Recombinant fused protein, preparation for immunotherapy based on thereof and method for immunotherapy of relapsing larynx papillomatosis

FIELD: medicine, biotechnology.

SUBSTANCE: invention relates to the development of a recombinant fused protein consisting of an oncoprotein E7 VPCH 11 type with a sequence SEQ № 2 and protein of "heat shock" Hsp 70 from M. tuberculosis cells prepared by using plasmid pQE30-E711-dnaK in E. coli cells. The preparation is used against relapsing human papillovirus comprises the recombinant fused protein in the effective amount and a pharmaceutically acceptable carrier. Method for immunotherapy of relapsing larynx papillomatosis involves intracutaneous administration of indicated preparation in a patient. The advantage of invention involves the development of a novel preparation showing improved immunological indices. Proposed preparative formulations can be used in immunotherapy of relapsing larynx papillomatosis in humans.

EFFECT: valuable medicinal properties of protein, improved method for treatment.

3 cl, 6 dwg, 7 ex

 

Recurrent (return) respiratory papillomatosis (RRP) is the most common tumor of the respiratory tract, occupying the first place among the causes of chronic obstruction of the larynx in children. Although the papilloma is a benign tumor in children because of frequent recurrence, rapid growth and anatomical narrow the lumen of the larynx disease clinically difficult, with symptoms of airway stenosis, which is absent timely assistance can lead to death.

Etiological factor RRS is the human papilloma virus (HPV). Currently identified more than 70 types of the virus, however, according to the literature, in the identification of HPV DNA in biopsy material most patients RRS determined mainly virus genotypes 6, 11, 16 and 18. The data presented in the literature on the relative prevalence of different subtypes of HPV in children, their relationship with disease severity and impact on the effectiveness of anti-relapse treatment is very controversial.

According to the literature, in the USA the incidence of RRP is around 2350 new cases per year in children and 3600 - adults (0.6 to 4.3 per 100000 and 1.6 to 3.8 per 100,000, respectively). In Denmark, the frequency of the RRS is 3.84 per 100,000, including children -3,62 per 100,000 adults - 3,94 per 100,000 in the population.

In the clinic, reconstructive surgery of the larynx DGKB St. Vladimir (clinical database research group SIC MMA. Sechenov) in 2002 was on treatment 182 children suffering from RRS; every year the clinic will be hospitalized 15-30 new patients. In General, in the Russian Federation annually about 4,000 new cases of laryngeal papillomatosis in children and adults.

Treatment of children with RRP is an unsolved problem. The primary method of treatment is surgical removal of papillomas, which consists in the removal of papillomas of the respiratory tract by micro tools, laser or ultrasound under the supervision of modern optics. However, the isolated use of surgical methods does not prevent the development of subsequent relapses. Moreover, re-operation often lead to the development of cicatricial stenosis of the larynx, leading to profound disability. In addition, the growth of warts on the vocal folds leads to obvious violations of speech, until aphonia, determining and many psychological and social problems, and also causing persistent against non-adaptable changes the personality of the child.

In this regard, in recent decades actively search for opportunities to improve the effectiveness of treatment RRS. Currently, the most effective one is by application, along with the surgical method, interferon (IFN). Data about the effectiveness of preparations contradictory. According to the results of various authors, the effect of IFN therapy ranges from 20% to 50%. In addition, the use of recombinant IFN has significant economic, social and health disadvantages: high cost of drugs, the need for intramuscular injection 3 times a week for 1-2 years and more, possible side effects.

The most promising appear to develop such new treatment methods RRS immunotherapy with vaccines.

Pharmaceutical company "Stressgen Biotechnology" (Stressgen Biotechnologies corp.) developed a therapeutic vaccine for the treatment of RRP. Based vaccine is a recombinant hybrid protein consisting of the oncoprotein E7 of HPV type 16 protein and "heat shock" of M.tuberculosis Hsp 65 (U.S. patent 6524825, Chu Nandal, Mizzen Lee, Woo Huacheng bill. Immune responses to HPV antigens, called tracks, which are HPV antigen and a stress protein or expression vector capable of expression of these proteins. (Chu N.Randall, Mizzen Lee, Wu Huacheng Bill. Immune responses against HPV antigens elicited by compositions comprising an HPV antigen and a stress protein or an expression vector capable of expression of these proteins). This solution can be specified as the closest analogue of the prototype.

Recently the published results of clinical trials of this vaccine. Triple vaccine is given in a dose of 500 µg allowed more than 40% increase in the interval between relapses.

However, full stop recurrence of the authors was not achieved. In our opinion, the failure of the vaccine because the vaccine composition includes oncoprotein E7 of HPV 16-th type, whereas the RRS is caused mainly HPV 11 and 6 types. Moreover, as was shown by us, special analyses, in biopsy material of patients RRS mainly detected HPV-11 type. In addition, HPV 11 type causes disease with a more aggressive course. Thus, the use of oncoprotein E7 16th-type pharmaceutical company "Stressgen Biotechnology" (Stressgen Biotechnologies corp.) it was not quite justified. Sufficient to compare the amino acid sequences of proteins E7 type 11 and 16, to verify the absence of any marked homology between them, and hence the cross-antigenic determinants. Therefore, it is difficult to expect a pronounced therapeutic effect in the treatment of RRP HPV-11 type, the vaccine is designed based on the protein type 16 E7. In our opinion, for immunotherapy RRS should be used E7 11 type that will allow you to achieve maximum effect. Moreover, a similar effect should be expected when using our vaccines in the case of therapy RRS due to HPV type 6, as between what they have expressed homology. To improve the immunogenic properties of the oncoprotein E7 company "Stressgen Biotechnology" (Stressgen Biotechnologies corp.) used protein "heat shock" Hsp 65.

The present invention is the preparation of recombinant protein drug based on it having improved immunological characteristics, as well as more effective immunotherapy.

The problem is solved a new drug created on the basis of recombinant protein consisting of oncoprotein E7 of HPV-11 type protein and "heat shock" of M.tuberculosis Hsp 70, and its use for immunotherapy of recurrent papillomatosis.

Recombinant hybrid protein is characterized by the fact that consists of oncoprotein E7 of HPV-11 type sequence SEQ No. 2 and protein "heat shock" of M.tuberculosis Hsp 70, and obtained using the plasmid pQE30-E711-dnaK in E. coli cells.

The drug contains synthesized specified recombinant hybrid protein in an effective amount and a pharmaceutically acceptable carrier. As the carrier can be any acceptable for introduction of immunogenic protein basis, in particular, can be used aluminium hydroxide, mannitol, polyoxidonium. Under the effective number should be same number, which should be sufficient for the treatment or prophylaxis. It will depend on age, sex, weight, condition of the patient. Effective the number is preferably from 100 to 2500 mcg. The drug has a high degree of standard, low reactogenicity.

There is also a method of immunotherapy of recurrent laryngeal papillomatosis, according to which the patient intradermally injected the drug against papillomavirus man in an amount of 0.1-2.5 mg protein per dose, if necessary, with the re-introduction of the drug in two weeks.

According to our data, Hsp 70 has the best immunomodulatory characteristics that do not depend on the HLA type. Developed the drug gives the best clinical results, namely: greatly increases the interval between relapses, and can be used for vaccination heterogeneous human population, as well as severe forms of the disease.

The invention can be illustrated by the following examples of its implementation.

Example 1.

Typing papilloma virus in patients with a diagnosis of RSD.

Postoperative material from patients diagnosed with RRS was used to determine the types of HPV. HPV DNA was able to identify all 26 children (including the primary outcome was confirmed by repeated trial, all 3 children surveyed twice). Isolated infection 11 type virus was detected in 15 patients (57,7%); isolated infection 6 type virus was detected in 7 children (26.9 per cent); the remaining 4 children (15.4 per cent) were in zirovanii and 6 and 11 HPV types simultaneously (Figure 1 and 2). Infection 16 and 18 HPV types in any case have been identified.

Figure 1 shows a photograph of agarose gels in the study warts HPV type 6. (The arrow shows the target bands. Because the primers were selected for the subsequent synthesis of the protein E7 of HPV 6 and 11 types, they gave additional bands with human genomic DNA).

Figure 2 presents a photograph of agarose gels in the study warts HPV 11 type. (The arrow shows the target bands. Because the primers were selected for subsequent protein synthesis E 7 HPV 6 and 11 types, they gave additional bands with human genomic DNA).

Example 2.

A comparative analysis of the amino acid sequence of the E7 oncoproteins of HPV 6, 11 and 16 types.

Alignment of E7 protein, HPV 6 and 11

E7 VPC SEQ No. 1

MHGRHVTLKDIVLDLQPPDPVGLHCYEQLVDSSEDEVDEVDGQDSQPLKQHYQIVTCCCG

E7 VPC SEQ No. 2

MHGRLVTLKDIVLDLQPPDPVGLHCYEQLEDSSEDEVDKVDKQDAQPLTQHYQILTCCCG

E7 VPC SEQ No. 3 CDSNVRLWQCTETDIREVQQLLLGTLNIVCPICAPKT

E7 VPC SEQ No. 4 CDSNVRLWECTDGDIRQLQDLLLGTLNIVCPICAPKP

Multiple alignment of E7 proteins, HPV 6, 11 and 16

E7 VPC

MHGRHVTLKDIVLDLQPPDPVGLHCYEQLVDSSEDEVDEVDGQDSQPLKQHYQIVTCC

E7 VPC

MHGRLVTLKDIVLDLQPPDPVGLHCYEQLEDSSEDEVDKVDKQDAQPLTQHYQILTCC

E7 VPC

MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFC

E7 VPC CGCDSNVRLWQCTETDIREVQQLLLGTLN1VCPICAPKT

E7 VPC CGCDSNVRLWECTDGDIRQLQDLLLGTLNIVCPICAPKP

E7 VPC CKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP

Example 3

Construction of recombinant vaccine that encodes the synthesis of a hybrid protein E7 11-Hsp 70 (see figure 3).

PR is measures 4

Getting a producer of hybrid protein containing the E7 peptide HPV 11 type and DnaK (HSP70) .tuberculosis.

1. PCR amplification E7 gene of human papillomavirus 11 type.

As the matrix was used provided a preparation of genomic DNA from clinical sample that has undergone a preliminary check for the presence of DNA 11 types of human papillomavirus.

PCR amplification was performed using primers:

E7-11-FGAAGATCTATGCATGGAAGACTTGTTAC
E7-11-RCGGGATCCTGGTTTTGGTGCGCAGATGG

The initiating codon is in the same reading frame with the sequence 6HIS tag termination codon is missing.

2. Cloning of the gene sequence of E7 in the recombinant vector-producer DnaK.

Has been used previously received vector pQESO-dnaK-Y (see the diagram on figure 4), providing for the expression of protein DnaK, fused with a 6HIS sequence at N-end. PCR fragment of the E7 gene was treated restrictase BamHI and BgIII and cloned in the BamHI site of the plasmid pQE30-dnaK-Y. Recombinants having the insert in the correct orientation were identified by restriction analysis. Circuit design is provided on the attached figure 4. It should be noted that the recombinant plasmid pQE30-E-dnaK provides products hybrid protein 6HIS-E7(type 11)-DnaK.

3. Grow the s strain producer of the protein E7 of HPV type 11 and DnaK(HSP70) M.tuberculosis.

For the cultivation of the producer strain using a nutrient medium Luria-Bertani composition:

10 l take 100 g of tryptone, 50 g yeast extract, 100 g of sodium chloride and bacto agar. To the sterilized medium add ampicillin to a final concentration of 50 mg/ml Incubated with strain at 37°With during the night.

4. Products E7(11)-DnaK

Synthesis of a hybrid protein E7(11)-DnaK induced using IPTG as follows:

Night culture DLT1270/ pQE30-E711-dnaK, grown in LB-broth, diluted 1:100 and grown in LB-broth (medium Luria-Bertani) on the rocking chair to the density OD600-0.5.

Then was made 0.1 mM IPTG and continued growing for 3 hours.

Production of the protein was monitored using SDS-PAGE. Watched the fusion protein of the expected molecular weight.

5. Selection of recombinant hybrid protein consisting of the E7 oncoprotein of HPV 11-type sequence SEQ No. 2 and protein "heat shock" of M.tuberculosis Hsp 70, and obtained using the plasmid pQE30-E711-dnaK in E. coli cells.

The raw material is biomass recombinant E.coli strain.

Recombinant protein, with the growth of the strain accumulated in the cytoplasm in the form of Taurus enabled.

Receiving and washing Taurus enabled.

The cell biomass of E. coli was added to the solution A: 50 mM Tris (121.14 g/mol) 0.5 M NaCl (58.44 g/mol), 5 mM EDTA (372.2 g/mol), 1% non-ionic detergent Nonidet P-40, ImM PMSF (174.2 g/the ol). pH=7.5±0.3

Received the homogeneous solution, which was subjected to sonication on the cage B.Braun (USA).

Processed ultrasound solution suspension was centrifuged in a centrifuge Beckman J2-21, at 10,000 RPM at +4°within 10 minutes the Supernatant was discarded. To the precipitate (bullock inclusion) solution was added In: 50 mM Tris (121.14 g/mol), 0.5 M NaCl (58.44 g/mol), 4 mM EDTA (372.2 g/mol), 2M urea (60.06 g/mol), 0.5 mM PMSF (174.2 g/mol), 0.05% Nonidet P-40 (w/w), pH=7.5±0.3, homogenized him and centrifuged in the same mode.

The processing solution was repeated 2 times with the same parameters.

Then, Taurus inclusion were treated 2 times so the same solution: 50 mM Tris (121.14 g/mol), 0.5 M NaCl (58.44 g/mol), 1.5 mM EDTA (372.2 g/mol), 2 M urea (60.06 g/mol).

Dissolving the washed Taurus on and refolding of the target product.

To the resulting precipitate (bullock inclusion) solution was added D: 25 mM Tris (121.14 g/mol), 0.25 M NaCl (58.44 g/mol), 8M urea (60.06 g/mol), 10 mM DTT (154.24 g/mol), pH=8.0±0.2 subsequent gomogenizirovannom.

The resulting mixture was treated with ultrasound on the cage B.Braun (USA).

After sonically, the solution decantation, and centrifuged in a centrifuge Beckman J2-21, rotor Beckman JA-14, at 12000 RPM at +4°within 20 minutes

The precipitate was discarded, and the supernatant decantation. The solution Taurus inclusions were placed on the magnetic stirrer, was fed a solution of PBS: .1 M Na 2HPO4(142 g/mol), 1.45 M NaCl (58.44 g/mol), pH=7.5±0.2 and stirred.

The resulting solution was subjected to tangential ultrafiltration with simultaneous dialysis in PBS solution. The solution was concentrated.

Figure 5 and 6 shows the expression of a hybrid protein consisting of the E7 oncoprotein of HPV 11-type sequence SEQ No. 2 and protein "heat shock" of M.tuberculosis Hsp 70, and obtained using the plasmid pQE30-E711-dnaK in E. coli cells.

Example 5

The manufacture of dosage forms of the vaccine.

Purified recombinant protein is mixed with a preparation of aluminium hydroxide (in the ratio of 1 mg of protein 0.4 mg aluminium hydroxide), thoroughly mixed under mild conditions for 20 minutes. After that, the suspension is centrifuged at 5000 revolutions/min, supernatant collected and used for measurement of residual protein. Sediment posing an aluminum hydroxide with adsorbed protein, suspendered in phosphate buffer and poured into bottles. The concentration of protein in a single dose is 0.5 mg

Example 6

Purified recombinant protein at a concentration of 2.5 mg per ml of phosphate buffer overflows in the vials 0.2 ml, containing 10 mg of mannitol, and liabilitiesa. Each vial contains 0.5 mg of protein.

Example 7

Method of immunotherapy of recurrent papillomatosis of the larynx.

Reaction to widediapasone drugs are extremely individual, due to the peculiarities of the organization of the main histocompatibility complex. In this regard, each case of drug requires an individual approach and objective laboratory criteria the effectiveness of therapy. As such the criteria used level antitelomerase in the serum of patients after administration of each dose and the rate of activation of the delayed-type hypersensitivity (GST) in respect of the E7 protein. This parameter was estimated using the skin test by intradermal injection of 10 μg of peptides containing antigenic determinants of the protein E7. 12 patients diagnosed with recurrent respiratory papillomatosis" vacciniavirus intradermally one dose of the drug obtained in example 1 and 4 patients drug in in example 2. Re-introduction of the drug were conducted over two weeks. All patients determined the titer of specific antibodies and assessed the level GST. In 8 patients recorded enough antitelomerase and level GST. In 8 patients, these figures were not expressed, in this regard, it was recommended re-introduction of the drug. After two vaccinations with the drug every 2 weeks performance antitelomerase and skin sample has reached the required level. Monitoring of patients during the year showed otsutstvie relapses.

1. Recombinant hybrid protein, characterized in that it consists of oncoprotein E7 of HPV-11 type sequence SEQ No. 2 and protein "heat shock" of M.tuberculosis Hsp 70, and obtained using the plasmid pQE30-E711-dnaK.

2. Drug against recurrent papillomavirus person, characterized in that it contains a recombinant hybrid protein according to claim 1 in an effective amount and a pharmaceutically acceptable carrier.

3. Method of immunotherapy of recurrent papillomatosis of the larynx, characterized in that the patient intradermally injected the drug against papillomavirus man according to claim 2 in an amount of 0.1-2.5 mg protein per dose, if necessary, with the re-introduction of the drug in two weeks.



 

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