Chimeric protein for malignant lymphoma treatment

FIELD: medicine, oncology, peptides.

SUBSTANCE: invention relates to a chimeric protein used in treatment of malignant lymphomas. Invention proposes chimeric protein consisting of two peptides representing inhibitor of cyclin kinases as an active fragment p16INK4a with amino acid sequence 84-13 or 84-106 as a therapeutic agent and the second peptide VP22 with sequence 140-301 of virus herpes simplex as a transporting agent. Advantage of the invention involves the development of preparation of high effectiveness of penetration into cells and possessing cytostatic and cytotoxic effects.

EFFECT: valuable medicinal properties of chimeric protein.

1 dwg, 2 ex

 

The invention relates to the field of medicinal products on the basis of chimeric (i.e. containing fragments of polypeptides of different proteins) penetrating proteins, intended for the treatment of neoplastic diseases of type malignant lymphoma.

One of the areas of treatment of neoplastic diseases is the use of inhibitors ticinovic kinases, for example protein family INK4a. While it is desirable that the molecular constructs were delivered into the cell "address" and worked on specific intracellular signal or function.

The study of transport properties of peptides with different structure (amino acid sequence) showed that depending on the structure of different peptides can be accumulated in different compartments of the cell. This property gives a theoretical possibility to construct a sequence with a target delivery to different organelles of the cell.

Known use as a transport agent vector Antp (penetratin) [Robin Fåhraeus, Jesús M. Paramio, Kathryn L. Ball, Sonia Laín, David P. Lane. Inhibition of pRb phosphorylation and cell-cycle progression by a 20-residue peptide from p16CDKN2/INK4A//Current Biology. 1996, 6:84-91].

Also known to use this as a viral vector and expression of the full-size protein [Schreiber, M., Muller, W.J., Singh, G., and Graham, F.L. Comparison of the effectiveness of adenovirus vectors expressing cyclin kinase inhibitors p16INK4A, p18NK4C, p19INK4D, p21Waf/Cip1 and p27Kip1 in inducing cell cycle arrest, apoptosis and inhibition of tumorigenicity //Oncogene. 1999, 18:1663-1676].

However, the disadvantages are the low expression level of protein and low efficiency of its penetration into cells.

None of the known chimeric proteins cannot be specified as the closest analogue of the invention.

The objective of the invention is the creation of a chimeric protein with high biological activity by improving the transport properties of the agent.

Accordingly, the effect of medico-biological effect, objectively manifested in the use of this chimeric protein consists of the solution of the stated problem.

The technical result is achieved by the fact that the chimeric protein for the treatment of malignant lymphomas contains two amino acid sequences, the first of which includes the inhibitor ticinovic kinases in the form of an active fragment of p16INK4a (amino acid residues 84-103 or 84-106) as a therapeutic agent, and the second includes the VP22 peptide (amino acid residues 140-301) of herpes simplex virus as a transport agent for transfer inhibitor ticinovic kinases inside the target cells.

The sequence of amino acids is as follows (underlined sequence of the P16):

D-A-A-T-A-T-R-G-R-S-A-A-S-R-P-T-E-R-P-R-A-P--R-S-A-S-R-P-R-R-P-V-E- D-A-A-R-E-G-F-L-D-T-L-V-V-L-H-R-A-G-A-R

Control and synchronization of events in the process of cell division is a large complex molecules. One of the key participants in this process are cycline, activating the so-called cyclin-dependent kinases (CDK). During the cell cycle is a sequential activation of transcription of specific cyclina with the subsequent formation of the active complex of the cyclin-CDK. Stop cell division in the control (restriction) points (G1, S, G2) is the inhibition of the relevant zaklinivalo complex through specific proteins. A family of proteins, inhibiting cyclin D (controlling the transition of G1-S). refers to the INK4a proteins.

Protein 16INK4a is the most studied of this group. This protein inhibits the activity of CDK4 and CDK6, included in the complex with cyclin D, which inhibits pRb phosphorylation and the release of E2F and leads to cell cycle arrest at the border of the G1-S transition. Structural and functional studies of this protein have revealed active fragment 16INK4a (amino acids 84-103 or 84-106), which is responsible for the inhibition of CDK4 and CDK6, included in the complex with cyclin D.

Protein 16INK4a plays an important role in the process of differentiation and aging of cells, and it is not functional or missing in many tumor tissues. For 40% of malignant the x lymphomas person is characterized by dysfunction of the protein 16INK4a. About 20% of cases with overexpression of Zilina D. I.e. more than half of all cases of malignant lymphomas have impaired control restriction point R1 (G1-S transition).

The ability to use natural inhibitors ticinovic kinases to control proliferation occurred after the discovery of the ability of some proteins to the TRANS-activation. I.e. proteins synthesized in the same cell, could go out and penetrate into a different cell, lead to the activation of certain genes. The study of the structure of these proteins allowed us to identify short (16 to 30 amino acids) amino acid sequences responsible for intracellular transport. It was shown that the addition of such a sequence in the structure of an arbitrary protein confers properties of intracellular and intranuclear internalization. Currently, there are about 30 known sequences of such peptides.

According to the invention was constructed chimeric protein molecule that carries internalize VP22 protein fragment, and a fragment of the protein p16INK4a inhibitor ticinovic kinases.

This sequence was synthesized directly on a peptide synthesizer, and the obtained genetic engineering methods. Was cloned gene and tested technique for hybrid protein product microbiological method.

Modica synthesis: peptides by solid state method [Stewart, J.M., Young, J.D Solid Phase Peptide Synthesis, 2nd edn., 1984, Pierce Chem.Co., Rockford, IL] ispolzovaniem Boc/Bzl strategy and the following protective groups for the side functions of amino acids: Asp(cHex), Glu(cHex), Arg(Tos), Met(O), Ser(Bzl), Thr(Bzl), His(Bom), Lys(2Cl-Z) and Thr(SNO). As the polymeric substrate was used is a copolymer of styrene with 1% divinylbenzene, containing of 0.53 mmol/g of Boc-Lys(2Cl-Z)-PAM, derivatives of amino acids, condensing agents and catalysts, solvents and triperoxonane acid (TFU). The release of the BOC-groups were carried out by the action of 50% TFU in dichloromethane (DCM) followed by neutralization with 5% solution of diisopropylethylamine in DCM. The syntheses were carried out in a flow reactor with a continuous recording of changes in the volume of peptidylarginine (svetlogradsky monitoring). Condensation was carried out using TBTU in the presence of 1-hydroxybenzotriazole (HOBt) and N-methylmorpholine [Reid, G.E. and Simpson, R.J. Automated solid-phase peptide synthesis: use of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate for coupling of tert-butyloxycarbonyl amino acids, Anal Biochem, 1992, v.200(2), pp.301-9]. The fullness of the passage of condensation reactions at each stage controlled by qualitative ninhydrin test [Kaiser, E., Colescott, R.L., Bossinger C.D. Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides, Anal Biochem, 1970, v.34(2), pp.595-8]. A four-fold excess of activated derivatives were used during the first and, if necessary, repeated condensations. The reaction was carried out in ECENA 20 hours. Just before cleavage of the peptide from the polymeric substrate was performed removal of Fmoc and/or formyl groups, blocked side functions tryptophan residues, processing resin 20%solution of piperidine in DMF (0°C, 2 hours). The peptides were tsalala from polymeric substrate with simultaneous removal of permanent protective groups of liquid hydrogen fluoride in two stages by the method of "low-high HF" [Tam J.P. and R.B. Merrifield Strong acid deprotection of the synthetic peptides: mechanisms and methods. - In: The Peptides, v.9 (S. Udenfriend, J. Meienhofer, eds.). New York: Academic Press, 1987, pp.185-248]. After lyophilization, the peptides were transferred to acetate by passing through a short column filled with ion exchange resin (3 g, acetate form). Was separated from by-products of ones nature chromatographytandem on the column. Was carried out by elution 1N AcOH. Front peak was collected and was liofilizovane. For all peptides obtained adequate values of molecular masses.

The strategy for cloning the chimeric gene was cloned gene fragment protein VP22 (amino acid residues 140-301) from the DNA of herpes simplex virus (HSV 1) in vector pR731, then chemical synthesis of a fragment of the gene p16INK4a (amino acid residues 84-103 or 84-106) and receiving vector pR760 with this fragment, then the merging of the two genes and obtain the cloned chimeric gene.

1) Polymerase chain reaction and cloning of DNA encoding the C-to the end of the protein VP22 (amino acid residues 140-301).

Using as a template DNA virus HSV1, by polymerase chain reaction using oligonucleotide primers composition

5'-ccg-gcg-gcg-gga-tcc-ace-cgc-ggc-agg

5'-ctg-ggt-aag-ctt-aag-atc-tct-cga-cgg gcc-gtc-tgg-ggc-gag,

corresponding to the middle and the end portion of the DNA that encodes a protein VP22, the amplified nucleotide sequence (underlined sites specific endonucleases BamHI, BglII and HindIII.

Polymerase chain reaction (PCR) was performed under the following conditions - 4.5 min - 94°; within 5 cycles: 64°C - 5 min, 72°s - 1 min and 94°C; 30 cycles: 64°C - 30 sec, 72°s - 1 min and 94°C - 30 sec; 64°C - 5 min; 72°- 10 minutes

PCR was performed in 25-50 μl reaction mixture containing 10 RMB each primer, 67 mm Tris-HCl buffer (pH 8.8 at 25° (C) 15 mm ammonium sulfate, 2.5 mm magnesium chloride 0.01% tween-20, the mixture of deoxynucleotides (dATP, dCTP, dTTP, DSTF to 2.5 mm) and 1 unit of the enzyme Taq polymerase.

To obtain plasmids expressing C-end of the protein, the DNA pCRVP22 size 510 i.e. cloned in plasmid pQE 16, using restriction endonucleases BamHI and HindIII, the DNA fragment pCRVP22 size 499 i.e. corresponding to the fragment of the gene VP22, combined with the DNA fragment of the plasmid pQE 16 size 3396 i.e. containing the promoter region of the phage T5. The obtained plasmid was named pR731. For this and subsequent stages for the TRANS is information used E.coli strain M15[REP4] (Nals, Strs, rifs, lac-, ara-, gal-, mtl-, F-, recA+, uvr+) (QUIAGEN). All molecular biology procedures, including DNA isolation, restriction, kierowanie oligonucleotides, ligation of DNA, transformation of DNA into E.coli cells. Vector pR731 used for producing the control protein VP22.

2) Synthesis and cloning oligonucleotide duplex encoding the peptide of the protein P16.

A fragment of the gene P16 (for the variant amino acid residue 84-103) received chemical-enzymatic method of the following oligonucleotides:

1.5'-gatccgacgc tgctcgcgaa ggtttcctg

2.5'-gacaccctggtagtactgca ccgtgctggt gctagatctt a

3.5'-accagggtgt ccaggaaacc ttcgcgagca gcgtcg

4.5'-agcttaagatctagcaccag cacggtgcag tact.

Sequence flanked by pulpitami specific endonucleases BamHI (ONT 1) and HindIII (ONT 4) (underlined), With the end before the HindIII site provides a stop codon. Inside oligonucleotide duplex provided by the sites restricts Nrul (ONT 1, 3), Seal (ONT 2, 4), BglII (ONT 2, 4) (all underlined).

Plasmid was obtained by cloning a pre-protonirovannykh and annealed oligonucleotides plasmid pQE16, which was taken by its fragment 3396 i.e. obtained by the restriction endonucleases BamHI and HindIII, and called pR760.

3) Cloning of the gene of the hybrid protein p16-VP22.

A plasmid expressing a hybrid protein containing a fragment of P16 gene and a fragment of the gene of the protein VP22, designed from FR is gment DNA plasmids pR731 size 3197 n, obtained by the restriction endonucleases BamHI and Pvul and vklyuchayushchego VP22 gene, and a DNA fragment of plasmid pR760 size 806 i.e. obtained by the restriction endonucleases BglII and Pvul and including a fragment of the gene P16.

The possibility of carrying out the invention with the implementation of the specified destination confirm the following examples.

Example 1. Analysis of cell proliferation.

A portion of the protein was conjugated with fluorescein-isothiocyanato (FITZ). During the reaction used a low concentration of FITZ and limited her time. Thus was achieved by partial labeling of the protein.

Method light fluorescence microscopy, it was shown that the protein binds to the cells and enters the cell lines Raji, Jurkatt, A, 293 and peripheral blood lymphocytes. The method of running cytofluorometry was shown the binding of the peptide to the cells and the absence of the effect of quenching fluorescence Trifanova blue. Which indicates the accumulation of the peptide inside the cell. In sum, the data obtained indicated the accumulation of the peptide inside the cell

Biological activity of the chimeric peptide containing the active fragment of the protein p16INK4a, was investigated in cell lines (Raji, Jurkatt, A549, 293). The study of cell division revealed the presence of the cytotoxic effect of this drug. The experiments were carried out on sin is ionizirovannykh in phase G0/1 cell lines Raji, Jurkatt and A549. Cell lines were synchronized by sowing in medium containing 0.2% fetal serum and, therefore, lacking in the required number of growth factors. Synchronized cell lines were cultivated in the medium containing 10% fetal serum. There was made the analyzed peptide to a concentration of 5 μm. Under the influence of growth factors, the cells entered mitosis and after 24, 48 and 72 hours were investigated concentration of cells was performed cell cycle analysis using the method of flow cytofluorometry. As a positive control was used 5-fluoro-uracil and Taxol. There was a decline in S, G2 and M phases of the cell cycle and the lag in the growth of cell lines from control. In addition, there was a significant increase in the number of cells included in apoptosis.

The most objective data on the accumulation of the peptide inside the cell were obtained using the method of scanning laser microscopy. Protein, labeled fluorescein isothiocyanates, was dissolved in 0.9% NaCl. Cell lines A549, 293 was grown on the subject sterile glasses and placed in a special chamber directly under the microscope eyepieces. The medium was replaced by medium with the test protein. The saturation of the protein was observed over time. In addition, the produced layer-by-layer scanning cells to determine the population of the localized accumulation of the peptide.

The analysis of the obtained images showed that the protein is distributed in the cell compartments unevenly. The penetration of the protein into the cell fast enough. And after 15 minutes of incubation, most of it is distributed in the cytoplasm.

The speed of his penetration into the peripheral blood lymphocytes, and lymphocyte cell lines (Raji, Jurkatt) was estimated using the method of flow cytofluorometry. This method allows you to capture large concentration of cells in time and is useful for evaluating the permeation rate of the investigated peptide. Measured fluorescence of lymphocytes under the influence of the protein P16-VP22-FITZ at pH 7.5 and pH 6.0. The principle of the method is based on a smaller quantum yield of fluorescence FITZ in solutions more acidic pH. As measurement speed fast enough, then the value of the pH inside the cell cannot be changed. Lymphocytes were incubated with the peptide for 1-15 minutes, then to him immediately added a 20-fold volume of phosphate buffer pH 6.0 and pH 7.5. It was estimated by the difference of fluorescence intensity. In most measurements, the fluorescence intensity did not differ significantly after 15 minutes of incubation. Thus, we can assume that after 15 minutes of incubation, the peptide fully penetrates into the cell. This presents graphs p is context menu a greater efficiency of the claimed chimeric protein.

Example 2. The study of the biological effect of a chimeric protein p16-VP22. Evaluation of cytotoxicity and antiproliferative activity of the drug on normal and tumor cell lines were evaluated for cell lines Raji, Jurkatt, A549, 293.

In experiments with cell lines was evaluated as the rate of proliferation and the percentage of cells entered apoptosis. Used for the analysis program FloMax 2.0 and ModFit LT 3.0. All cell lines was shown cytotoxic effect of protein p16-VP22. The experiments were carried out as in synchronized and non-synchronized in phase G0/1 cell lines Raji, Jurkatt, A549, 293. The maximum effect of protein p16-VP22 was achieved on synchronized cell lines. So the percentage subdiploid peak in the DNA histogram characterizing the level of apoptosis, reached 80% at a protein concentration of 10 μm. The ratio of cells in S, G2 and M phases of the cell cycle, cells in G0/1 phase, characterizes the degree of cell proliferation. The ratio control is significantly higher than in the case of using protein p16-VP22.

Example 3. Three groups of mice BLRB transplanted into the abdominal cavity short-line breast cancer obtained from spontaneous cancer the same line. One group received no treatment, the second experimental peptide, and a third peptide, and having inoculate replacement it is not active.

Survival analysis shows the increase in life expectancy in the experimental group and 10 to 30% and increase the proportion of animals with large lifetimes.

Thus, shows a significant cytostatic and cytotoxic effect of the claimed chimeric protein, due to the synergetic effect of its components.

Chimeric protein for the treatment of malignant lymphomas containing two amino acid sequences, the first of which includes the inhibitor ticinovic kinases in the form of an active fragment of p16INK4a (amino acid residues 84-103 or 84-106) as a therapeutic agent, and the second includes the VP22 peptide (amino acid residues 140-301) of herpes simplex virus as a transport agent for transfer inhibitor ticinovic kinases inside the target cells.



 

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30 cl, 1 tbl, 69 ex

FIELD: medicine.

SUBSTANCE: method involves carrying out 5-10 sessions of general magnetotherapy beginning from 3-rd day after surgical intervention in static mode: magnetic field rotation frequency is equal to 100 Hz, magnetic field intensity of 30 oersted, magnetic field shape is sinusoid half-cycle, raising time being equal to 30 s, time of recession 30 s; the number of procedures is equal to 15. Then, remote radiation therapy is administered 4 weeks later after surgical intervention. The treatment is applied to removed kidney bed and lymphatic collectors in single stage in classical fractioning mode at a doze of 2 Gy 5 times a week within 5 weeks up to reach total doze of 50 Gy with general magnetotherapy being concurrently applied in 1 session per day mode, the number of procedures being equal to 25. The first biotherapy course with recombinant human alpha tumor necrosis factor (TNF-α) is given at a dose of 2 mln units 2 weeks later after the radiation therapy being done. Total З biotherapy courses are to be given with 21 days long pause available between the courses.

EFFECT: enhanced effectiveness of treatment; reduced risk of postoperative complications; increased immune activity.

FIELD: medicine.

SUBSTANCE: method involves excising basic tumor as one-stage operation by means of high-energy СО2 laser or cytoreduction operation is carried out (basic tumor component removal) using laser output power of 20-40 W at the first stage. Remote photodynamic therapy is applied by irradiating the whole vulva surface with radiation dose of 100-150 J/cm2, power density of 50-70 mW/cm2 and irradiating tumor localization zone with radiation dose of 200-300 J/cm, power density of 150-200 mW/cm at the second stage. Additional interstitial laser irradiation with radiation dose of 200-300 J is optionally carried out at radiation power of 200-300 mW in tumor invasion zone.

EFFECT: enhanced effectiveness of organ-retaining and sparing treatment; accelerated treatment course; reduced risk of postoperative complications.

2 cl

FIELD: medicine, oncology.

SUBSTANCE: during the first day of therapy it is necessary to affect the lesion focus with alternating magnetic field at induction being 5-160 mTl, frequency of 50-100 Hz for about 7-10 min. Out of peripheral vein one should sample blood into two vials with hemoconservant per 100 ml/each; one vial should be supplemented with doxorubicin at the dosage of 50 mg/sq. m, the second vial - with 5-fluorouracil 750 mg/sq. m. Both vials should be incubated. Then it is necessary to inject intravenously by drops the content of the first, and then of the second vial for a patient. During the period since the first day up to the seventh one it is necessary to introduce cyclophosphan intramuscularly per 200 mg/sq. m. Since the first up to the tenth day of therapy one should affect with alternating and direct magnetic field. On the eighth day after seances of magnetic therapy one should repeatedly introduce anti-tumor chemopreparations at the same dosages and sequence. In two weeks it is necessary to repeat the above-mentioned curative impacts. In two weeks after the last introduction of chemopreparations one should fulfill surgical removal of lesion focus. The innovation enables to avoid side toxic manifestations of chemopreparations and notching of sutures and, also, healing due to secondary tension.

EFFECT: higher efficiency of therapy.

1 ex

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

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