Fusion protein consisting of a fragment of il-ra and the fc fragment of igg4

Abstract

The present invention relates to the field of biomolecular pharmacology, biotechnology and genetic engineering and describes nucleic acid molecules and novel recombinant IL-1RA-hFc polypeptides encoded thereby, said recombinant polypeptides being human IL-1 antagonists capable of effectively blocking IL-1 signalling (pro-inflammatory response). The obtained recombinant polypeptides can be used in the treatment of inflammatory and autoimmune diseases mediated by dysregulation of IL-1 signalling.

Description

[0001] FUSION PROTEIN CONSISTING OF A FRAGMENT OF IL-1RA AND THE FC PART OF IGG4

[0002] 5 Field of technology

[0003] The invention relates to the field of biomolecular pharmacology, biotechnology and genetic engineering and concerns nucleic acid molecules and new soluble proteins encoded by them - functional antagonists of human IL-1, capable of blocking the proinflammatory response, as well as a method for obtaining them. The invention is based on the use of the IL-1 RA protein (lnterleukin-1 receptor antagonist protein), which is a natural functional antagonist of IL-1, as part of a decoy receptor molecule.

[0004] State of the art

[0005] Cytokines comprise a class of proteins that mediate inflammation and modulate immunity. One group of cytokines, the interleukins, are so named because they were thought to

[0006] 15 that they mediate signaling between leukocytes (hence the name interleukins) (Dinarello et al., 2011). Cytokines play a crucial role in the development of many inflammatory diseases (Feldmann, 2008).

[0007] Interleukin IL-1 is one of the first cytokines to be recognized. The IL-1 gene encodes two related but functionally distinct isoforms: IL-1a and IL-1 p (Dinarello, 2011). This pair

[0008] 20 mediators exert multiple effects on host defenses and play a role in the pathogenesis of a wide range of diseases. Notable effects of IL-1 on many cell types include the following events: induction of prostaglandin production via induction of cyclooxygenase-2; production of nitric oxide via increased levels of the inducible isoform of NO; ​​induction of expression of many cytokines, including increased transcription of its own genes; increased expression of leukocyte adhesion molecules and thrombus mediators; and activation of cells involved in innate immunity, primarily mononuclear phagocytes (Dinarello, 2009)

[0009] The action of IL-1 is controlled by several levels of regulation. The IL-1 family includes the structurally related IL-1 receptor antagonist (IL-1 RA). The balance between

[0010] 30 proinflammatory isoforms IL-1a and IL-1p and this endogenous inhibitor (IL-1RA) represents one of the important levels of control. IL-1a and IL-1p isoforms have different functional profiles. IL-1a is normally located on the cell surface and transmits signals over short distances through direct contact. In contrast, IL-1p can act at a distance (Dinarello, 2011). Two receptors bind the main members of the IL-1 family. IL-1 receptor I (IL-1 RI) transmits the IL-1 signal. In contrast, IL-1 receptor II (IL-1 RII) binds ligands but does not transmit a signal because it lacks a cytoplasmic domain. IL-1 RII thus acts as a “bait” or decoy receptor, providing another level of negative regulation of IL-1 signaling. IL-1 can induce self-gene expression in many cell types (Beltrami-Moreira, Vromman et al., 2016).

[0011] The IL-1 cytokine family plays an important role in innate immune responses, in particular IL-1a and IL-1|3 are the main mediators of autoinflammatory diseases and proinflammatory activity (Dinarello, 2009; Dinarello, 2011). IL-1|3 is considered a key cytokine in the pathogenesis of acute gouty arthritis and a number of other autoinflammatory diseases, and blocking IL-1|3 signaling has a therapeutic effect (Schlesinger, 2014).

[0012] IL-1β mediates the proinflammatory response in rheumatoid arthritis through multiple mechanisms, including activation of osteoclasts, synovial fibroblasts, and endothelial cells (Mclnnes and Schett, 2011). An IL-1β receptor antagonist, anakinra, has been described. Anakinra is a recombinant non-glycosylated IL-1β RA protein that matches the native IL-1β sequence with an additional methionine at the N-terminus, produced by production in E. coli. The drug is approved for the treatment of rheumatoid arthritis but has relatively modest efficacy. Anakinra, canakinumab, and rilonacept have potent anti-inflammatory activity in inherited systemic autoinflammatory diseases characterized by increased IL-1β production. Anakinra is effective in some patients with systemic inflammatory diseases, including systemic-onset juvenile idiopathic arthritis, adult-onset Still's disease, and other autoinflammatory conditions.Clinical trials have shown encouraging results in arthritis caused by abnormal accumulation of salt crystals, such as gout and chondrocalcinosis. Promising results have also been reported with anakinra in type 2 diabetes mellitus and smoldering / indolent multiple myeloma (Igel, Toprover et al., 2019).

[0013] Fc-fusion proteins have proven themselves as therapeutic and prophylactic agents. Proteins obtained using this technology have an effector part associated with the Fc domain, which significantly extends the half-life of proteins in blood plasma, which prolongs their therapeutic activity, and also leads to slower renal clearance for larger molecules. In addition, the molecules have fairly low immunogenicity, since their constituent parts are natural proteins of the human body. At the same time, such molecules have significant therapeutic potential, since they are able to bind the necessary ligands and, thus, block signal transduction chains. Recently, active development of therapeutic agents based on Fc-fusion proteins has been underway (see, for example, RU2689522, 28.05.2019; WO2023063842, 20.04.2023 (RU2787060)).

[0014] The aim of the invention is to develop effective IL-1 antagonists based on Fc-fusion protein technology.

[0015] The essence of the invention

[0016] The objective of the present invention is to expand the arsenal of technical means for treating inflammatory and autoimmune diseases. In particular, the objective concerns the production of polypeptide preparations - IL-1 antagonists, possessing high affinity for the IL-1RI receptor; the objective also concerns the development of nucleotide sequences encoding such polypeptides and being the expression basis for their production.

[0017] The solution to the problem is achieved by creating a fusion protein IL-1 RA-hFc, which is an antagonist of IL-1, the structure of which includes human IL-1 RA, represented by amino acids 26-177, corresponding to the natural sequence of IL-1 RA (SEQ ID NO: 1), as well as the constant part of the heavy chain (Fc fragment) of human IgG4, represented by the sequence SEQ ID NO: 3. In this case, the Fc fragment of human IgG4 is attached to the fragment of the IL-1 RA protein from the C-terminus via the linker sequence RS.

[0018] In some embodiments of the invention, the fusion protein further comprises a signal sequence located at the N-terminus of the fusion protein. In particular embodiments of the invention, the signal sequence is presented in the sequence SEQ ID NO: 4.

[0019] In some particular embodiments of the invention, the fusion protein has an amino acid sequence represented by SEQ ID NO: 2: MYRMQLLSCIALSLALVTNSRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKI DWPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFES AACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDERSPPCPSCPAPEFLGGPSVFLFPPK PKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQ DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSPGK

[0020] The stated task is also solved by creating an isolated nucleic acid molecule encoding such a fusion protein. In some particular embodiments of the invention, the nucleic acid molecule has the sequence SEQ ID NO: 5.

[0021] The said task is also solved by creating an expression vector carrying a nucleotide sequence corresponding to the sequence of an isolated nucleic acid molecule encoding such a fusion protein, under the control of regulatory elements necessary for the expression of this nucleotide sequence in a host cell; as well as by creating a host cell comprising the expression vector described in the present application, ensuring the efficient production of such a fusion protein using the above-mentioned nucleic acid molecule. In some embodiments of the invention, such cells are mammalian cells (in some particular embodiments of the invention, Chinese hamster ovary (CHO) cells).

[0022] The solution of the set task can also be achieved by using the above-mentioned fusion protein for the treatment and prevention of a wide range of inflammatory conditions and autoimmune diseases. The result of the invention is the creation of an IL-1 inhibitor, which is achieved by using a fragment of IL-1 RA in the fusion protein IL-1 RA-hFc, which is a decoy receptor aimed at reducing the ability of IL-1 isoforms to interact with the cellular receptor IL-1 RI and activate signal transmission. The IL-1 RA-hFc protein can be administered to a patient in a therapeutically effective amount, preferably as part of a pharmaceutical composition.

[0023] The following technical results are achieved by implementing the invention:

[0024] - a variant of a therapeutic agent based on the fusion (hybrid) protein IL-1 RA- hFc has been created, containing the functional part of IL-1 RA fused with the constant part (Fc domain) of human immunoglobulin IgG4, and capable of blocking the signal transmission of IL-1 isoforms (proinflammatory response), as well as

[0025] - nucleotide sequences have been developed that encode such fusion proteins and serve as the expression basis for their production;

[0026] - due to the design features, the developed recombinant polypeptide has a potentially high degree of safety for therapeutic use in combination with improved pharmacokinetic properties.

[0027] Brief description of the drawings

[0028] Fig. / . Scheme of an expression vector containing the nucleotide sequence of SEQ ID NO: 5 encoding a fusion protein having the amino acid sequence of SEQ ID NO: 2. Definitions and terms

[0029] The following terms and definitions are used in this document unless otherwise explicitly stated. References to techniques used in describing this invention refer to well-known techniques, including variations of these techniques and their replacement by equivalent techniques known to those skilled in the art.

[0030] In the documents of this invention, the terms "includes", "including", etc., as well as "contains", "comprising", etc. are interpreted to mean "includes, among other things" (or "contains, among other things"). These terms are not intended to be interpreted as "consists only of".

[0031] The terms "polypeptide," "protein," and "peptide" are used interchangeably herein and all refer to a polymer of amino acid residues. These terms may also be used interchangeably herein to refer to the final product resulting from expression of a nucleic acid sequence in a host cell.

[0032] By "fusion protein" ("hybrid protein", "recombinant protein", "fusion polypeptide", "recombinant polypeptide", "hybrid construct") is meant a construct of two or more parts of a polypeptide nature, covalently linked to each other, formed as a result of the expression of a recombinant DNA molecule in which the coding regions of two or more different genes or their fragments are connected to each other in one reading frame.

[0033] The term "isolated" has its generally accepted meaning, known to a person of ordinary skill in the art, and when used in relation to an isolated nucleic acid or an isolated polypeptide, is used without limitation to denote a nucleic acid or polypeptide that, due to man, exists separately from its native environment and is therefore not a product of nature. An isolated nucleic acid or polypeptide may exist in purified form or may exist in a non-native environment, such as, for example, a host cell.

[0034] The term "synonymous substitution" refers to a nucleotide sequence having a nucleotide sequence that may differ from a reference nucleic acid sequence by one or more substitutions that do not change the amino acid sequence of the protein encoded by the nucleic acid molecule. Since the genetic code is "degenerate", i.e. triplets of different nucleotide sequences can encode the same amino acid, synonymous substitutions in the nucleotide sequence do not change the amino acid sequence of the protein. The term "decoy receptor" refers to hybrid proteins consisting of the extracellular domain of the molecule (receptor) and the Fc domain of immunoglobulin. The extracellular domain of the receptor is responsible for target binding, and the Fc domain is responsible for dimerization of the hybrid protein.The latter is necessary to increase the binding efficiency and ensure greater stability of the high-molecular complex. In the context of the present invention, the "trap receptor" consists of a functional fragment of the human IL-1 RA protein and the constant part of the human |gG4 heavy chain.

[0035] The term "treatment" means curing, slowing down, stopping, or reversing the progression of a disease or disorder. As used herein, "treatment" also means alleviating symptoms associated with a disease or disorder. The fusion protein of the invention can be used for use in the treatment of IL-1-mediated diseases. In particular, such a disease can be selected from arthritis, enteritis, asthma, pulmonary fibrosis, glomerulonephritis, graft-versus-host disease, acute lung injury, rheumatoid arthritis, atopic dermatitis, etc.

[0036] The term "prophylaxis", "prevention", "preventive therapy" covers the elimination of risk factors, as well as preventive treatment of subclinical stages of the disease in humans, aimed at reducing the likelihood of the clinical stages of the disease. Patients for preventive therapy are selected based on factors that, based on known data, entail an increased risk of developing clinical stages of the disease compared to the general population. Preventive therapy includes a) primary prevention and b) secondary prevention. Primary prevention is defined as preventive treatment in patients who have not yet reached the clinical stage of the disease. Secondary prevention is the prevention of recurrence of the same or a similar clinical state of the disease.

[0037] The term risk reduction refers to therapy that reduces the incidence of clinical disease. Examples of risk reduction include primary and secondary disease prevention.

[0038] By "therapeutically / prophylactically effective amount (therapeutic dose)" is meant the amount of the medicinal product administered to the patient, at which the expected therapeutic (prophylactic) effect will most likely manifest. The exact required amount may vary from subject to subject depending on numerous factors, such as the severity of the disease, age, body weight, general condition of the body, combined treatment with other drugs, etc. The administration of the medicinal product according to the invention to the subject in need of treatment and / or prevention of the disease or condition is carried out in a dose sufficient to achieve the therapeutic effect.When carrying out treatment and / or prevention, the administration can be carried out either once or several times a day, more often in the form of a course of administration over a period of time sufficient to achieve a therapeutic effect (from several days to a week, several weeks and up to months), while the courses of administration of the medicinal product can be carried out repeatedly. In particular, in moderate forms of the disease, the single dose, frequency and / or duration of administration of the medicinal product according to the invention can be increased. Preferably, the fusion protein according to the invention is administered to the patient as part of a pharmaceutical composition including, in addition to the active component, pharmaceutically acceptable carriers and / or fillers.

[0039] Unless otherwise defined, technical and scientific terms in this application have the standard meanings generally accepted in the scientific and technical literature.

[0040] The numbers of the nucleotide and amino acid sequences referred to in this application correspond to the number in the Sequence Listing (SEQ ID NO) according to Standard ST.26, which is part of this description of the invention. In the event of discrepancies in the structure of the sequences between the mention in the text of the description and the corresponding sequence in the Sequence Listing according to Standard ST.26, the data given in the text of the description shall prevail.

[0041] Detailed description of the invention

[0042] The present invention provides DNA constructs encoding fusion proteins (recombinant polypeptides) - IL-1 antagonists that block the proinflammatory response induced by IL-1 a and p isoforms.

[0043] More specifically, the invention relates to nucleotide constructs encoding polypeptides comprising IL-1 RA fused to the constant portion (Fc domain) of human immunoglobulin IgG4 (IL-1 RA-hFc).

[0044] The natural sequence of the human IL-1 RA protein consists of 177 amino acids (aa) (SEQ ID NO: 1). The native protein contains a signal peptide (aa 1–25 of SEQ ID NO: 1) and a functional domain (aa 26–177 of SEQ ID NO: 1).

[0045] Within the framework of the present invention, in the process of developing new polypeptide drugs - inhibitors of the action of IL-1, a sequence corresponding to the functional part of IL-1RA was cloned and fused in one reading frame with a sequence encoding the constant part of the human IgG4 heavy chain. When constructing the hybrid protein of the invention, special attention was paid to the linker connecting two active fragments of the protein construct. It is known that protein linkers should provide the necessary interval between the domains of the hybrid protein, maintaining the correct folding of the protein in the case when the interactions of the N or C ends are critical for folding. In addition, it is preferable that the protein linker used enhances the stability of the molecule. In addition, when constructing the hybrid protein of the invention, special attention was paid to the safety of the protein construct when it is administered to a patient during therapy.As is known, the manifestation of undesirable immunogenicity of biotechnological drugs based on fusion proteins is a serious problem that significantly affects their use in therapy, especially in the treatment of diseases that require long-term systemic administration of drugs based on them (Avdeeva Zh.I. et al. Problems associated with undesirable immunogenicity of biotechnological drugs (therapeutic proteins) / / Immunology v.40, no.3, 2019, pp.51-64). Therefore, when constructing a hybrid protein, attention was paid not only to its stability and therapeutic efficacy, but also to reducing the risk of potential immunogenicity.In analyzing the possible risks of immunogenicity development, the inventors drew attention to the fact that despite the fact that fragments of IL-IRA molecules and the Fc fragment of IgG are of human origin and should naturally have a low immunogenic profile, the sequence obtained as a result of the fusion of these molecules is not always safe. Thus, long flexible linker peptides used as linkers usually consist of small non-polar amino acid residues, such as glycine, and polar amino acid residues, such as serine and threonine: several glycine residues give the linker an unstructured conformation, which ensures its flexibility, and serine or threonine provide a polar surface area to limit hydrophobic interactions in the peptide or with constituent fragments of the fusion protein.However, such glycine-rich peptide linkers, such as GGGGS repeats, share a common motif with prion domains (see, for example, Kun-Hua Yu et al. The Effect of Octapeptide Repeats on Prion Folding and Misfolding И Int J Mol Sci. 2021 Feb; 22(4): 1800; Kim S. et al. Transmembrane glycine zippers: Physiological and pathological roles in membrane proteins / / PNAS, October 4, 2005 vol. 102 no. 40 p. 14278 -14283), which can cause undesirable structural changes in the protein, its aggregation, and immunogenicity. Unstructured long flexible peptides used as linkers may have multiple surface neoepitopes, which may be formed by regions of the linker peptide or regions of molecules involved in the formation of the fusion protein adjacent to the linker at the fusion site. Such surface neoepitopes, accessible for recognition by the immune system, may also cause undesirable immunogenicity.For example, the sequences GGGGSGGGGS, GGGGSGG, GGGGSAE and EPKSSDK formed in the linker portion of the IL-1 RA-hFc fusion proteins known from the prior art are present in a large number of different types of bacteria, fungi and parasitic protozoa (such as Acinetobacter sp., Cryptosporangium parvum, Bacteroides fragilis, Dermabacter hominis, Toxoplasma gondii, Chryseobacterium sp., Rhodotorula toruloides, Actinomyces oris, Plasmodium vivax, Plasmodium ovale curtisi, etc.), many of which are pathogenic or opportunistic for humans. Exposure of humans to such a peptide may cause cross-reactivity with the IL-1 RA-hFc fusion protein and a subsequent immune reaction.

[0046] As a result of the work carried out, it was found that the use of the RS linker not only ensures the necessary stability of the molecule, but also, due to at least its length, ensures the production of a fusion protein with a low risk of potential immunogenicity. In this case, the necessary flexibility of the fusion protein molecule of the invention is ensured by including in the design a fragment of the hinge region PPCPSCP of the constant part of the human IgG4 heavy chain, represented by aa 1-7 of SEQ ID NO: 3, which allows for the necessary rotation and rigidity without excessive unstructured ™, due to which the functional domain of the IL-1 RA protein and the Fc domain included in the molecule can act independently of each other due to increased flexibility in this region. Thus, due to the design features, the recombinant polypeptide of the invention simultaneously has potentially improved pharmacokinetic properties and a low risk of potential immunogenicity.

[0047] The use of the 1g64 isotype Fc fragment in the fusion protein according to the invention provides additional advantages: minimal effector function for antibody-dependent cellular cytotoxicity (ADCC) and the absence of complement-dependent cytotoxicity (CDC), which reduces the possible risk of inflammatory reactions and sensitization when using a therapeutic agent based on the IL-1 RA-hFc hybrid protein.

[0048] Thus, the hybrid protein of the invention IL-1 RA-hFc (in a particular embodiment of the invention, represented by the sequence SEQ ID NO: 2), consisting of the functional domain of the human IL-1 RA protein, represented by amino acids 26-177 of SEQ ID NO: 1, and the Fc fragment of human IgG4, represented by the sequence SEQ ID NO: 3 and attached to the fragment of the IL-1 RA protein from the C-terminus via the linker sequence RS, has a high degree of safety for therapeutic use.

[0049] The IL-1 RA-hFc fusion protein of the invention may also optionally comprise a signal sequence, which may include any sequence known to a person skilled in the art of directing the secretion of a polypeptide or protein from a cell, and may include natural or synthetic sequences. Typically, the signal sequence is located at the N-terminus of the fusion protein of the present invention. In particular embodiments of the invention, the signal peptide has the sequence SEQ ID NO: 4.

[0050] The fusion protein (recombinant polypeptide) which is the subject of the present invention can be obtained as follows.

[0051] Soluble recombinant IL-1 antagonist polypeptides are produced by expressing nucleotide sequences encoding them in eukaryotic cell lines, followed by purification of the synthesized recombinant proteins using affinity, ion exchange or hydrophobic chromatography, used individually or in various combinations with each other, as well as using other methods for purifying proteins. The corresponding nucleotide sequences are obtained by combining DNA regions encoding selected IL-1 RA domains with a DNA sequence encoding the Fc fragment of human IgG4. In some particular embodiments of the invention, nucleic acid molecules encoding such IL-1 antagonist polypeptides have a nucleotide sequence corresponding to SEQ ID N0: 5.

[0052] The said problem is also solved by creating an expression vector containing the given nucleic acid molecule under the control of regulatory elements necessary for the expression of the given nucleic acid in a host cell. In preferred embodiments of the invention, the host cell containing such an expression vector can be Chinese hamster ovary cells CHO (for example, cell lines CH0-K1 or CHO DG44), adapted for the production of therapeutic proteins. In the present invention, the expression vector is preferably selected for the expression of heterologous sequences in mammalian cells, but in some embodiments of the invention, the expression vector can be selected for expression in other systems, such as, for example, insect cells, yeast or bacterial cells.Accordingly, each expression vector has its own set of regulatory elements that allow expression of a heterologous sequence (product) in the host cell, such as promoters and / or enhancers, Kozak sequences, polyA sequences and other regulatory sequences. It may also have sequences encoding leader (signal) peptides that provide secretion of recombinant polypeptides into the extracellular environment. Termination of protein synthesis from a given isolated nucleic acid sequence is determined by adding one or more stop codons to it from the 3'-end in one reading frame. After transfection of the vector into cells of a eukaryotic cell line, the recombinant polypeptide is synthesized and secreted into the culture serum-free medium.The resulting recombinant polypeptide is purified from the medium using, as a rule, affinity chromatography for protein A or protein G, but other methods of protein purification can also be used.

[0053] The following examples are provided for the purpose of disclosing the characteristics of the present invention and should not be construed as limiting the scope of the invention in any way.

[0054] Example 1. Construction of plasmids

[0055] To construct plasmids with the claimed nucleotide sequences, the sequence encoding the human IL-1 RA protein obtained from plasmid RG218518 (OriGene, IL-1 RA, Human Tagged ORF clone) was used. The sequence encoding IL-1 A was used for cloning. The following primers were selected for cloning the region corresponding to amino acids 26-177 for SEQ ID NO: 2:

[0056] IL-1 RA_SENSE (TAATGAATTCGCGACCCTCTGGGAGAAAATC) (SEQ ID NO: 6) IL-1 RA_ANTISENSE (TAATAGATCTCTCGTCCTCCTGGAAGTAGA) (SEQ ID NO: 7)

[0057] PCR was performed under the following conditions: 98°C 1 min, 30 cycles (98°C 10 sec, 59°C 10 sec, 72°C 1 min), 72°C 5 min. Reaction components: polymerase - Phusion (Thermo), buffer containing Mg2+ for Phusion polymerase (Thermo), 10 pmol of each primer and 0.25 mM nucleotide triphosphate mixture (Thermo). Amplification was performed using the RG218518 sequence (Origene). After PCR: PCR product was purified using the GeneJet PCR purification kit (Fermentas). Concentration and compliance with the predicted molecular weight were checked using gel electrophoresis in 1% agarose (TAE buffer). The obtained PCR products were used for further construction.

[0058] The pFUSE-hlgG4e-Fc2 vector (InvivoGen) was used for cloning the target sequences. It allows obtaining sequences fused with the Fc domain of lgG4 and studying their properties. The EcoRI restriction site (IL-1 RA_SENSE) was included in one of the primers, and the Bglll restriction site (IL-1 RA_ANTISENSE) in the other, which after amplification and restriction allowed obtaining a DNA fragment with sticky EcoRI, Bglll ends. The vector and PCR product were treated with EcoRI (Thermo) and Bglll (Thermo) restriction enzymes for 1 hour at 37°C, then purified using the GeneJet PCR purification kit (Fermentas) and ligated using 1 U (IU) of ligase (Thermo) according to the manufacturer's protocol. The ligation mixture was used to transform competent E. coli XL1-Blue cells and plated on LB medium containing zeocin.

[0059] The dishes were incubated overnight in a thermostat at 37°C. The next day, 20 colonies from each ligation mixture were tested for the presence of the desired insert by diluting a portion of the colony in 20 μl of water and boiling for 5 minutes. After cooling, the mixture was spun down and 1 μl was used as a template in the PCR reaction. PCR was performed using Taq DNA polymerase (Fermentas) and PROMF2 and FC primers. The presence of the insert was tested after electrophoresis of the PCR products. Plasmid DNA was isolated from two colonies containing the insert, the lengths of the restriction fragments were verified using restriction endonucleases EcoRI and Hindlll, and sequenced using PROMF2 and FC primers on an Applied Biosystems 3500 sequencer according to the manufacturer's instructions, using PROMF2 (forward) and FC (reverse) primers.

[0060] PROMF2: GCCTGACCCTGCTTGCTCAACT (SEQ ID NO: 8)

[0061] FC: CTCACGTCCACCACCACGCA (SEQ ID NO: 9)

[0062] The resulting constructed plasmid containing the target nucleotide sequences is shown in Fig. 1.

[0063] Example 2. Production of a hybrid protein

[0064] To produce the hybrid protein, CHO cells were transiently transfected with a constructed plasmid encoding the hybrid protein.

[0065] The CHO cell line was cultured in Dynamis medium supplemented with Glutamax (6 mM), Antidumping reagent B (0.5%) and zeocin (500 μg / ml). Cultivation was carried out in a Multitron Cell shaker-CO2 incubator (Infers, Switzerland) in an atmosphere of 5% CO2 at a temperature of 37 °C and a relative humidity of 95% in 125 ml Ernlenmeyer flasks (Corning, USA) (stirring 120 rpm).

[0066] For transfection of CHO cells, ultrapure ("transfection grade") plasmid DNA was isolated using the EndoFree Plasmid MaxiKit (Qiagen) according to the manufacturer's protocol. Linearization of the constructed plasmid was performed using the Notl site (Thermo). For transfection, 20 μg of linearized plasmid were added to 100 μl of cell suspension at a concentration of 5x10 7 cells / ml. Electroporation was carried out according to the protocol: 3 pulses of 1130 V with a duration of 20 ms.

[0067] Selection of transfected cells began 48 hours after transfection by adding the antibiotic zeocin at a concentration of 500 μg / ml and continued during further passages.

[0068] Protein production was analyzed by standard SDS-PAGE. The secreted fusion protein was visualized using rabbit antibodies against the human Fc domain (Jackson Immunoresearch, USA). The target IL-1 RA-hFc protein was also purified from the culture fluid by FPLC chromatography on a HiTrap ProteinA HP column (Cytiva) and assessed by gel electrophoresis. Cultivation of these pools confirmed the production of IL-1RA-hFc fusion polypeptides and their secretion in the culture fluid.

[0069] Thus, as a result of the conducted studies, a nucleotide sequence encoding the IL-1 RA-hFc fusion protein was developed and is the expression basis for its production, and a fusion protein IL-1RA-hFc was obtained, containing the functional domain of IL-1RA fused with the constant part (Fc-domain) of human immunoglobulin IgG4, capable of effectively blocking IL-1, which can be used in the therapy and prevention of the development of inflammatory and autoimmune diseases. This invention has a number of improved properties compared to analogues and therefore expands the range of available candidates for the treatment of inflammatory and autoimmune diseases, in the pathogenesis of which IL-1 is involved.

[0070] Although the invention has been described with reference to the disclosed embodiments, it will be apparent to those skilled in the art that the specific experiments described in detail are provided merely for the purpose of illustrating the present invention and should not be considered as limiting the scope of the invention in any way. It will be understood that various modifications can be made without departing from the spirit of the present invention.

Claims

Invention formula 1. A fusion protein - an IL-1 antagonist, comprising the functional domain of human IL-1 RA, represented by the amino acid sequence corresponding to positions 26-177 of the sequence SEQ ID NO: 1, and the Fc fragment of human IgG4, represented by the amino acid sequence SEQ ID NO: 3 and attached to the functional domain of human IL-1 RA from the C-terminus via a linker sequence RS.

2. The fusion protein according to claim 1, further comprising a signal sequence localized at the N-terminus of the fusion protein.

3. The fusion protein according to claim 2, wherein the signal sequence is represented by SEQ ID NO:

4.

4. The fusion protein according to item 3, represented by the amino acid sequence SEQ ID NO:

2.

5. An isolated nucleic acid molecule encoding a fusion protein according to any one of paragraphs 1-4.

6. An isolated nucleic acid molecule according to claim 5, represented by the sequence SEQ ID NO:

5.

7. An expression vector containing a nucleic acid sequence according to any of paragraphs 5-6 under the control of regulatory elements necessary for its expression in a host cell.

8. A cell capable of expressing the fusion protein according to any of paragraphs 1-4, which is not a human embryonic cell and is transfected with the expression vector according to paragraph 7.

9. The cell according to claim 8, which is a Chinese hamster ovary cell. (SNO).

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