of reports have been made that the chemical conjugate of heat shock proteins of mycobacteria hsp65 or hsp70 (or hsp65 or hsp70 of Escherichia coli bacteria) with part of the malarial antigen is capable of inducing the formation of specific antibodies (humoral immunity) in the absence of or adjuvant. (Barrios at all Eur. J. Immunol. 22.1365-1372, 1994. Barrios et all Clin. Exp. Immunol. 98: 224-228, 1994). K.Suzue and R. Young ("The journal of immunology", "Adjuvant-free hsp70 fusion protein system elicits memoral and cellular immune responses to HIV1h24") show that the conjugate induces 10 times more in the absence of any adjuvant the intensive cellular and humoral component of immunity than the antigenic p24 HIV1 protein in its free form.

This approach was used in international application 99/07860 (adopted by us for the prototype), which proposed the composition of some HPV protein (as an antigenic determinant) combined with a stress protein (heat shock protein) with an acceptable pharmacological component. The fusion protein composition is capable of inducing a cellular, cell-mediated cytolytic immune response. Most of the actual application material is devoted to compositions based on fused or recombinant proteins E7 and / or E6 HPV 16 with heat shock protein Hsp65, which belongs to heat shock proteins of the Hsp60 class. Conjugates can be obtained by expression of the corresponding genes in a vector under the control of the P T7 promoter in E. coli.

Examples 5, 6, 8 show that the regression of induced tumors in mice immunized with these compositions by the introduction of TC-1 cancer cells expressing the E7 and E6 proteins by them, occurs with the same or slightly lower efficiency as when they were immunized with the E7 protein in Freud's complete adjuvant (the so-called positive control).

This is a very important result. Methods of modern biochemistry, genetic engineering allow you to get almost any antigen, but make it work, i.e. to make sure that it is not excreted by macrophages of the liver and kidneys, which do not form an immune response, but is presented to immunocompetent cells so that a chain of cellular interrelated reactions or events is formed, i.e. for cellular immunity to form - this requires no less effort and is the subject of many scientific works and inventions (see, for example, US Pat. No. 20010031262, 5997900, 6419920, 5965125, 5906817, PCT 01/41809 and many others). In fact, this is the subject matter of the prototype invention: a working construct has been developed in general to develop the cellular component of the immune response for human papillomavirus proteins as antigenic determinants. However, the breadth of protection is the flip side of the lack of development of private solutions that pose more specific, specific tasks. Its use directly for immunotherapy and vaccination of cancer is doubtful due to the fact that, as noted earlier, cancer cells are able to lose many of their determinants, which are also weak immunogens.

An inventive task is to create, within the framework of the proposed designs, such recombinant proteins, their compositions and a set of reagents for immunotherapy and vaccination of tumor diseases of the anogenital sphere that would really meet the requirements for such drugs: they were effective for the vast majority of patients, they were distinguished by the manufacturability and for which they would a treatment and vaccination regimen has been developed.

The inventive task is solved by the fact that a composition of recombinant proteins is proposed for immunotherapy and prophylactic vaccination of tumor diseases of the anogenital sphere associated with human papilloma viruses, the difference in which is that it contains an effective amount of hybrid proteins consisting of amino acid sequences of the human papillomavirus E7 oncoprotein 16 and 18 types that are covalently attached to the amino acid sequence of heat shock protein 70 (Hsp70) from M. tuberculosis with following ratio E7 protein 16-Hsp70 and E7 18-Hsp70 - (1-2): 1.

The proposed composition may additionally contain pharmacologically acceptable components.

The inventive task is also solved by the fact that a pharmaceutical kit of reagents for immunotherapy and prophylactic vaccination of tumor diseases of the anogenital sphere associated with human papillomavirus is proposed, containing a composition of recombinant proteins E7 16-Hsp70 and E7 18-Hsp70 from M. tuberculosis in the ratio (1-2 ): 1 and BCG TB vaccine.

A method for producing a recombinant protein composition is also provided, characterized in that the expression of the corresponding genes is carried out in the vector plasmids pTHE 716 and pTHE 718 under the control of the T7 promoter.

A method of immunotherapy and prophylactic vaccination of tumor diseases of the anogenital sphere associated with human papillomaviruses is also provided, characterized in that they pre-stimulate the immune system by administering the BCG TB vaccine to the patient, after which the composition of recombinant proteins E7 16-Hsp70 and E7 18- is introduced into the revaccination period Hsp70 (from M. tuberculosis).

The invention is based on two new substances, the recombinant proteins E7 16-Hsp70 (from M. tuberculosis) and E7 18-Hsp70 (from M. tuberculosis), which, in the experiment with induced tumors, showed greater efficacy than the E7 16 protein in complete adjuvant Freud (see example 6), i.e. these substances turned out to be more effective antigenic constructs than the corresponding hybrid proteins in the prototype method. Since heat shock proteins are found in any tissue and in any organism, the Hsp70 class is found in mammalian, bacterial, and mycobacterial cells, such as M.leprae, M. tuberculosis, M.vaccae, MSmegmetis, M. bovis, and the relationship between the structure of a chemical compound and its properties is established only empirically, the invention already at this stage meets the criterion of inventive step.

As the antigenic determinant of HPV in the composition, only E7 protein is used (in the prototype, E6 protein is equally used along with E7). Our experience, as well as the analysis of relevant scientific and clinical data, indicates that only the E7 protein is a reliable target for an immunological attack in cervical cancer and other tumor diseases of the anogenital sphere. The composition includes two hybrid oncoproteins: E7 16 and E7 18, and only in this case the composition will be effective for a larger patient population (up to 70-80% versus 40-50% of the prototype).

The selection of Hsp70 from M. tuberculosis not only led to a more effective antigenic construct than in the prototype, but it was not accidental also because the applicant hoped to use the well-known BCG anti-TB vaccine (approved in Russia and made by the newborn) to stimulate the immune response to Hsp 70 M. tuberculosis, due to which there is a sharp increase in the immune response to the entire hybrid protein. As it turned out, this approach was justified, as shown in example 6. It is implemented through a pharmacological kit and method of immunotherapy and vaccination, which are new and meet the criterion of “inventive step”.

It should be noted that the resulting hybrid proteins meet the requirement of manufacturability: at the N-terminus, they contain a sequence of 6His, which facilitates their purification and ensures the required purity of the reagent. A plasmid and promoter was also selected that provided expression of the corresponding genes with the highest level of protein synthesis. The level of synthesis of hybrid proteins in E. coli cells under the control of the T7 polymerase promoter was 30% of the total cellular protein, while the promoter P _L (bacteriophage λ) and lac promoter, also used in experiments to optimize the synthesis of hybrid proteins, produced protein synthesis 12 and 15, respectively % As is clear, the recombinant protein composition does not need adjuvant due to conjugation with Hsp70, which is also very convenient to use, because Freud's adjuvant often gives complications in the form of abscesses, fistulas and remaining scars, and practical medicine has not yet received new adjuvants (progress in this area has mainly concerned only vaccines for veterinary purposes).

The invention is illustrated by the following examples. Examples 1,2,3 are devoted to the production and isolation of fusion proteins. Examples 4 and 5 describe the composition and pharmacological set of reagents. Example b shows the efficacy of using the E7 16-Hsp70 fusion protein and its combined use with BCG vaccine as compared with the “positive control” in experimental immunotherapy of tumors in mice. Example 7 shows the effectiveness of using a composition of hybrid proteins in a group of volunteers (based on the CCV dispensary).

Example 1. The sequence of cloned genes (see the end of the text).

Example 2

Creating Producer DnaK (HSP70)

The M. tuberculosis dnaK gene was amplified using dnaK-1 primers (gc gga tcc gct cgt ggg gtc ggg atc g) and dnaK-2 (gtc cgt cac ttg gcc tcc). The resulting fragment was digested with BamHI restriction enzyme and cloned between the BamHI and Smal sites of the pQE30 expression vector (Qiagen). This construct allows the expression of DnaK fused to 6 histidines at the N-terminus under the control of a lacI-regulated promoter. The presence of 6 histidine amino acids facilitates protein purification. The obtained recombinant plasmid pQESO-dnaK was maintained in the E. coli strain DLT1270 containing a chromosomally integrated copy of the lacI repressor.

The correctness of the cloning procedure was confirmed by restriction analysis and PCR.

DnaK synthesis was induced using IPTG. An overnight culture of DLT1270 / pQE30-dnaK grown in LB broth was diluted 1: 100 and grown in a LB broth on a shaker to OD ₆₀₀ = 0.5. IPTG (optimal dose of 0.1 mM) was then added and cultivation was continued for 3-4 hours. Protein production was monitored using SDS-PAGE, product yield under these conditions was 40-70% soluble proteins. (Figure 1, which shows the electrophoresis of proteins in SDS page 13% with SDS, where

1. Molecular weight markers.

2. Lysate of E. coli cells containing plasmid pQE30 (hsp70 + 6 His gene).

3. Supernatant from a recombinant E. coli strain with plasmid pQE30 after destruction by ultrasound.

4. Sediment from a recombinant E. coli strain containing plasmid pQE30 after destruction by ultrasound.

5. Protein Hsp70 after single-step chromatography on a chelate carrier.

6. Hsp70 protein after the second purification step.)

pSKE16

Contains the E7 Human papillomavirus type 16 gene, amplified from the DNA of clinical isolates using gene-specific primers. Fragment 303 bp long cloned by EcoRi-BamHI saigas in plasmid pBluescriptSK (+) (Stratagene). The gene sequence is consistent with GenBank data (e.g., AF125673). Design features: 1) ATS triplet is stored in front of the initiating codon to optimize eukaryotic expression; 2) the absence of a termination codon, since the gene was planned to produce a hybrid protein.

pSKE18

Contains the E7 Human papillomavirus type 18 gene, amplified from the DNA of clinical isolates using gene-specific primers. Fragment 324 bp cloned by SaRI EcoRI-BamHI in plasmid pBluescnptSK (+) (Stratagene). The sequence contains nucleotide substitutions compared to GenBank (for example, HPU89349; PAPHPV18), however, the amino acid sequence of the protein is retained.

A termination codon is also absent. Before the initiating codon is an AGT triplet.

rTE716

The plasmid was obtained by cloning an EcoRI-BamHI fragment of the pSKE16 plasmid containing the HPV16 E7 gene together with a BamHI-HinDIII fragment of the pQE-HSP70 plasmid (a new variant with the correct 3'-terminal sequence of the hsp70 gene) into the vector obtained by cleavage of the plasmid pKS70 EcoRI and HRI and selection of the fragment corresponding to the vector part. In fact, the plasmid pT7-7 was used as a vector. Expression of the E7-HSP70 hybrid gene is under the control of the T7 promoter and is induced by IPTG in E. coli BL21 (DE3) cells - see Figure 2.

RTNE716

obtained by cloning the oligonucleotide duplex His1 + His2 into a vector obtained by cleavage of plasmid pTE716 NdeI and EcoRI.

Encodes the H16 fusion protein: Met (His) ₆ -GluPheIle-E7 / 16-GlySer-Hsp70 (736 aa, 79.3 kDa, IEI 4.7).

rTNE718

obtained by cloning ÅCORI-BamHI. a fragment of plasmid pSKE18 containing the HPV18 E7 gene, together with His1 + His2 oligonucleotide duplex, into a vector obtained by cleaving plasmid pTE716 Ndel and BamHI. Encodes the fusion protein H18: Met (His) ₆ -GluPheSer-E7 / 18-GlySer-Hsp70 (743 aa, 80.3 kDa, IEI 4.8).

Example 3. The selection of proteins H16 and H18

E. coli cells of strain BL21 (DE3) were transformed with DIC of plasmid pTHE716 (718) and grown overnight at 37 ° C in LB medium with the addition of ampicillin (100 μg / ml) and glucose (0.2%). Then the night culture was diluted 25 times with fresh LB medium with ampipillin and grown for 2-3 hours until the turbidity was about 0.5, after which IPTG solution was added to 0.2 mM. They grew another 3 hours, centrifuged the culture at 5 thousand rpm for 10 minutes, discarded the supernatant.

After centrifugation, the precipitate was suspended in 20 mM Tris-HC1 buffer, pH 8, 0.3 M NaCl (1 ml per sediment from 50 ml of culture) and 6 pulses were announced for 15 s each. Centrifuged at 10 thousand rpm, 10 min, the precipitate was washed with the same buffer with the addition of 0.1% Triton X-100 (0.5 ml), and then suspended using a homogenizer in 5 ml of buffer A (10 mm Tris-HCl , pH 8, 6M urea, 0.4M NaCl, 10 mM β-mercaptoethanol, 5 mM imidazole). It was stirred for 1 hour at room temperature, the supernatant was separated by centrifugation at 10 thousand rpm for 10 minutes, the precipitate was discarded.

A column of IDA agarose (1 ml) was charged with a solution of NiSO ₄ , balanced with buffer A with 5 mM imidazole. The supernatant was applied to the column, washed with 5 ml of buffer A, then 10 ml of buffer B (buffer A with 20 mM imidazole). Elution was performed with buffer C (10 mM Tris-HC1, pH 8, 2 M urea, 0.4 M NaCl, 10 mM β-mercaptoethanol, 0.3 M imidazole). This buffer was added in 0.5 ml, collecting the appropriate fractions. Typically, H16 and H18 proteins were detected in fractions 1-3.

The combined fractions were dialyzed overnight against a 100-fold volume of 10 mM Tris-HC1 buffer, pH 8, 150 mM NaCl. (Figure 3, which shows the electrophoresis of proteins in SDS page 13% with SDS, where

1.8 - Molecular Weight Markers

2 - Lysate of E. coli cells containing plasmid pQE30 before IPTG induction

3 - Lysate of E. coli cells containing plasmid pQE30 after induction of IPTG

4 - Lysate of E. coli cells containing plasmid pTHE716 after induction of IPTG

5 - Lysate of E. coli cells containing plasmid pTHE718 after induction of IPTG

6 - Hybrid protein E716-Hsp70 after single-stage chromatography

7 - E718-Hsp70 Hybrid Protein after One-Step Chromatography.)

Example 4

The composition of the hybrid proteins containing proteins E7 16-Hsp70 (M.tuberculosis) and E7 18-Hsp70 (M.tuberculosis) in the ratio (1-2): 1.

The mixture is a white powder, highly soluble in water, in physiological saline and phosphate-buffered saline Dilbecco. The composition is stored under aseptic conditions in sealed glass capsules in the absence of acid, in dry form or dissolved in these solvents.

The composition with a protein ratio of 1: 1 is used for immunotherapy according to example 7.

Example 5

Pharmaceutical kit

The pharmaceutical kit consists of two reagents: the composition of the hybrid proteins described in example 4, and the well-known BCG vaccine. The BCG vaccine is a live mycobacterium of the BCG vaccine strain modified in a 1.5% solution of sodium glutaminate. The vaccination dose contains 0.05 mg in 0.1 ml of solvent. The drug is stored at a temperature not exceeding 8 ° C.

Example 6

Immunotherapy of experimental tumors expressing HPV-16 E7 protein using E716 fusion protein - Hsp 70.

) и Е7- Hsp 70 (

) в дозе 100 мкг. На третий день после иммунизации мышам подкожно вводили клетки ТС-1 в количестве 1,3×10⁵. Наблюдение за мышами вели в течение 50 дней.For the induction of tumors, the TC-1 mouse mouse line containing the integrated HPV-16 genome, in which the viral oncogenes E6 and E7 are actively expressed, was used. To assess antitumor immunity, C57B1 / 6 mice were injected subcutaneously with 100 μg of purified E7 proteins (in complete Freud's adjuvant), Hsp 70, and E7-Hsp 70 in Dilbecco's phosphate-buffered saline. On the third day after immunization, 1.3 × 10 ⁵ TC-1 cells were administered. Within 7 days, all experimental animals developed subcutaneous palpable tumor. The Figure 4 presents the summarizing results of these experiments, which demonstrated the prophylactic immunization with recombinant E7 proteins in Freud's complete adjuvant (

) and E7- Hsp 70 (

) at a dose of 100 mcg. On the third day after immunization, mice were injected subcutaneously with TS-1 cells in an amount of 1.3 × 10 ⁵ . Mice were monitored for 50 days.

As follows from the data presented, by day 30, all mice immunized with the E7-Hsp 70 fusion protein had complete tumor regression. Partial regression (10-15% of animals) was observed in the case of immunization with protein E7 and Hsp 70 (data not shown). In the control group (non-immunized mice), progressive tumor growth was observed.

) и E7-Hsp 70 (

) и E7-Hsp70 на фоне предварительной иммунизации БЦЖ (

). Доза для всех реагентов 15 мкг. На третий день после иммунизации мышам подкожно вводили клетки ТС-1 в количестве 1,3×10⁵. Наблюдение за мышами вели в течение 50 дней.Immunotherapy of experimental tumors with the E7-16-hsp70 hybrid protein was carried out against the background of preliminary immunization with BCG with a hybrid protein concentration 6.5 times (15 μg) lower than in experiments without preliminary sensitization of BCG animals. The data are presented in Figure 5, which shows the prophylactic immunization with recombinant E7 proteins in Freud's complete adjuvant (

) and E7-Hsp 70 (

) and E7-Hsp70 against the background of preliminary immunization with BCG (

) The dose for all reagents is 15 mcg. On the third day after immunization, mice were injected subcutaneously with TS-1 cells in an amount of 1.3 × 10 ⁵ . Mice were monitored for 50 days.

Example 7

Two groups of volunteers (30 women each with a diagnosis of grade I cervical dysplasia) were injected subcutaneously twice with an interval of 14 days with a composition of E7 16-Hsp70 and E7 18-Hsp70 proteins and E7 16-Hsp70 protein, respectively, in physiological saline at a dose of 0 , 3 mg per 1 injection. After a month of observation, the first and second groups underwent a routine examination. Positive dynamics was observed in both groups. However, in the first group this percentage was 75%, while in the second it was observed only in 54% of patients.

Claims (6)

1. The composition of recombinant proteins for immunotherapy and prophylactic vaccination of tumor diseases of the anogenital sphere associated with human papilloma viruses, based on hybrid proteins of the human papillomavirus associated with heat shock proteins, characterized in that it contains an effective amount of hybrid proteins consisting of amino acid sequences of the oncoprotein E7 of human papillomavirus types 16 and 18, respectively, which are covalently linked to the amino acid sequence of the protein heat th shock with molecular 70 kDa (Hsp70) from M. tuberculosis, E716-Hsp70 and E718-Hsp70 with a ratio of (1-2): 1. 2. The composition according to claim 1, characterized in that it further comprises pharmacologically acceptable components. 3. The composition according to claim 1, characterized in that the recombinant proteins contain peptide fragments at the N-terminus of 6-His. 4. A method of obtaining a recombinant protein composition for immunotherapy and prophylactic vaccination of tumor diseases of the anogenital sphere associated with human papillomaviruses, characterized in that the E716-Hsp70 and E718-Hsp70 fusion proteins are obtained by expression of the corresponding genes in plasmid pTE 716 under the control of the T7 promoter. 5. A pharmaceutical kit of reagents for immunotherapy and prophylactic vaccination of tumor diseases of the anogenital sphere associated with human papillomaviruses containing, as reagents, a composition of hybrid proteins E716-Hsp70 and E718-Hsp70 in the ratio of (1-2): 1 and BCG tuberculosis vaccine. 6. A method of immunotherapy and prophylactic vaccination of tumor diseases of the anogenital sphere associated with human papillomaviruses, characterized in that they pre-stimulate the immune system by administering BCG tuberculosis vaccine to the patient, after which the composition of the hybrid proteins E716-Hsp70 and E718-Hsp70 from M. tuberculosis.

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