RU2677264C1 - Immunotoxin for multiple sclerosis therapy, method for preparation and application thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine and concerns an immunotoxin for the treatment of multiple sclerosis, which is a recombinant protein, including the A-subunit of viscumin and a fragment of myelin-oligodendrocyte glycoprotein linked by a linker for binding these fragments. Group of inventions also relates to recombinant plasmid DNA for expression in Escherichia coli cells of the gene encoding said immunotoxin; concerns a method for producing an immunotoxin; concerns a method of treating multiple sclerosis, consisting in the intravenous administration of a pharmaceutical composition containing the indicated immunotoxin, in a therapeutically effective amount.

EFFECT: group of inventions provides immunotoxin for the treatment of multiple sclerosis with high specificity and low toxicity.

19 cl, 9 dwg, 3 tbl, 1 ex

Description

Technical field

The invention relates to the field of biochemistry, in particular to a recombinant immunotoxin that can be used in medicine for the treatment of multiple sclerosis, a method for its preparation and use. The inventive immunotoxin is a polypeptide chain, which consists of a delivery vector (fragment of myelin-oligodendrocyte glycoprotein) and the active part (A-chain of plant viscumin toxin).

State of the art

The treatment of multiple sclerosis (PC) remains one of the most serious problems of modern medicine. If previously therapeutic care for patients with PC was limited only to symptomatic treatment, then by now there are ways to influence the course of the disease. Unfortunately, a drug has not yet been developed that is able to completely stop the development of the disease. Modern treatment for PC includes the use of drugs, "changing the course of the PC" (PITRS). The drugs available to doctors reduce the frequency and severity of exacerbations, as well as slow down the rate of accumulation of neurological deficits, as evidenced by magnetic resonance imaging of the brain and spinal cord [J van der Star B, YS Vogel D, Kipp M, Puentes F, Baker D, Amor S. In vitro and in vivo models of multiple sclerosis. CNS & Neurological Disorders-Drug Targets (Formerly Current Drug Targets-CNS & Neurological Disorders). 2012 Aug 1; 11 (5): 570-88; Freedman, M.S., et al., Efficacy of disease-modifying therapies in relapsing remitting multiple sclerosis: a systematic comparison. Eur Neurol, 2008. 60 (1): p. 1-11; Fisher E, Nakamura K, Lee JC, You X, Sperling B, Rudick RA. Effect of intramuscular interferon beta-la on gray matter atrophy in relapsing-remitting multiple sclerosis: A retrospective analysis. Multiple Sclerosis Journal. 2015 Aug 3: 1352458515599072]. Approved PITRS include drugs: interferon-beta (INFB), glatiramer acetate (Copaxone), mitoxontron, fingolimod, natalizumab, daclizumab, teriflunomide, dimethyl fumarate, alemtuzumab [Loma, I., & Heyman, R. (2011) . Multiple Sclerosis: Pathogenesis and Treatment. Current Neuropharmacology, 9 (3), 409-416]. The first two drugs are most actively used in the remitting course of PC, however, they are not effective in all cases, and then they switch to the use of newer and stronger, but with side effects, drugs.

For PC therapy, both nonspecific and more targeted drugs are at different stages of development. The following drugs that have passed different stages of clinical trials can be classified as non-specific: 1) Fingolimod (an inhibitor of sphingosine-1-phosphate receptors; stops the release of T and B cells from secondary lymphoid organs, preventing the migration of lymphocytes into the central nervous system) [Brinkmann, V ., Sphingosine 1-phosphate receptors in health and disease: mechanistic insights from gene deletion studies and reverse pharmacology. Pharmacol Ther, 2007.115 (1): p. 84-105]; 2) Teriflunomide (inhibits T-cell proliferation) [Korn, T., et al., Suppression of experimental autoimmune neuritis by leflunomide. Brain, 2001. 124 (Pt 9): p. 1791-802]; 3) Tecfidera (also known as BG-12; contains dimethyl fumarate; it is an immunomodulator with an unclear mechanism of action; reduces the relapse rate in a relapsing-remitting form of PC by 2 times according to the data of stage III clinical trials) [Robert J. Fox et al. Placebo-controlled Phase 3 study of oral BG-12 or glatiramer in multiple sclerosis. N Engl J Med, 2012.367: p. 1087-1097]; 4) Roquinimex (immunostimulant; increases the activity of natural killers, reduces angiogenesis and TNFα production; prevents the formation of new foci of inflammation in the brain, which was confirmed during stages II and II of clinical trials) [Wolinsky, JS, et al., Linomide in relapsing and secondary progressive MS: part II: MRI results. MRI Analysis Center of the University of Texas-Houston, Health Science Center, and the North American Linomide Investigators. Neurology, 2000.54 (9): p. 1734-41]; 5) Laquinimod (a structural analogue of Roquinimex; blocks the ability of T cells and macrophages to move through the blood-brain barrier, and also switches the phenotype from Tx-2 to Tx-3) [Yang, JS, et al., Laquinimod (ABR-215062) suppresses the development of experimental autoimmune encephalomyelitis, modulates the Th1 / Th2 balance and induces the Th3 cytokine TGF-beta in Lewis rats. J Neuroimmunol, 2004.156 (1-2): p. 3-9]; 6) Cladribine (an inhibitor of deoxycytidine kinase that affects DNA repair and indirectly induces the death of lymphocytes; passed stage III clinical trials for the treatment of PC, after which its introduction was stopped) [Thone, J. and G. Ellrichmann, Oral available agents in the treatment of relapsing remitting multiple sclerosis: an overview of merits and culprits. Drug Healthc Patient Saf, 2013.5: p. 37-47]. Due to the nonspecific effect, these drugs have various serious side effects.

An alternative approach to PC therapy is the use of monoclonal antibody preparations directed against specific molecules involved in the pathogenesis of the disease [Buttmann, M. and P. Rieckmann, Treating multiple sclerosis with monoclonal antibodies. Expert Rev Neurother, 2008.8 (3): p. 433-55]. In clinical practice, Natalizumab is used, which is a monoclonal antibody to α4β1 integrins [Steinman, L. (2012). The discovery of natalizumab, a potent therapeutic for multiple sclerosis. The Journal of Cell Biology, 199 (3), 413-416]. It blocks the adhesion of mononuclear cells on the surface of stromal endothelial cells, which prevents the migration of autoreactive lymphocytes through the blood-brain barrier and into inflamed tissues. The effectiveness of this drug in the treatment of PC was higher than the effectiveness of interferon beta [Ransohoff, R. M., Natalizumab for multiple sclerosis. N Engl J Med, 2007.356 (25): p. 2622-9].

Rituximab (Rituximab) is intended for the treatment of various autoimmune diseases, including PC. Rituximab is an IgG antibody specific for CD20 (a membrane protein present on the surface of all mature B cells). The drug induces apoptosis of cells carrying CD20, which leads to a systemic effect on antibody production, the cytokine network, the presentation of antigen by B cells, the activation of T cells and macrophages [Cross, A. N., et al., Rituximab reduces B cells and T cells in cerebrospinal fluid of multiple sclerosis patients. J Neuroimmunol, 2006.180 (1-2): p. 63-70; Duddy, M. and A. Bar-Or, B-cells in multiple sclerosis. Int MS J, 2006.13 (3): p. 84-90; Hauser, S. L., et al., B-cell depletion with rituximab in relapsing-remitting multiple sclerosis. N Engl J Med, 2008.358 (7): p. 676-88; Uchida, J., et al., The innate mononuclear phagocyte network depletes B lymphocytes through Fc receptor-dependent mechanisms during anti-CD20 antibody immunotherapy. J Exp Med, 2004.1999 (12): p. 1659-69; Liossis, S.N. and P.P. Sfikakis, Rituximab-induced In cell depletion in autoimmune diseases: potential effects on T cells. Clin Immunol, 2008.127 (3): p. 280-5]. Data obtained during the second phase of clinical trials showed that the administration of 2000 mg of Rituximab significantly reduces the number of foci of demyelination within 48 weeks compared to the control group [Bar-Or, A., et al., Rituximab in relapsing-remitting multiple sclerosis: a 72-week, open-label, phase I trial. Ann Neurol, 2008.63 (3): p. 395-400]. A serious drawback of Rituximab is the non-specific removal of the vast majority of the circulating B-cell population.

Despite the large number of existing approaches to the treatment of PC, the idea of directed suppression of a population of autoreactive lymphocytes remains relevant and relevant. Using the selective elimination approach for autoreactive B cells, most of the problems described earlier can be avoided [O'Connor PW, Oh J. Disease-modifying agents in multiple sclerosis. Handb Clin Neurol. 2014 Feb 5; 122: 465-501], characteristic of non-specific therapy of autoimmune diseases. The ideal candidates for the role of targeted molecules are immunotoxins, the extensive experience of which in cancer therapy creates a significant basis for the creation of therapeutic drugs for autoimmune diseases [Arkfeld, D.G., The potential utility of B cell-directed biologic therapy in autoimmune diseases. Rheumatol Int, 2008.28 (3): p. 205-15; Kreitman, R.J., Immunotoxins for targeted cancer therapy. AAPS J, 2006.8 (3): p. E532-51].

Molecules of immunotoxins consist of two key fragments: the signal sequence of targeted delivery to the target cell and the active part, leading to cell death. The signal sequence may be: antibodies, Fab antibody fragments, hormones, cytokines, cell factors, surface T- or B-cell receptor ligands [Zocher, M., et al., Specific depletion of autoreactive B lymphocytes by a recombinant fusion protein in vitro and in vivo. Int Immunol, 2003.15 (7): p. 789-96; Frankel, A.E., Increased sophistication of immunotoxins. Clin Cancer Res, 2002.8 (4): p. 942-4]. Almost all immunotoxins contain antibody fragments, with the exception of the ONTAK preparation, which includes the human interleukin-2 molecule as the domain responsible for binding to target cells. As an active ingredient, bacterial (e.g. diphtheria toxin, pseudomonas exotoxin A) or plant (e.g. ricin) toxins are typically used.

M., Sandvig K. Ricin and Ricin-Containing Immunotoxins: Insights into Intracellular Transport and Mechanism of action in Vitro // Antibodies. 2013. Vol.2, №2. P. 236-269; Becker N., Benhar I. Antibody-Based Immunotoxins for the Treatment of Cancer // Antibodies. 2012. Vol. 1, №3. P. 39-69; Pastan I. et al. Immunotoxin treatment of cancer // Annu. Rev. Med. 2007. Vol. 58. P. 221-237]. Именно первый этап (связывание с поверхностными рецепторами) обеспечивает специфичность действия иммунотоксинов и направленную элиминацию клеток-мишеней.The mechanism of action of immunotoxins is as follows: first, the immunotoxin binds to a specific receptor on the surface of the target cells, then through the receptor-mediated endocytosis, the immunotoxin enters the cell, followed by intracellular transport and the interaction of the toxin with the target. In the case of diphtheria toxin and pseudomonas exotoxin A, the toxic effect is based on the modification of the eukaryotic elongation factor (eEF-2), which leads to the cessation of protein synthesis. Ribosomine activating proteins, which include ricin, also block protein synthesis, but act directly on the ribosomes. Despite the differences in the catalytic mechanism of the toxins used, the penetration of immunotoxins into the cells is generally the same [Srivastava S., Luqman S. Immune-o-toxins as the magic bullet for therapeutic purposes // Biomed. Res. Ther. 2015. Vol. 2, No. 1. P. 169-183;

M., Sandvig K. Ricin and Ricin-Containing Immunotoxins: Insights into Intracellular Transport and Mechanism of action in Vitro // Antibodies. 2013. Vol. 2, No. 2. P. 236-269; Becker N., Benhar I. Antibody-Based Immunotoxins for the Treatment of Cancer // Antibodies. 2012. Vol. 1, No. 3. P. 39-69; Pastan I. et al. Immunotoxin treatment of cancer // Annu. Rev. Med. 2007. Vol. 58. P. 221-237]. It is the first stage (binding to surface receptors) that provides the specificity of the action of immunotoxins and the targeted elimination of target cells.

International application WO 8906968 discloses a method for the preparation and use for the treatment of autoimmune diseases of an immunotoxin consisting of a full-sized monoclonal antibody to a CD5 membrane protein present on 85-100% mature T-lymphocytes and ricin A-chain. The connection of the two components is carried out by chemical conjugation. The disadvantages of the described method include the preparation of the ricin A chain by isolating ricin from a plant source (castor seeds), restoring the disulfide bond between the A and B chains of the toxin, and purifying the A chain of the B chain and full length ricin by chromatography. This method of producing ricin A chain requires special safety measures during production (ricin is a biological weapon and is extremely toxic to humans) and careful monitoring of the completeness of purification. In addition, the CD5 receptor is present in 20-30% of B cells in healthy people, which causes the side effect of the described immunotoxin.

The solution presented in European application EP365087A1 discloses a method for producing immunotoxins for the treatment or prevention of autoimmune diseases (including multiple sclerosis), which are conjugates of peptides recognized by T lymphocytes and the active part (ricin A chain or chelating agent complexes with radioactive isotopes ⁹⁰ Y, ¹⁵³ Sm, ⁴³ Sc, ⁵⁷ Cu, ¹⁸⁶ Rh, ¹⁸⁸ Rh, ²¹² Bi, ²¹¹ At, ¹⁰³ Pd). For the treatment of multiple sclerosis, it is proposed to use peptides corresponding to fragments of the main myelin protein 1-16, 1-37, 59-74, 68-88, 89-169, 114-122. The disadvantages of the described method are: the complexity and high cost of chemical synthesis of the corresponding specific peptide; the need to isolate the ricin A chain from a natural source or the synthesis of a chelator complex with a radioactive isotope (in both cases, special safety measures are required during production) loss of intermediates in the synthesis of the conjugate of the specific and active part of the immunotoxin.

Thanks to scientific progress in the field of genetic engineering of proteins, the trend towards the development of recombinant immunotoxins has recently prevailed due to the possibility of obtaining more homogeneous preparations of a high degree of purity. In the described invention, this approach was applied.

Disclosure of invention

The present invention is the development of an immunotoxin for the treatment of multiple sclerosis and a method for its preparation.

The technical result of the claimed invention is the preparation of an immunotoxin for the treatment of multiple sclerosis, which has high specificity and low toxicity. In the claimed invention, the active ingredient for the treatment of multiple sclerosis is characterized by a selective effect on autoreactive B-lymphocytes, without affecting healthy cells. The claimed immunotoxin has low toxicity, as evidenced by the absence of toxic effects in toxicological studies in animal models. The inventive method of obtaining allows to obtain the claimed immunotoxin, without resorting to additional safety measures. Also, the inventive method allows you to control the homogeneity of the composition at all stages of the synthesis.

The problem is solved by an immunotoxin for the treatment of multiple sclerosis, which is a recombinant protein that includes the A-subunit of viscumin and a fragment of myelin-oligodendrocyte glycoprotein, which binds to target cells connected by a linker to bind the mentioned fragments, while the A-subunit of viscumin is a polyp a sequence of amino acids not less than 95% homologous to SEQ ID NO: 1, and a fragment of myelin-oligodendrocyte glycoprotein is a peptide with It has been consistent for at least 90% homologous to SEQ ID NO: 2. Preferably, the immunotoxin is a protein having a sequence at least 95% homologous to SEQ ID NO: 3.

Also, the problem is solved by recombinant plasmid DNA for expression in Escherichia coli cells of a gene encoding the claimed immunotoxin, which is a commercial vector modified by a fragment encoding the claimed immunotoxin, including the following fragments:

a. XhoI-NcoI fragment;

b. bacteriophage T7 promoter;

c. bacteriophage T7 RNA polymerase terminator;

d. replication start region ColE1 / pMB1 / pBR322 / pUC;

e. an area encoding an antibiotic resistance enzyme gene.

In this case, it is preferable to use plasmids pET-22b (+), pET-26b (+), pET-28a-c (+) as a commercial vector.

When pET-28b (+) is used as a plasmid vector for modification, the modified pET-28b (+) _ ML1A-MOG vector has a size of 6127 bp, in which the sections have the following coordinates:

a. XhoI-NdeI fragment of pET-28b (+) vector;

b. bacteriophage T7 promoter;

c. bacteriophage T7 RNA polymerase terminator;

d. replication start region ColE1 / pMB1 / pBR322 / pUC;

e. the region encoding the gene of aminoglycoside-Z'-phosphotransferase, providing resistance to the antibiotic kanamycin;

f. unique recognition sites by restriction endonucleases: XhoI - 158, BamHI - 233, AscI - 468, AgeI - 686, SacII - 793, NdeI - 996.

The problem is also solved by a strain of Escherichia coli - the producer of the claimed immunotoxin, transformed with recombinant plasmid DNA.

Also, the problem is solved by a method for producing the claimed immunotoxin, which consists in cultivating a producer strain to obtain biomass containing inclusion bodies, which is then subjected to destruction by ultrasonic disintegrator, followed by isolation of inclusion bodies by centrifugation. The obtained inclusion bodies are washed by the method of sequential dissolution of impurities to obtain an immunotoxin in undissolved form, which is then converted into a dissolved form using a solution of a chaotropic agent, after which the inactive dissolved immunotoxin is subjected to renaturation by dilution, diafiltration and concentration using tangential filtration. In this case, the destruction of biomass using an ultrasonic disintegrator is carried out 3-4 times for 3 minutes with 2-minute breaks at a frequency of 22 kHz and cooling on ice, and inclusion bodies are separated from soluble cell components by centrifugation. Purification of inclusion bodies is carried out using solutions containing detergents and chaotropic agents that remove impurities of DNA, RNA, lipids and impurity proteins. The transfer of immunotoxin from an insoluble inactive form to a soluble inactive form is carried out using a solution containing a chaotropic agent, while translation (renaturation) is carried out by dilution in a solution containing arginine and having an acidic pH, then the active immunotoxin is concentrated after renaturation using tangential filtration.

It is preferable to use solutions of urea, thiourea, guanidine hydrochloride as a chaotropic agent, and sodium dodecyl sulfate, deoxycholate, sarcosyl, Tween 20, Tween 80, Triton X-100 as a detergent.

The problem is also solved by a pharmaceutical composition for the treatment of multiple sclerosis, comprising as the active substance the claimed immunotoxin in a therapeutically effective amount and a pharmaceutically acceptable base.

Also, the problem is solved by the method of treatment of multiple sclerosis, which consists in the intravenous administration of a pharmaceutical composition containing the claimed immunotoxin in a therapeutically effective amount.

Brief Description of the Drawings

In FIG. Figure 1 shows the physical map of plasmid pET-24d (+).

In FIG. 2 shows a physical map of the plasmid pET-28b (+).

In FIG. Figure 3 shows the physical map of the pET-24d (+) _ ML1A-MOG genetic construct.

In FIG. Figure 4 shows a physical map of the genetic construction of pET-28b (+) _ ML1A-MOG.

In FIG. 5 shows examples of electrophoregrams of an immunotoxin substance.

In FIG. Figure 6 shows the melting curve for the immunotoxin substance obtained by the claimed method (temperature dependence of the derivative of fluorescence intensity).

In FIG. 7 is a graph of the relative luminescence intensity (proportional to the amount of luciferase expressed) versus the concentration of immunotoxin obtained by the claimed method.

In FIG. 8 is a graph showing the development of signs of experimental autoimmune encephalomyelitis in experimental animals after administration of different doses of the immunotoxin obtained by the claimed method.

In FIG. 9 shows the life expectancy of mice with experimental autoimmune encephalomyelitis in the experimental groups after administration of different doses of the immunotoxin obtained by the claimed method.

The implementation of the invention

The inventive immunotoxin for the treatment of multiple sclerosis (ITRS) as a delivery vector contains a fragment of myelin-oligodendrocyte glycoprotein (MTF). The high specificity and affinity of antibodies present on the B-lymphocyte membrane underlies the specific recognition of the MTF peptide and the binding of ITRS on the surface of autoreactive B-cells. The active component of the immunotoxin is the A-chain of the plant viscumin toxin. The viscumin A chain possesses N-glycosidase activity and catalyzes the depurinization of ribosomes in eukaryotic cells (the adenine residue at position 4324 in the 28S rRNA of the large ribosome subunit), which stops protein synthesis.

Synthesis of a genetic construct (sequence SEQ ID No. 3) encoding the A-subunit of viscumin (sequence SEQ ID No. 1) and a fragment of MTF, which is a peptide with the sequence SEQ ID No. 2, connected by a linker, which ensures their crosslinking, which can have a minimum length ( from two amino acids), was carried out using standard well-known methods of genetic engineering: cloning, restriction, ligation, transformation [Green, MR, Sambrook, J. (Ed.). Molecular Cloning: A Laboratory Manual (Fourth Edition). 2012. Cold Spring Harbor].

Any commercially available plasmid for expression of proteins in Escherichia coli (E. coli) under the control of the T7 promoter, in which there are NcoI and XhoI restriction sites, for example, plasmids pET-24d (+), is used to subclone the obtained immunotoxin-encoding sequence. (Novagen), pET-26b (+) (Novagen), pET-28a-c (+) (EMD Biosciences) (https://www.addgene.org/vector-database/query/?q_vdb=*+** *% 20vector_type: Bacterial). Ha FIG. 1 and FIG. 2, for example, physical maps of the vectors pET-24d (+) and pET-28b (+) are shown. In FIG. 3 and FIG. Figure 4 shows physical maps of plasmids pET-24d (+) and pET-28b (+) containing the region encoding the described immunotoxin (in the figures, ML1A-MOG is indicated).

A plasmid carrying the genetic construct encoding the described immunotoxin is transformed with an E. coli strain suitable for expression of proteins under the control of the T7 bacteriophage promoter (expressing T7 RNA polymerase, preferably carrying the lacIq gene or lysY gene, more preferably both of these genes), for example, E. coli BL21 (DE3) or E. coli Lemo (DE3). After transformation, E. coli cells are seeded on an antibiotic LB nutrient medium, depending on the plasmid used (eg, kanamycin or ampicillin) [Green, M.R., Sambrook, J. (Ed.). Molecular Cloning: A Laboratory Manual (Fourth Edition). 2012. Cold Spring Harbor].

The obtained single colonies of transformed E. coli are cultured in LB medium with an antibiotic, depending on the plasmid used (for example, kanamycin or ampicillin), and the resulting E. coli cell suspension is used as a seed (inoculum) for the fermenter.

To obtain biomass containing ITRS, an auto-induced medium with the addition of an antibiotic is used depending on the plasmid used (for example, kanamycin or ampicillin). For this purpose, seed (sterile) is sterilely transferred to the prepared fermenter, filling approximately 1/3 of the fermenter volume (or sterilizing the medium directly in the fermenter), the inoculum is aseptically introduced through the appropriate port (1 / 20-1 / 10 of the volume of the nutrient medium) . The biomass is incubated for at least 10 hours. After incubation, the synthesis of ITRS is checked by enzyme-linked immunosorbent assay or by analytical electrophoresis. Confirmation of the presence of the target protein is the detection of a signal (for example, absorption at a certain wavelength), a certain intensity during the enzyme-linked immunosorbent assay, or the detection of a band corresponding to a protein of a certain mass on the gel after staining. Using any method, a control sample containing a known amount of purified immunotoxin is used.

During electrophoresis under denaturing conditions, the main spot corresponding to ITRS should migrate as a protein with a mass of 28 ± 1 kDa. Based on the previously constructed calibration curve for the spot area on the electrophoregram, its amount in the analyzed samples is determined depending on the amount of applied ITRS protein.

The resulting biomass from the culture fluid is separated by centrifugation or filtration. Supernatants (culture fluid) are removed, and the resulting cell pellet is stored at -20 ° C until use in the next steps.

ITSS is expressed in E. coli cells in an insoluble form (as inclusion bodies). To isolate inclusion bodies, the biomass of cells is destroyed by ultrasound, after which the inclusion bodies are separated by centrifugation (remain in the sediment) and purified from various impurities by washing with solutions of a certain composition containing detergents and chaotropic agents that remove DNA, RNA, lipids and impurities proteins.

As a chaotropic agent, solutions of urea, thiourea, guanidine hydrochloride can be used.

As detergents, sodium dodecyl sulfate, deoxycholate, sarcosyl, Tween 20, Tween 80, Triton X-100 can be used.

To isolate and wash inclusion bodies, four solutions are used (A, B, C, D). All solutions are prepared in deionized water (type I) and used freshly prepared.

Solution A contains 1.5-2.5 M NaCl, 1-5 mM dithiothreitol, 5-15 mM EDTA and 20-100 mM Tris-HCl or phosphate buffer, while the pH of the solution should be 8.0-9, 0. Solution B for washing inclusion bodies includes 1-3% Triton X-100, 25-150 mM NaCl, 1-5 mM dithiothreitol, 20-100 mM Tris-HCl or phosphate buffer, while the pH of the solution should be pH 8.0- 9.0. Solution B is similar in composition to solution B, but does not contain Triton X-100. Solution G for washing inclusion bodies from impurity proteins includes 3.5-4.5 M urea, 1-5 mm Dithiothreitol, 20-100 mm Tris-HCl or phosphate buffer, while the pH of the solution should be 8.0-9.0 .

For washing (cleaning) inclusion bodies, solution A is poured onto the previously obtained cell pellet at the rate of 1: 5-1: 10 (biomass volume: solution A volume). Cell pellet resuspended. Cells are destroyed by ultrasound at a frequency of 22 kHz when cooled on ice to avoid overheating of the suspension. Ultrasound treatment is repeated 3-4 times for 3 minutes with 2-minute breaks. Then the suspension is centrifuged at 5000-10000 g and 15 ± 5 ° C to separate the inclusion bodies from the soluble impurities of the destroyed cells. The supernatant is removed. The treatment of the precipitate with solution A is repeated another 1-2 times.

Then, solution B is added to the resulting precipitate at a rate of 1: 5-1: 10 (sediment volume: volume of solution B). The precipitate is resuspended and homogenized with ultrasound on ice 2-3 times for 3 minutes with 2-minute breaks. The resulting suspension is centrifuged at 5000-10000 g and 15 ± 5 ° C to precipitate inclusion bodies. The supernatant is removed, and the precipitate is washed 1-2 times more with solution B.

Solution B is added to the resulting precipitate at a rate of 1: 5-1: 10 (sediment volume: solution volume B). The precipitate is resuspended and homogenized with ultrasound on ice for 2-3 minutes with 2-minute breaks. The resulting suspension is centrifuged at 5000-10000 g and 15 ± 5 ° C to precipitate inclusion bodies. The supernatant is removed, and the precipitate is washed with solution B another 1-2 times (or until the formation of foam ceases).

The supernatant is removed, solution G is added to the precipitate at the rate of 1: 1-1: 2 (the volume of the precipitate is the solution B). The precipitate is resuspended and homogenized with ultrasound on ice 2-3 times for 3 minutes with 2-minute breaks. The resulting suspension is centrifuged at 15 ± 5 ° C. The supernatant is removed, and the precipitate is washed 1-2 more times with solution G.

To the precipitate obtained, a solution is added to solubilize inclusion bodies at the rate of 1: 5-1: 10 (sediment volume: solution volume).

The solubilization solution includes 5.5-6.0 M guanidine hydrochloride, 1-5 mm dithiothreitol, 25-75 mm Na-acetate buffer, while the pH of the solution is 4.5-5.5. The solution for solubilization of inclusion bodies is used freshly prepared.

After pouring the solution for solubilization to the washed inclusion bodies, the solution must be mixed to uniformly dissolve the precipitate (it is necessary to completely separate the precipitate from the bottle wall without foaming the solution). You can mix manually or using a magnetic stirrer. The solutions are incubated at +2 - + 8 ° C for 1.5 hours and with periodic stirring.

From the obtained opalescent transparent viscous solution, undissolved inclusions are separated. To remove insoluble protein, the solubilizate is filtered using a vacuum filtration unit through a 0.22 μm filter into sterile bottles. The solution is immediately used for refolding.

For refolding, the resulting filtrate is diluted with a refolding solution. The refolding solution includes 0.45-0.5 M arginine, 25-75 mm Na-acetate buffer, while the pH of the solution should be 4.5-5.5. The resulting solution was placed on a magnetic stirrer and stirred at room temperature for the time required to fold the immunotoxin (20-30 hours).

To remove dithiothreitol and guanidine hydrochloride, diafiltration is carried out. The diafiltration solution contains 0.45-0.5 M arginine, 40-60 mm Na-acetate buffer, while the pH of the solution should be 5.0 ± 0.2. Before diafiltration, the solution is cooled to +2 - + 8 ° C. Diafiltration is carried out using a tangential filtration unit (the filter material is polyethersulfone, mass cut-off is 10,000 Da).

The resulting solution was concentrated 20-25 times and sterilized. Concentration is carried out by tangential filtration (the filter material is polyethersulfone, mass cut-off is 10,000 Da). The concentrate is sterilized by the filtration method (the filter material is polyethersulfone, pore size 0.22 μm).

The inventive immunotoxin is proposed as an active ingredient for the preparation of a pharmaceutical composition for intravenous administration in the treatment of multiple sclerosis, which may contain an effective amount of an immunotoxin (up to 50 mass%, preferably up to 20 mass%, more preferably up to 10 mass%, even more preferably up to 0.1-0.5 mass%), and the rest is additional pharmaceutically acceptable additives (excipients). By pharmaceutically acceptable excipients are meant diluents, auxiliary agents (target additives) and / or carriers, stabilizers, pH regulators, buffers, antioxidants, preservatives, compounds that prolong the action of the drug, etc., for example, selected from the group of: benzalkonium chloride, sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, sodium citrate, sodium edetate, sodium bisulfite, sodium thiosulfate, sodium carbonate, magnesium stearate, mannitol, benzalkonium chloride, chlorhexidine, f enethyl alcohol, boric acid, hyetellose (hydroxyethyl cellulose), hydroxymethyl cellulose, sodium hydroxide, citric acid, polyethylene glycol, glycerin, dextrose, dextran 40, dextran 70, polyvinylpyrrolidone, tween-80, starch, lactose and other pharmaceutically acceptable components.

For the treatment of multiple sclerosis, a pharmaceutical composition comprising an immunotoxin is administered intravenously in a therapeutically effective amount, the frequency and duration of administration being chosen depending on the nature and severity of the disease.

The present invention is illustrated by specific examples of execution, which are not the only possible, but clearly demonstrates the possibility of achieving the desired technical result.

1. Obtaining a genetic engineering construct.

The plasmid pET-22b (+) with the ML1A gene was used as the basis for the preparation of a plasmid carrying the ML1A gene with the MOG gene fragment at the C-terminus (pET-28b (+) _ ML1A-MOG). For cloning into a new plasmid with different antibiotic resistance, the ML1A gene was amplified using the following primers (restriction sites are underlined):

Moreover, restriction sites BamHI (GGATCC) and NcoI (CCATGG) were introduced at the ends of the sequence encoding ML1A.

The MOG fragment was amplified using primers:

At the same time, XhoI (CTCGAG) and BamHI (GGATCC) restriction sites were introduced at the ends of the sequence encoding the MOG fragment.

The resulting fragments 778 and 92 bp long. purified from impurities by electrophoresis on a 2% agarose gel, followed by extraction from the gel using the innuPREP Gel Extraction Kit.

To generate a sufficient amount of the matrix, the plasmid pET-28b (+) was transformed into E. coli strain DH5α. Transformed cells were plated on LB agar medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 2% agar agar) supplemented with 32 μg / ml kanamycin. The resulting colonies were transferred to test tubes with 5 ml LB growth medium supplemented with 32 μg / ml kanamycin. After overnight incubation (16 hours) at 37 ° C and stirring at 180 rpm, the cells were pelleted by centrifugation for 5 minutes at 5000 g. After that, using the irmuPREP Plasmid Mini Kit, plasmid pET-28b (+) was isolated from them.

The purified fragments encoding ML1A and the MOG fragment (insert_ML1A and insert_MOG), as well as the plasmid pET-28b (+), were subjected to restriction with the corresponding enzymes: insert_ML1A - BamHI and NcoI, insert_MOG - BamHI and XhoI, pET-28b (+) -NcoI and XhoI . Then, all fragments were ligated using T4 bacteriophage DNA ligase (ratio matrix: insert_ML1A: insert_MOG = 1: 2: 2). The resulting ligase mixture was transformed into E. coli strain DH5α. Transformed cells were plated on LB agar medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 2% agar agar) supplemented with 32 μg / ml kanamycin. As a result, 23 colonies were obtained. Four colonies were plated in tubes with 5 ml LB growth medium supplemented with 32 μg / ml kanamycin. After incubation overnight at 37 ° C and stirring at a speed of 180 rpm, the cells were pelleted by centrifugation (5 min, 5000 g). After that, plasmids were isolated from them with the innuPREP Plasmid Mini Kit. Since the concentration of plasmids was insufficient for sequencing, additional PCR was performed using universal primers T7_for (5'-TAATACGACTCACTATAGGG-3 ') and T7_rev (5'-GCTAGTTATTGCTCAGCGG-3'). The resulting fragments weighing 1022 bp purified on an agarose gel and transferred for sequencing. Thus, as a result of genetic engineering, a plasmid carrying the gene of the target protein ML1A-MOG was obtained. The obtained plasmid transformed E. coli strain BL21 Star (DE3).

Then, 8 single E. coli BL21 Star (DE3) _ML1A-MOG colonies were selected from Petri dishes. These colonies were transferred under sterile conditions into tubes with 5 ml of LB culture medium with kanamycin (32 μg / ml). The tubes were incubated overnight (16 hours) at 34 ° C and 180 rpm. Then, 1.35 ml of a sterile 50% glycerol solution was added to the resulting cell suspension and mixed. 200 μl aliquots were transferred to cryovials and frozen at -70 ° C.

A target expression of the target protein was performed for each clone: 25 μl of the museum culture was transferred into a test tube with 10 ml of LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl) with kanamycin (32 μg / ml) and cultivated for 6 hours at 37 ° C and 180 rpm, after which IPTG was added to a final concentration of 0.5 mM to induce expression of ML1A-MOG and incubated for 3 hours at 34 ° C and 130 rpm. Analytical electrophoresis of samples taken before and after expression induction showed that ML1A-MOG was produced in all clones.

2. Growing seed

Preparation and sterilization of a nutrient medium

210 ml of deionized water was added to a 500 ml glass beaker; weighed portions of yeast extract (1.5 g), tryptone (3.0 g), sodium chloride (3.0 g) were added and mixed until the components were completely dissolved. The volume of the solution was adjusted to 300 ml with deionized water. The prepared solution was transferred into six conical flasks with a capacity of 250 ml (50 ml each), closed with a cotton-gauze plug and sterilized at 0.5-0.8 atm. for 60 minutes in an autoclave.

0.75 g of glycerol and 0.048 g of kanamycin sulfate were added to a 2 ml microtube on a balance, the total weight was adjusted with deionized water to 1.5 g. The resulting solution was thoroughly mixed until the components were completely dissolved. The solution was filtered through a 0.22 μm filter into a 2 ml sterile microtube. Under sterile conditions, 50 μl of kanamycin solution was transferred into conical flasks with a nutrient medium.

Inoculum cultivation

50 μl of seed in glycerol, packaged in cryovials (cell suspension of E. coli producer strain BL21 Star (DE3) _ML1A-MOG) was sterilely added to conical flasks with a kanamycin culture medium. Cultivation was carried out in a shaker-incubator at a temperature of (34 ± 1) ° С and stirring at a speed of 130 rpm for 16 hours. The resulting suspension of E. coli cells was used as a seed (inoculum) for the fermenter.

3. Obtaining biomass containing ITRS

Preparation and sterilization of incomplete culture medium

4 l of deionized water was added to a 5 l glass. Samples of yeast extract (30 g), tryptone (60 g), NaCl (17.53 g), Na ₂ HPO ₄ * 12H ₂ O (53.72 g), KH ₂ PO ₄ (20.41 g), MgSO ₄ * 7H ₂ O (2.9578 g) and (NH ₄ ) ₂ SO ₄ (3.9642 g) were also transferred to a beaker and dissolved on a magnetic stirrer. The resulting solution was transferred to a fermenter. Another 1.5 L of deionized water was poured into the same glass and also transferred to the fermenter.

All open exits of the fermenter were covered with aluminum foil. They blocked all the supply and selection lines, installed plugs on unused ports and started the autoclaving program.

Sugar Solution Preparation

The solution was prepared in a 250 ml glass beaker. A portion of glycerol (30 g) was made in a glass beaker. 100 ml of deionized water were added there. The solution was stirred using a magnetic stirrer. Then, glucose (3 g) and lactose (12 g) were weighed into the resulting solution. After the Sugars were dissolved, the solution was sterilized by filtration through a 0.22 μm filter and transferred sterile to the fermenter (first make sure that the medium had cooled after autoclaving).

Preparation of antibiotic solution

A solution of the antibiotic (kanamycin) was prepared in a 15 ml polypropylene tube. For this, kanamycin sulfate (0.6 g) was hung in a test tube and the weight was adjusted with deionized water to 12.0 g. The solution was thoroughly mixed with a vortex. After complete dissolution of kanamycin, the solution was sterilized by filtration through a 0.22 μm filter into a 15 ml sterile tube. The solution was sterilely transferred to a fermenter.

Biomass cultivation

A probe for measuring temperature was connected. All unused ports were plugged. Water was connected to the jacket of the refrigerator to return condensate. Glycerin was poured into the cell for the thermoprobe; the thermoprobe was installed. We turned on the device in the network.

The temperature was set at + 37 ° C. The motor was turned on and stirring was set to 200 rpm. Using a peristaltic pump, 6 ml of antifoam was supplied to the fermenter flask. Probe cables were connected to determine the pH and dissolved oxygen to the instrument. The oxygen sensor was calibrated. The pH value was set to 6.9. Set aeration to 40%.

Aseptically introduced the inoculum through the appropriate port. Incubated biomass for 20 hours

4. Checking the level of expression of immunotoxin (ITRS)

To assess the expression level, the content of ITRS protein in cells was determined using analytical electrophoresis under denaturing conditions in a polyacrylamide gel. 1-2 ml of cell suspension were taken from the fermenter into a special collector. Transfer 0.1 ml of the suspension to a 1.5 ml microtube. 0.2 ml of deionized water was added to the sample and resuspended by pipetting. 0.01 ml of the resulting suspension was transferred to a new microtube and 0.01 ml of sample buffer with β-mercaptoethanol was added. The sample was incubated for 5 min at 95 ° C. Then centrifuged for 1 min at 5000 g. For analytical polyacrylamide gel electrophoresis, 0.020 ml of samples and 5 μl of a mixture of molecular weight markers were added to the wells.

After visualization of the proteins in the gel, it was photographed in a chamber for documenting ChemiDoc MP gels and the electrophoregram was evaluated using ImageLab Software, a program for processing the results. The main spot corresponding to ITRS should migrate as a protein with a mass of 28 ± 1 kDa. Based on a previously constructed calibration curve for the spot area on the electrophoregram, its amount in the analyzed samples, which should be 1 ± 0.2 μg, was determined depending on the amount of applied ITRS protein.

5. Biomass separation

Disable aeration, mixing, temperature and pH control. Disconnected the device from the network. The suspension was poured through a special port into a 10-liter plastic bottle.

The biomass from the culture fluid was separated by centrifugation: the cell suspension was transferred into 6 centrifuge polypropylene bottles with a capacity of 1000 ml and centrifuged for 20 min at 3000 g. Supernatants (culture fluid) were removed by decantation and sent for autoclaving (after that - into drains). The obtained cell pellet was stored at -20 ° C until use in the following steps.

After removing the cell suspension, the fermenter was cleaned in accordance with the manufacturer's instructions.

6. Isolation and purification of inclusion bodies

To isolate and wash inclusion bodies, four solutions were used (A, B, C, D). All solutions were prepared in deionized water (type I).

Preparation of solution A for washing inclusion bodies

Solution A (2 M NaCl, 5 mM dithiothreitol, 5 mM EDTA, 25 mM Tris-HCl pH 8) was prepared in a 5,000 ml beaker. 3000 ml of deionized water were poured into a glass and weighed portions of NaCl (566.84 g), dithiothreitol (3.7418 g), tris (hydroxymethyl) aminomethane hydrochloride (10.7670 g) and tris (hydroxymethyl) aminomethane (6.4263 g) were dissolved. . After that, a portion of EDTA (9.0271 g) was dissolved. The solution was transferred to a 5000 ml graduated cylinder, the volume was adjusted to 4850 ml with deionized water and transferred back to the beaker. Solution A was used freshly prepared.

Preparation of solution B for washing inclusion bodies

Solution B (2% Triton X-100, 50 mM NaCl, 1 mM dithiothreitol, 25 mM Tris-HCl pH 8) was prepared in a 5000 ml volumetric beaker. 3000 ml of cold distilled water was poured into a glass and weighed portions of NaCl (106.58 g), dithiothreitol (0.5630 g), tris (hydroxymethyl) aminomethane hydrochloride (8.1030 g) and tris (hydroxymethyl) aminomethane (4.8363 g) were dissolved. ) After complete dissolution of the salts, Triton X-100 (73 ml) was added to the solution and thoroughly mixed on a magnetic stirrer. After dissolution of the Triton X-100, the solution was transferred into a 5000 ml volumetric cylinder, the volume was adjusted to 3650 ml with deionized water and transferred back to the beaker. Solution B was used freshly prepared.

Preparation of solution B for washing inclusion bodies

Solution B (1 mM dithiothreitol, 25 mM Tris-HCl pH 8) was prepared in a 5,000 ml beaker. 3000 ml of deionized water was poured into a glass and weighed portions of dithiothreitol (0.5630 g), tris (hydroxymethyl) aminomethane hydrochloride (8.1030 g) and tris (hydroxymethyl) aminomethane (4.8363 g) were dissolved. After complete dissolution of the salts, the solution was transferred to a 5000 ml volumetric cylinder, the volume was adjusted to 3650 ml with deionized water and transferred back to the glass. Solution B was used freshly prepared.

Preparation of solution G for washing inclusion bodies

Solution G (4 M urea, 1 mM dithiothreitol, 25 mM Tris-HCl pH 8) was prepared in a 5,000 ml beaker. 2500 ml of deionized water were poured into a glass and weighed portions of urea (876.88 g), dithiothreitol (0.5630 g), tris (hydroxymethyl) aminomethane hydrochloride (8.1030 g) and tris (hydroxymethyl) aminomethane (4.8363 g) were dissolved. . The solution was transferred to a 5000 ml volumetric cylinder, the volume was adjusted to 3650 ml with deionized water and transferred back to the beaker. Solution G was used freshly prepared.

Cleaning inclusion bodies

400 ml of solution A were added to centrifuge bottles with biomass. Cell pellet was resuspended. Cells were destroyed by ultrasound while cooling on ice (3 times for 3 minutes with 2-minute breaks). The bottles were centrifuged at 8000 g, 15 ° C for 15 minutes. The supernatant was removed. 400 ml of solution A were again added to the precipitates, resuspended and sonicated. The bottles were centrifuged at 8000 g, 15 ° C for 15 minutes.

The supernatant was removed, and 300 ml of solution B was added to the precipitates. The precipitates were resuspended and homogenized by ultrasound on ice (2 times for 3 minutes with 2-minute breaks). The bottles were centrifuged at 8000 g, 15 ° C for 15 minutes. The supernatant was removed, and the precipitate was washed again with solution B. The bottles were centrifuged at 8000 g, 15 ° C for 15 min.

The supernatant was removed, 300 ml of solution B was added to the precipitates. The precipitates were resuspended and ultrasonically homogenized on ice (2 times for 3 minutes with 2-minute breaks). The bottles were centrifuged at 8000 g, 15 ° C for 15 minutes.

The supernatant was removed, 30 ml of solution G was added to the precipitates. The precipitates were resuspended and ultrasonically homogenized on ice (2 times for 3 minutes with 2-minute breaks). The bottles were centrifuged at 8000 g, 15 ° C for 15 minutes. The supernatant was removed, and the precipitate was washed again with solution B. The bottles were centrifuged at 8000 g, 15 ° C for 15 min.

The supernatant was removed, and the precipitates were sent for solubilization.

Supernatants from all stages were combined for further disposal.

7. Solubilization of Taurus inclusion. Preparation of a solution for solubilization of inclusion bodies

Preparation of solubilization solution

A solution (6 M guanidine hydrochloride, 1 mM dithiothreitol, 25 mM Na-acetate buffer pH 5) was prepared in a 10 L plastic bottle. 3380 ml of deionized water (type I) were poured into the bottle (ultrapure water with a specific resistance of 18.18 MΩ * cm at 25 ° C, i.e. type 1 water according to ASTM, NCCLS, ISO 3696, CAP) and weighed portions of guanidine hydrochloride (3467.74 g), dithiothreitol (0.9332 g) and CH ₃ COONa⋅3H ₂ O (20.5814 g). An anchor for a magnetic stirrer was lowered into the bottle and transferred to a magnetic stirrer. Using glacial acetic acid (about 2.9 ml), the pH of the solution was adjusted potentiometrically to 5.0 ± 0.5. The solution for solubilization of inclusion bodies was used freshly prepared.

Solubilization

300 ml of solubilization solution were added to centrifuge bottles with washed inclusion bodies. The precipitate was carefully resuspended (it is necessary to completely separate the precipitate from the wall of the bottle without foaming the solution). Another 700 ml of solubilization solution was added to the bottles. Large anchors for the magnetic stirrer were lowered into the bottle and covered with lids. The solutions were incubated at +2 - + 8 ° C for 1.5 hours and with periodic stirring on a magnetic stirrer.

Department of undissolved inclusions

To remove insoluble protein, the solubilizate was filtered using a vacuum filtration unit through a 0.22 μm filter into 1000 ml sterile bottles. The solution was immediately used for refolding.

8. Obtaining active ITRS

Preparation of a solution for refolding ITRS

A refolding solution (0.5 M arginine, 50 mM Na-acetate buffer pH 5) was prepared in three plastic bottles with a capacity of 50 L. Weighed portions of arginine (4180.8 g) and CH ₃ COCNa⋅3H ₂ O (326.592 g) were transferred to a bottle and 43.45 L of deionized water (type I) was poured. Large anchors for the magnetic stirrer were placed in the bottle and transferred to the stirrer. After dissolving the salts, the pH of the solution was adjusted potentiometrically to 5.0 ± 0.2 (about 1440 ml of glacial acetic acid per bottle).

ITRS refolding

The bottles with the refolding solution were placed on a magnetic stirrer and, with continuous stirring, 2000 ml of solubilized inclusion bodies were slowly added (after the stage of separation of the insoluble protein). The procedure was repeated for all 3 bottles. The solutions were incubated for 20-24 hours at room temperature.

9. Obtaining substance ITRS

Preparation of diafiltration solution

A diafiltration solution (0.5 M arginine, 50 mM Na-acetate buffer pH 5) was prepared in a 50 L plastic pyrogen-free bottle. Weighed portions of arginine (4355 g) and CH ₃ COCNa⋅3H ₂ O (340.2 g) were transferred to a bottle and 45.27 L of water for injection was poured. A large anchor for a magnetic stirrer was placed in the bottle and transferred to a magnetic stirrer. After dissolving the salts, the pH of the solution was adjusted potentiometrically to 5.0 (about 1500 ml of glacial acetic acid). Before diafiltration, the solution was cooled to +2 - + 8 ° C.

Concentration and diafiltration of refolding medium

After refolding, the ITRS solution was concentrated on a KMPi System tangential filtration unit (membrane - mPES, 10000 NMWL) to about 5 l. After that, 12.5 L of diafiltration solution was added to the concentrate bottle. The resulting solution was again concentrated to about 5 L. The procedure was repeated 3 more times. In the last stage, the solution was concentrated to 2.3 ± 0.3 L.

Sterilization of substance ITRS

ITRS concentrate was sterilized under aseptic conditions (local zone of purity class A) by filtration through a polyethylene sulfone filter with a pore size of 0.22 μm.

Verification of the authenticity and purity of the substance

The purity and molecular weight of the immunotoxin were confirmed by electrophoretic separation in 13% polyacrylamide gel under non-reducing conditions (without mercaptoethanol). The bands were visualized using Coomassie G-250 staining followed by densimetry. In this case, the main band corresponding to a protein with a mass of 28 ± 1 kDa should be present on the electrophoregram, and the sum of the intensities of other bands should not exceed 5% of the total intensity of all bands.

For three series of immunotoxin substance obtained by the claimed method, the% content of immunotoxin and its molecular weight were determined. Examples of the obtained electrophoregrams are presented in FIG. 5. Precision Plus Protein Unstained Standards (Bio-Rad) were used to determine molecular weight. After staining, the gels were scanned, and the content and molecular weight of the target protein were determined using ImageLab Software (Bio-Rad). Using Microsoft Excel, the standard deviation and relative standard deviation were calculated. The results are presented in Table 1.

The formation of the tertiary structure was confirmed by determining the melting temperature. The melting point was determined by Thermofluor method using a SYPRO Orange fluorescent dye. The melting temperature corresponds to the maximum on the curve of the dependence of the first derivative of the fluorescence intensity on temperature and should be 39 ± 2 ° C.

In FIG. 6 shows an example of a melting curve for the substance ITRS obtained by the claimed method.

An example demonstrating the possibility of using immunotoxin as an active component.

From the immunotoxin obtained by the above method, a preparation solution of the following composition was prepared:

Immunotoxin (ITS) 0.05 mg

Excipients:

Arginine 87.1 mg Sodium Acetate Trihydrate 6.8 mg Glacial acetic acid 31.5 mg Water for injections up to 1 ml

For this, using the prepared dilution solution, the resulting purified immunotoxin solution was adjusted to a concentration of 0.05 mg / ml.

The dilution solution (pH 5.0 ± 0.2) has the following composition:

Arginine 87.1 mg Sodium Acetate Trihydrate 6.8 mg Glacial acetic acid 31.5 mg Water for injections up to 1 ml

The drug in liquid dosage form was a colorless transparent solution with no visible particles.

Intended use in the clinic: 4 single daily intravenous doses at a dose of 2 mcg / kg. Repeat a course in a month. The intended route of administration is intravenous infusion: the calculated single dose of the drug is taken from the vial using a syringe and dissolved in a 100 ml vial with an isotonic solution; first, the drug is administered at a low speed (6 ml / h) to identify a possible hypersensitivity reaction, after 15 minutes - 12 ml / h, then every 15 minutes the speed increases by 12 ml / h to 72 ml / h.

Specific Activity Studies

A study of specific activity was carried out in vitro and in an animal model.

The properties of an immunotoxin are determined by two main parameters: the manifestation of the catalytic activity of the toxin and the interaction with specific receptors on the surface of target cells. The catalytic activity of ITRS is due to the presence of viscumin in the A subunit of the fusion protein, which catalyzes the depurinization of the adenine residue at position 4324 (A4324) in 28 S rRNA, which leads to inactivation of ribosomes and to a halt in protein synthesis. A cell-free luciferase expression system was chosen as a model system for studying translation suppression. Lucifer is an enzyme capable of oxidizing luciferin substrate, and the reaction is accompanied by the emission of light. Thus, the amount of model protein (luciferase) synthesized in a cell-free system can be estimated by the luminescence intensity. When ribosomes are inactivated, a decrease in the detected signal occurs. According to the dependence of the luminescence intensity on the amount of GLF introduced into the reaction mixture, it is possible to determine the inhibitory concentration of ITRS (IC ₅₀ value). IC ₅₀ for immunotoxin determined by the described method is 5 ± 2 nM. An example of an immunotoxin inhibition curve obtained by the claimed method of protein translation in a cell-free expression system is shown in FIG. 7.

To assess the ability of an immunotoxin to bind to the surface of B-lymphocytes, an in vitro model based on a hybridoma producing specific antibodies was used. To evaluate the binding, the immunotoxin immobilized on the surface of the tablet was incubated with a suspension of cells containing specific immunotoxin receptors (line A) and not containing (line B). In both cases, the specificity of the immunotoxin was judged by the number of bound cells. In the absence of immunotoxin, both cell lines practically did not bind to the plate (average 179 cells / well for line A and 209 cells / well for line B). If the surface was modified by immunotoxin, line A cells bind to the plate (2032 cells / well), and line B cells did not (346 cells / well).

Testing the activity of the immunotoxin preparation was also carried out using an animal model. A common animal model of multiple sclerosis is experimental autoimmune encephalomyelitis (EAE), which is most often induced in mice or rats in studies. Although EAE does not fully reflect all aspects of the development of multiple sclerosis in humans, this model is used to evaluate the effectiveness of new drugs.

For the study, 46 female mice of the C57BL / 6 strain of 8 weeks of age were used. Animals were divided into 4 groups of 10 animals and one group of 6 animals (control group No. 1; mice received injections of saline instead of EAE-inducing agents and the study drug). In the remaining groups, EAE was induced by double administration (with an interval of 1 day) of PTX pertussis toxin (2000 ng / mouse, intravenously in POC) and mouse spinal cord homogenizate (GSM; 5 mg / mouse, subcutaneously). One of these groups was control No. 2 - a carrier was introduced instead of the study drug, and the remaining groups received different doses of the study drug (0.05, 0.5, and 5 μg ITRS / mouse).

Signs of the development of EAE began to appear in animals from 1 day after the start of the experiment. For each experimental group, the mean values for evaluating the development of EAE were calculated (see Fig. 8).

In the group that did not receive the ITSS study drug, the signs of the disease manifested themselves earlier and accumulated faster in the group, while for all groups receiving ITSS, a later time of the disease manifestation can be noted.

Also, when animals were administered ITSS, an increase in life expectancy was observed. The death of animals from the experimental groups occurred due to complete paralysis and subsequent dehydration and respiratory depression (see Fig. 9).

The most effective dose was 0.5 μg / mouse, since at this concentration of ITSS the manifestation of signs of EAE occurred later, and the average life expectancy was maximum. The experimental results obtained in animal models can be extrapolated to the further use of ITRS in humans (Guidelines for preclinical studies of drugs. Edited by A. N. Mironov. Part 1. - M., 2012).

Acute Toxicity Study

The study of acute toxicity of immunotoxin was carried out in two animal species: nonlinear white mice (9-10 weeks old, 18-20 g body weight) and nonlinear white rats (9-10 weeks old, 180-200 g body weight). Used doses of immunotoxin: 50, 200, 500, 750, 1000 μg / kg (10 animals for each group, 5 males and 5 females), physiological saline was used. The route of administration is intravenous injection. Observation of the animals was carried out within 14 days after administration. The general condition of the animals was assessed by the condition of the coat and mucous membranes, and body weight. After 14 days, the animals were euthanized with carbon dioxide, an autopsy was performed, the morphological state of the organs and mucous membranes was visually determined, and the mass coefficients of organs were determined (heart, lungs with trachea, thymus, liver, spleen, kidneys, adrenal glands, brain, ovaries or testicles) .

During the study, mortality of animals (both mice and rats) from the drug was absent. There were no signs of intoxication during examination. Immediately after the administration of large doses of the drug, the animals experienced slight shortness of breath and slight inhibition. These clinical signs passed within 1-2 hours. Body weight and mass ratios of organs of animals treated with ITSS did not differ from the control group. There were no visible morphological changes in organs and mucous membranes. Chronic toxicity

A study of the chronic toxicity of immunotoxin was carried out on two animal species: nonlinear white rats (9-10 weeks old, 180-200 g body weight) and Chinchilla rabbits (2-3 months old, 2200-2500 g body weight). The studied doses for rats were 12 and 80 μg / kg (10 animals per group, 5 males and 5 females), for rabbits - 6 and 80 μg / kg (6 animals in the group, 3 males and 3 females). Also, control groups of animals that received saline were formed. The introduction was carried out intravenously daily for 30 days.

The list of indicators determined during the study and the observed changes are shown in Tables 2 and 3. There was no mortality of animals in all experimental groups.

Immunotoxicity study

In the study, a standard animal model was used - hybrid mice (CBAxC57BL / 6) Fl, age 6-8 weeks, body weight 18-20 g. The drug was administered in a regime similar to that expected in the clinic (4 single administrations, one each day); into the tail vein. The studied doses were 200 and 400 mcg / kg (10 animals per group, 5 males and 5 females); control groups received saline.

To assess the effect on humoral immunity, the number of antibody-forming cells (AOKs) was determined upon immunization with a T-dependent antigen (ram erythrocytes). An hour after drug administration, all animals were immunized with an intraperitoneal injection of sheep erythrocytes at a suboptimal dose of 5 × 10 ⁷ red blood cells per animal. The number of AOKs in the spleen of mice was calculated on the 5th day after immunization. The reaction was set on slides without a supporting medium according to Canningham. By the number of erythrocyte hemolysis zones around individual antibody-forming cells determined under a microscope, the number of AOKs was calculated per 10 ⁶ splenocytes. No differences in the amount of AOK were revealed, which indicates the absence of the effect of immunotoxin on the humoral immunity of laboratory animals.

To study the effect on cellular immunity 1 hour after the last injection of the drug or control solution, all mice were sensitized by subcutaneous injection of ram erythrocytes into the interscapular region. On day 5 after sensitization, an evaluation of delayed-type hypersensitivity was performed. To do this, all the animals were injected with red sheep erythrocytes into the cushion of the hind paw (permitting injection). Saline was injected into another hind paw. After 24 hours, the local inflammatory response was assessed by the difference in the mass of the hind legs. No impact on the delayed-type hypersensitivity to sheep erythrocytes was detected, which indicates the absence of the effect of immunotoxin on the cellular immunity of laboratory animals.

The effect on the phagocytic activity of peritoneal macrophages was investigated 24 hours after the last injection. For this, the animals were euthanized with carbon dioxide, and the abdominal cavity of the euthanized animals was washed with the culture medium. The concentration of peritoneal exudate cells was adjusted to 2 mln / ml. Then, 2 ml were transferred into 40 mm diameter Petri dishes and incubated in an incubator for 1 h at 37 ° C. Unattached cells were washed. Opsonized yeast was added to the medium. Upon completion of the reaction, the cells were fixed and stained according to Romanovsky-Giemsa. The number of phagocytosed and non-phagocytized macrophages was counted under a microscope. The percentage of phagocytized macrophages in relation to the total number was determined. The average number of phagocytosed yeast cells was determined. In females, no differences between the groups were detected. In males, in response to the introduction of ITRS (200 and 400 μg / kg), an increase in the phagocytic index occurred (from 46.0 ± 4.5 to 70.8 ± 4.3 and 73.0 ± 5.0, respectively). This may indicate an immunostimulating effect of the drug.

The study of allergenic properties

The allergenic properties of the drug were evaluated by the development of a general anaphylaxis reaction and a slow type hypersensitivity reaction.

To assess the sensitizing properties in the reaction of general anaphylaxis, nonlinear guinea pigs were used (age 4-6 weeks, body weight 240-260 g). The studied doses: 10 and 100 mcg / kg (10 animals per group, 5 males and 5 females); animals of the control group received saline. The animals received three injections of ITRS (the first - subcutaneously, the next two - intramuscularly). 14 days after the first administration, the animals were given an intravenous resolving dose of ITRS. The same dose was administered to animals in the control group. After that, an intensity of anaphylactic shock was assessed. No signs of anaphylactic shock were recorded in any of the groups (reaction index 0 on the Weigle scale).

The determination of delayed-type hypersensitivity was carried out on white non-linear mice (age 9-10 weeks, body weight 18-20 g). The investigated doses: 200 and 400 mcg / kg (10 animals in the group, 5 males and 5 females); animals of the control group received saline. Mice were sensitized by subcutaneous administration of ITRS (200 or 400 μg / kg), 1 injection for 5 days. Animals of the control group were injected phys. solution. To detect sensitization 5 days after the last injection, all mice were injected with 50 μl of the test drug into the hind paw pad. 50 μl of solvent was injected into the pad of the other hind paw. 24 hours after this, the animals were euthanized and both hind legs were cut off. Then, the HRT reaction was assessed by the mass difference between the control and experimental paws. There were no significant differences between the experimental groups.

Thus, the drug in studies on laboratory animals did not exhibit allergenic properties.

Mutagenicity study

To assess the mutagenicity of the study drug, a test was conducted on the induction of gene mutations. Salmonella typhimurium (strains TA 97, TA 98 and TA 100), which is a standard bacterial test system, was used as a model object for the Ames mutation test. Mutations to histidine prototrophy were taken into account under the action of compounds inducing mutations such as substitution of bases or reading frame shifts in the genome of this organism. Solvent treatment was used as a negative control. Mutants inducing mutations in the corresponding strains of Salmonella typhimurium were used as a positive control. In the Ames test, the drug did not exhibit mutagenic properties.

To assess the mutagenicity of the test drug on eukaryotic cells, the methodology recommended by the Organization for Economic Co-operation and Development (OECD), “Test No. 476: In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes. " The principle of the method is to register mutations (point substitutions, reading frame shifts, deletions or insertions) leading to inactivation of the Hprt gene encoding hypoxanthine-guanine-phosphoribosyltransferase. The activity of this enzyme determines the cytotoxicity of 6-thioguanine, which becomes toxic only after phosphorylation. Normal cells expressing Hprt are sensitive to the cytostatic effect of 6-thioguanine (6-TG), while mutant cells with an inactivated Hprt gene are able to proliferate in the presence of 6-TG. The test object was CHO-K1 cells. In this test, the drug also did not exhibit mutagenic properties.

To assess the genotoxicity of the test drug on eukaryotic cells, the methodology recommended by the Organization for Economic Cooperation and Development (OECD) was used - Test No. 487: In Vitro Mammalian Cell Micronucleus Test, 2016. Counting micronuclei in cells is a genotoxicity test that determines the proportion of cells containing micronuclei in the cytoplasm. The test object was a cell line Saso-2 and HepaRG. There were no differences with control.

Reproductive Toxicity Study

A study of the effect on reproductive function was carried out on the outbred stock of Wistar rats (age 10–13 weeks, body weight 220–300 g). The studied doses of the drug: 8 and 40 mcg / kg; control groups received the media. Introduction: intravenously into the lateral vein of the tail; to females - during 15 days before mating and within 20 days during pregnancy; males - for 49 days before mating. The experiment consisted of several parts: 1) determination of the effect of ITRS on the generative function of females (administration of the drug to female rats and sitting with intact males; pregnancy registration); 2) determination of the effect of ITRS on the generative function of males (administration of the drug to male rats and seeding with intact female rats; pregnancy registration); 3) determination of the embryotoxic potential of the drug (administration of ITRS to females during pregnancy; registration of fetal viability, physical development of offspring).

During the study, there was no animal mortality. In all animals treated with the carrier and the drug, a local reaction to the introduction of tail cyanosis of varying severity was observed. Other manifestations of intoxication in animals were not observed. In a study of the effect of the ITRS drug on the reproductive function of females, it was determined that the drug administered to females intravenously at a dose of 40 mcg / kg over three estrous cycles before shedding had a negative effect on the fertility of females, significantly lowering it relative to control animals (fertility index decreased from 0.95 to 0.60). It was shown that the drug administered to males during the formation of gametes did not affect their generative function. It was also shown that offspring born to females who received the drug before and during pregnancy did not differ either in survival or in the dynamics of weight gain from the offspring of control females.

Thus, we can talk about the presence of reproductive toxicity of the drug in relation to the generative function of females and the absence of reproductive toxicity in relation to males. You can also talk about the lack of fetotoxic effects of the drug.

Claims (24)

1. An immunotoxin for the treatment of multiple sclerosis, which is a recombinant protein that includes the A-subunit of viscumin and a fragment of myelin-oligodendrocyte glycoprotein, which provides binding to target cells connected by a linker to link the mentioned fragments. 2. The immunotoxin according to claim 1, characterized in that the A-subunit of viscumin is a polypeptide with a sequence of at least 95% homologous to SEQ ID NO: 1. 3. The immunotoxin according to claim 1, characterized in that the myelin-oligodendrocyte glycoprotein fragment is a peptide with a sequence that is not less than 90% homologous to SEQ ID NO: 2. 4. The immunotoxin according to claim 1, which is a recombinant protein with a sequence of not less than 95% homologous to SEQ ID NO: 3. 5. Recombinant plasmid DNA for expression in Escherichia coli cells of the gene encoding the immunotoxin according to claim 1, which is a vector modified by a fragment encoding the immunotoxin according to claim 4, including the following fragments: a) an XhoI-NcoI fragment; b) T7 bacteriophage promoter; c) T7 bacteriophage RNA polymerase terminator; d) the region of origin of replication ColE1 / pMB1 / pBR322 / pUC; e) an area encoding an antibiotic resistance enzyme gene. 6. Plasmid DNA according to claim 5, characterized in that pET-22b (+), pET-26b (+), pET-28a-c (+) are used as the vector. 7. The strain Escherichia coli is the producer of the immunotoxin according to claim 1, transformed with the recombinant plasmid DNA according to claim 5. 8. The method of producing an immunotoxin according to claim 1, characterized in that the producer strain according to claim 7 is cultivated to obtain a biomass containing inclusion bodies, which is destroyed by ultrasonic disintegrator, followed by isolation of inclusion bodies by centrifugation, which are then washed by the method sequential dissolution of impurities, to obtain an immunotoxin in undissolved form, which is then converted into a dissolved form using a solution of a chaotropic agent, after which inactive dissolved The first immunotoxin is subjected to renaturation by dilution, diafiltration and concentration using tangential filtration. 9. The method according to p. 8, characterized in that the destruction of biomass using an ultrasonic disintegrator is carried out at a frequency of 22 kHz and cooling on ice. 10. The method according to p. 9, characterized in that the ultrasonic treatment is carried out 3-4 times for 3 minutes with 2-minute breaks. 11. The method according to p. 8, characterized in that the inclusion bodies are separated from the soluble components of the cells by centrifugation. 12. The method according to p. 8, characterized in that the purification of inclusion bodies is carried out using solutions containing detergents and chaotropic agents that remove DNA, RNA, lipids and impurity proteins. 13. The method according to p. 8, characterized in that the transfer of the immunotoxin from an insoluble inactive form to a soluble inactive form is carried out using a solution containing a chaotropic agent. 14. The method according to PP. 12 and 13, characterized in that solutions of urea, thiourea, guanidine hydrochloride are used as a chaotropic agent. 15. The method according to p. 12, characterized in that the detergent is sodium dodecyl sulfate, deoxycholate, sarcosyl, Tween 20, Tween 80, Triton X-100. 16. The method according to p. 8, characterized in that the transfer of immunotoxin from a soluble inactive form to a soluble active form (renaturation) is carried out by dilution in a solution containing arginine and having an acidic pH value. 17. The method according to p. 8, characterized in that the concentration of the active immunotoxin after renaturation is carried out using tangential filtration. 18. A pharmaceutical composition for the treatment of multiple sclerosis, comprising, as an active ingredient, the immunotoxin according to claim 1 in a therapeutically effective amount and a pharmaceutically acceptable base. 19. The method of treatment of multiple sclerosis, which consists in the intravenous administration of the pharmaceutical composition according to p. 18 in a therapeutically effective amount.

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