WO2025042305A1 - Nucleotide sequence encoding a pdgfra-hfc fusion protein - Google Patents

Abstract

The present invention relates to the field of biomolecular pharmacology, biotechnology and genetic engineering and describes nucleic acid molecules and novel recombinant polypeptides encoded thereby, said recombinant polypeptides being PDGFR antagonists and being capable of effectively blocking the tyrosine kinase activity of PDGFR pathways. The obtained recombinant polypeptides can be used in the treatment of oncological and fibrotic diseases.

Description

NUCLEOTIDE SEQUENCE ENCODING THE PDGFRA-HFC FUSION PROTEIN

Field of technology

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 PDGFRa (Platelet-derived growth factor receptor A), capable of blocking PDGFR signaling pathways, as well as a method for obtaining them.

State of the art

The PDGFR signaling pathway is an important RTK (receptor tyrosine kinase) pathway that is activated in various tumors and regulates tumor tissue growth and vascularization (Cavalcanti, Ignazzi, De Michele, & Caruso, 2019). PDGFR proteins are transmembrane receptors that are able to dimerize and phosphorylate upon binding to the PDGF ligand (Kashishian, Kazlauskas, & Cooper, 1992), thereby activating various downstream signaling pathways such as PI3K/AKT/PKB (phosphatidylinositol 3-kinase/protein kinase B pathway), MAPK/ERK (mitogen-activated protein kinase/extracellular signal-regulated kinase, JAK (Janus kinase), STAT (transducer and activator of transcription) signaling pathway, and the Notch pathway (Huang et al., 2021 ; Zou et al., 2022).

The PDGFR family of tyrosine kinase receptors includes two related receptors: PDGFRa and PDGFRp. The PDGF ligands that bind to these receptors have different specificities: depending on the specific PDGF isoform, different homo- or heterodimers of PDGFRa and PDGFRp receptors will be formed (Reigstad, Varhaug, & Lillehaug, 2005). Five PDGF isoforms are expressed in tissues in different and partially overlapping patterns. PDGFs are secreted by endothelial cells, macrophages, and epithelial cells and are present in platelets, being released upon degranulation. PDGF-C has greater structural similarity to VEGF than to PDGF-B (Reigstad et al., 2003), while VEGF has approximately 25% sequence similarity to all PDGFs and has been reported to bind PDGFRa but fail to efficiently activate it (Pennock, Kazlauskas, et al., 2012).

Inhibition of PDGFR suppresses cancer proliferation, metastasis, invasion, and angiogenesis, and improves the action of anticancer drugs (Balamurugan et al. aL, 2021 ; Thies et al., 2021). There are a number of novel therapeutic strategies based on PDGFR pathway inhibition for cancer treatment (Sun, Yue, Xu, & Hu, 2022).

PDGF signaling is also associated with fibrotic diseases, which involve progressive scarring and loss of tissue function due to mesenchymal cell proliferation and extracellular matrix deposition. Liver fibrosis can develop as a reversible reaction during wound healing following injury, but if it becomes chronic, it can lead to liver cirrhosis with a poor prognosis. The PDGFR pathway is considered one of the key pathways in the initiation of fibrosis of various origins (Zhao et al., 2022). Liver fibrosis involves the activation of hepatic stellate cells and portal fibroblasts (Friedman, 2008, Gressner and Weiskirchen, 2006), the proliferation and migration of which depend on PDGF-B and -D (Borkham-Kamphorst et al., 2007, Pinzani et al., 1989). However, expression of PDGF-A or single-linked C transgenes in mouse liver also induced fibrosis, which for PDGF-C also led to tumor development (Thieringer et al., 2008). Increased expression of PDGF and PDGFR has been observed in both experimental models and human diseases (Borkham-Kamphorst et al., 2008).

There are three main approaches to inhibiting the PDGF/PDGFR pathway: 1) sequestration of the ligand with neutralizing antibodies, soluble extracellular portions of the receptors acting as ligand decoys, or DNA/RNA aptamers, 2) disruption of the interaction between ligand and receptor by blocking the receptor with receptor-specific antibodies or small molecule inhibitors, and 3) use of small molecule inhibitors to block the kinase activity of PDGFR. These three approaches are discussed in more detail below.

The development of selective therapeutic antibodies that sequester PDGF is problematic because there is functional redundancy in the activation of PDGFR by their ligands. In addition, PDGFRa and PDGFRp constitute a group of class III receptor tyrosine kinases that are similar in structure and function to the cell surface receptors Kit, FLT3, and CSF1 R (Blume-Jensen and Hunter, 2001). There are also structural similarities to class IV and class V tyrosine kinases, further complicating the development of specific small molecule inhibitors that specifically target the PDGFR pathway.

A more promising avenue for the development of selective PDGFR inhibitors is PDGFR-blocking antibodies. Olaratumab (IMC-3G3, Lartruvo™) is a recombinant human IgG1 monoclonal antibody against PDGFRa that specifically binds to PDGFRa, blocking PDGF-AA, PDGF-BB, and PDGF-CC binding and receptor activation without cross-reacting with PDGFR[3 (Loizos et al., 2005). Olaratumab reduces proliferation of several tumor models both in vitro and in vivo (Gerber et al., 2012, Loizos et al., 2005, Matei et al., 2006, Russell et al., 2010, Stock et al., 2007). Based on the results of a phase Ib/II trial in advanced soft tissue sarcoma, where combination therapy with olaratumab and doxorubicin showed a significant improvement in median overall survival of 11.8 months compared with doxorubicin alone (Tap et al., 2016), oralatumab received the first global approval for the treatment of soft tissue sarcoma in the US in October 2016 (Shirley, 2017). It is currently undergoing a confirmatory international phase III clinical trial in soft tissue sarcoma (ANNOUNCE study: NCT02451943) and has received conditional approval for use in the EU (European Medicines Agency, 2016) for the treatment of patients with advanced soft tissue sarcoma that is refractory to curative treatment with surgery or radiotherapy. However, despite promising results in preclinical studies in glioblastoma and leiomyosarcoma xenografts (Loizos et al., 2005), oralatumab was ineffective in other solid tumors such as glioma, prostate cancer, or ovarian cancer. Moreover, two phase II studies of olaratumab were terminated: a study in advanced non-small cell lung cancer (NCT00918203) and unresectable and/or metastatic gastrointestinal stromal tumors (GIST) (NCT01316263), which did not show a clear effect on progression-free survival in patients without PDGFRa mutations (Wagner et al., 2017). In June 2017, a new phase I/II study was initiated testing the effects of oralatumab in combination with nab-paclitaxel and gemcitabine in patients with metastatic pancreatic cancer (NCT03086369).

An effective approach to block PDGFR signaling is the use of small molecules that can penetrate cells and block its enzymatic activity. Most current anticancer drugs to inhibit PDGFR kinase activity are based on multi-targeted small molecules, collectively called tyrosine kinase inhibitors (TKIs), of which imatinib was the first, approved for clinical use in 2001 (Iqbal Nida, Iqbal Naveed, 2014). These drugs bind to the ATP-binding pocket of kinases, thereby competing with ATP and stopping receptor activation and signaling. Due to the conserved structure of the ATP pocket, the inhibitor usually inhibits kinases beyond their intended target. In addition, adverse cellular responses to TKI treatment, such as activation of the apoptosis inhibitor XIAP by imatinib, activation of FAK and p130 in a Cas AN-independent manner by imatinib and nilotinib (Frolov et al, 2016), will also shape the response to TKIs and may explain the failure of the drugs to inhibit oncogenic transformation. Other drugs in this class include nilotinib, dasatinib, ponatinib, sunitinib. Notably, all TKIs have multiple targets and there is no selective inhibitor that blocks only PDGFR.

Fc-fusion proteins have proven themselves as therapeutic and prophylactic agents. Proteins obtained using this technology have great therapeutic potential, as they are linked to the Fc domain, which significantly prolongs the half-life of proteins in blood plasma, which prolongs therapeutic activity, and also leads to slower renal clearance for larger molecules. 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)).

The aim of the invention is to develop effective PDGFRa antagonists based on Fc-fusion protein technology.

The essence of the invention

The objective of the present invention is to expand the arsenal of technical means for treating oncological and fibrotic diseases. In particular, the objective concerns obtaining new highly effective polypeptide drugs - PDGFRa antagonists (inhibitors of signal transmission through the PDGFRa receptor), possessing high affinity for PDGFRa receptor ligands and having a high degree of safety of therapeutic use; the objective also concerns the development of nucleotide sequences encoding such polypeptides and being the expression basis for their production.

The solution to the problem is achieved by creating a PDGFRa-hFc fusion protein, which is a PDGFRa antagonist, the structure of which includes (a) a fragment of the soluble extracellular part of the human PDGFRa protein, represented by amino acids 24-528 of SEQ ID NO: 1 and including five immunoglobulin-like repeats of type C2, included in the natural sequence of PDGFRa, as well as (b) the constant part of the heavy chain (Fc fragment) of human IgG4. The fusion PDGFRa-hFc protein does not contain the tyrosine kinase domain of PDGFRa, as well as other intracellular domains of PDGFRa. In this case, the Fc fragment of human IgG4 is attached to the fragment of the PDGFRa protein from the C-terminus via the linker sequence RS. In preferred embodiments of the invention, the PDGFRa-hFc fusion protein of the invention is represented by the amino acid sequence SEQ ID NO: 3. In some embodiments of the invention, the fusion protein further comprises a signal sequence located at the N-terminus of the fusion protein, which ensures secretion of the fusion protein into the extracellular environment during its synthesis. In particular embodiments, the signal sequence is represented by SEQ ID NO: 4. In some particular embodiments of the invention, the fusion protein has the amino acid sequence of SEQ ID NO: 2.

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.

The said problem 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).

The solution to the stated problem can be achieved by using the above-mentioned fusion protein for the treatment of a wide range of human oncological and fibrotic diseases, where inhibition of PDGFRa tyrosine kinase activity is required. The result of the invention is the creation of a selective PDGFR inhibitor, which is achieved by using the extracellular part of PDGFRa in PDGFRa-hFc, which is a decoy receptor aimed at reducing the ability of PDGF ligands to interact with the cellular PDGFRa receptor and activate signal transmission. The PDGFRa-hFc protein can be administered to a patient in a therapeutically effective amount, preferably as part of a pharmaceutical composition.

The following technical results are achieved by implementing the invention:

- a variant of a therapeutic agent based on the fused (hybrid) protein PDGFRa-hFc has been created, containing the functional domains of the extracellular part of PDGFRa fused with the constant part (Fc domain) of human immunoglobulin IgG4, and capable of blocking the tyrosine kinase activity of the PDGFR pathways, and nucleotide sequences encoding such fused proteins have been developed, which are the expression basis for their production;

- the developed recombinant polypeptide has a high degree of safety for therapeutic use, the basis of which is the features of its design, namely: minimal effector function for antibody-dependent cellular cytotoxicity (antibody-dependent cellular cytotoxicity - ADCC) and the absence of complement-dependent cytotoxicity (CDC), due to the use of the constant part of human immunoglobulin IgG4 isotype; low immunogenicity, due to the design of the recombinant polypeptide at the fusion site of the PDGFRa and IgG4 fragments: the region of the hybrid protein formed with the participation of the RS linker has a minimal risk of cross-reactivity of the immune system; increased protein stability and reduced cross-reactivity, due to the presence of an additional amino acid at the N-terminus of the hybrid structure, represented by isoleucine (I).

Brief description of the drawings

Fig. 1. 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

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.

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".

The term "and/or" means one, more, or all of the listed elements.

The term "optional" / "optional" (or "optional") as used in this document means that the subsequently described event or circumstance may, but does not necessarily, occur, and that the description includes instances in which the event or circumstance occurs and instances in which it does not occur.

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.

By "fusion protein"("hybridprotein","recombinantprotein","fusionpolypeptide","recombinantpolypeptide","hybridconstruct") is meant a construct of two or more parts of a polypeptide nature, covalently linked between itself, 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.

The terms "PDGFRa", "PDGFRa" and "PDGFR-alpha" are interchangeable and refer to the platelet-derived growth factor receptor A.

The abbreviation "ac" stands for "amino acids".

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.

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 result in a change in 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 may encode the same amino acid, synonymous substitutions in the nucleotide sequence do not result in a change in 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 performs 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 "decoy receptor" consists of certain fragments of the soluble extracellular portion of the human PDGFRa protein and the constant portion of the human IgG4 heavy chain.

The term “treatment” means to cure, slow, halt, or reverse the progression of a disease or disorder. As used herein, “treatment” also means to alleviate the symptoms associated with the disease or disorder.

A "therapeutically effective amount (therapeutic dose)" is the amount of a drug administered to a patient that is most likely to produce the desired therapeutic effect. The exact the 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, combination therapy with other drugs, etc. The administration of the medicinal product of the invention to a subject in need of treatment and/or prevention of a disease or condition is carried out in a dose sufficient to achieve a therapeutic effect. During treatment and/or prevention, the administration may 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 may be repeated. In particular, in moderate forms of the disease, a single dose, frequency and/or duration of administration of the medicinal product of the invention may be increased. Preferably, the fusion protein of the invention is administered to a patient as part of a pharmaceutical composition comprising, in addition to the active component, pharmaceutically acceptable carriers and/or excipients.

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

Detailed description of the invention

The present invention provides DNA constructs encoding fusion proteins (recombinant polypeptides) - PDGFRa antagonists that block the PDGFRa signaling pathway.

In particular, the invention relates to nucleotide constructs encoding polypeptides containing the extracellular portion of PDGFRa (including five immunoglobulin-like repeats of type 02) fused with the constant portion (Fc domain) of human immunoglobulin IgG4, PDGFRa-hFc.

The natural sequence of the human PDGFRa protein consists of 1089 amino acids (aa) (SEQ ID NO: 1). The native protein contains a signal peptide (1-23 aa BEO ID NO: 1), five immunoglobulin-like repeats of type C2 (24-113, 117-201, 202-306, 319-410, 414-517 aa), a transmembrane domain (529-549 aa), and a tyrosine kinase domain (593-954 aa). The protein is divided into an extracellular (extracellular) part (24-528 aa SEQ ID NO: 1) and a cytoplasmic part (550-1089 aa SEQ ID NO: 1).

Within the framework of the present invention, in the process of developing new polypeptide drugs - inhibitors of tyrosine kinase activity of PDGFRa, the sequence corresponding to the extracellular domain of PDGFRa and containing five immunoglobulin-like repeats of type C2 was cloned and fused in one reading frame with the sequence encoding the constant part of the heavy chain of IgG human. When constructing the hybrid protein of the invention, special attention was paid to the linker connecting the two active fragments of the protein construct. It is known that protein linkers should provide the necessary spacing between the domains of the hybrid protein, maintaining the correct folding of the protein in the case where 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 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) I Immunology v. 40, no. 3, 2019, pp. 51-64). Therefore, when constructing the hybrid protein, attention was paid not only to its stability and therapeutic efficacy, but also to reducing the risk of potential immunogenicity. Analyzing the possible risks of developing immunogenicity, the authors of the invention drew attention to the fact that despite the fact that fragments of PDGFRa 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, when using the Fc fragment of the IgG1 isotype in combination with the GS linker, the neoepitope GSDKTHT is formed in the junction site, which is present in a large number of different types of bacteria and fungi (Candida dubliniensis, Budvicia aquatica Pediococcus parvulus, Stagonospora sp. SRC1lsM3a, Jiangella sp. DSM 45060, Jiangella alkaliphila, Jiangella alba, Catenaria anguillulae PL171, Methylosinus spohum, Phenylobacterium deserti, Aeromicrobium phragmitis, Aeromicrobium camelliae, Jiangella rhizosphaerae, Budvicia aquatica, Flavobacterium sp. 90, Jiangella aurantiaca, Methylosinus sp. C49, Methylocystis heyeri, Tripterygium wilfordii (Thunder God vine), Tripterygium wilfordii (Thunder God vine), Pedobacter puniceum, Anseranas semipalmata, Bradyrhizobium sp. CCBAU 53421, Colletotrichum plurivorum, Kiritimatiellaceae bacterium, Hyphomicrobiales bacterium, Anaerolineae bacterium, Methylobacterium sp. (strain 4-46), Beijerinckia indica subsp. indica (strain A TCC 9039 / DSM 1715 / NCIMB 8712), Methylobacterium nodulans), many of which are pathogenic or opportunistic for humans, and some are used for food preparation (fermentation). Human exposure to such a peptide may cause cross-reactivity with the PDGFRa-hFc fusion protein and a subsequent immune response. As a result of the work, it was unexpectedly discovered that the use of the RS linker not only ensures the necessary stability of the molecule, but also ensures the production of a fusion protein with a low risk of potential immunogenicity, provided that the IgG4 isotype (and not IgG1) is used as the Fc fragment. As the analysis showed, this combination not only solves the problem of reducing the immunogenicity of the neoepitope formed in the junction site (the resulting neoepitope RSYGPPC was not detected in pathogenic and opportunistic bacterial species for humans - this peptide is present only in a few organisms that are not pathogenic for humans: Uncinula necator, Molossus molossus and Streptosporangium becharense), but also achieves additional advantages that ensure increased safety of the hybrid protein, which are associated with the use of the IgG4 isotype of the Fc fragment - minimal effector function for antibody-dependent cellular cytotoxicity and the absence of complement-dependent cytotoxicity, which reduces the possible risk of inflammatory reactions and sensitization when using a therapeutic agent based on the PDGFRa-hFc hybrid protein.

Another feature of the fusion protein design according to the invention is the presence of an additional amino acid represented by isoleucine (I) at the N-terminus of the hybrid design. As follows from the analysis of the natural sequence of the human PDGFRa protein, the terminal amino acid at the N-terminus of the fragment of the soluble extracellular part of the protein (24-528 aa SEQ ID NO: 1) is glutamine (Q). It is known that glutamine often cyclizes to form pyroglutamate at the N-terminus of secreted proteins, in particular antibodies (Nakayoshi et al. 2020). This process is especially pronounced in the production of antibodies in cell culture and in vitro purification. The rate of glutamine conversion in cell culture is about 1.41% per hour (Liu H. et al. 2014). Because of this process, when obtaining drugs based on recombinant proteins with Q (or E) at the N-terminus, most of them are represented by a mixture of modifications containing pyroglutamate at the N-terminus. It is known that in some cases, such a modification at the N-terminus of proteins and peptides can have serious functional consequences. In particular, it is known that pyroglutamate at the N-terminus of the truncated peptide amylode beta (Abeta) plays a fundamental role in the development of Alzheimer's disease. Vaccines and antibodies against such pyroglutamate-Abeta modifications are currently being developed (Alawode D. et al. 2021). Studies conducted by the authors of the invention indicate that recombinant proteins with pyroglutamate at the N-terminus have potential cross-reactivity with such drugs and vaccines, which can affect both the efficacy and safety of therapy in sensitized patients. The addition of isoleucine (I) at the N-terminus of the hybrid construct of the invention eliminates the formation of N-terminal pyroglutamate and thus thus preventing the occurrence of cross-reactivity with drugs aimed at recognizing pyroglutamate at the N-terminus of polypeptide molecules. Moreover, as studies have shown, such a modification also contributes to increased stability of the developed protein structure.

Thus, the hybrid protein of the invention, represented by the sequence SEQ ID NO: 3 and consisting of a fragment of the soluble extracellular part of the human PDGFRa protein, represented by amino acids 24-528 of SEQ ID NO: 1, and the Fc fragment of human IgG4, attached to the fragment of the PDGFRa protein from the C-terminus via the linker sequence RS, and having an additional isoleucine at the N-terminus of the hybrid construct, has a high degree of safety for therapeutic use.

The PDGFRa-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 controlling 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 sequence is represented by SEQ ID NO: 4.

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

Soluble recombinant PDGFRa 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 PDGFRa domains with a DNA sequence encoding the Fc fragment of human IgG4. In some particular embodiments of the invention, nucleic acid molecules encoding such PDGFRa antagonist polypeptides have a nucleotide sequence corresponding to SEQ ID NO: 5.

The said task 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 comprising such the expression vector may be Chinese hamster ovary CHO cells (for example, CHO-K1 or CHO DG44 cell lines) adapted for the production of therapeutic proteins. In the present invention, the expression vector is preferably selected for expression of heterologous sequences in mammalian cells, but in some embodiments of the invention, the expression vector may 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 a host cell, such as promoters and/or enhancers, Kozak sequences, polyA sequences and other regulatory sequences. And 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 the addition of one or more stop codons to it at the 3'-end in one reading frame.

Expression of the hybrid construct in mammalian cells is possible by creating stable producer clones after transfection of cells with this construct. Transfection by electroporation or using a transfection reagent such as Lipofectamine 2000 can be used. This hybrid construct can also be used for introduction into a lentiviral construct and subsequent infection of cells. To increase the yield of the hybrid protein in mammalian cells, various approaches can be used. Optimization methods are known to those skilled in the art and are described, for example, in (Almo SC, Love JD. Better and faster: improvements and optimization for mammalian recombinant protein production // Curr Opin Struct Biol. 2014 Jun; 26:39-43).

After transfection of the vector into the cells of the 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.

The developed product is a previously uncreated combination of nucleotide sequences of the human genome and is intended to be an expression basis for a fusion protein with predicted improved pharmacological properties for obtaining new highly effective polypeptide drugs - PDGFRa antagonists (inhibitors of signal transduction through the PDGFRa receptor), with high affinity for PDGFRa ligands, effective for the treatment of oncological and fibrotic diseases. After passing animal safety tests and clinical trials, the hybrid protein of the present invention can be included in a pharmaceutical composition for the treatment of human cancer and fibrotic diseases.

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.

Example 1. Construction of plasmids

To construct plasmids with the claimed nucleotide sequences, the sequence encoding the human protein PDGFRa obtained from the plasmid RG211740 (OriGene, PDGFRa, Human Tagged ORF clone) was used. The PDGFRa fragment was selected based on bioinformatics analysis of the sequence and the corresponding nucleotide sequence encoding it was used for subsequent cloning. To clone the region corresponding to amino acids 24-528 of SEQ ID NO: 1 (with the aim of subsequently obtaining a protein having the amino acid sequence presented by SEQ ID NO: 2 and a protein having the sequence of SEQ ID NO: 3), the following primers were selected:

PDGFRA_S: p-CCAGCTTTCATTACCCTCTATCCTT (SEQ ID NO: 6)

PDGFRA_R: TAAGATCTAGCCACCGTGAGTTCAGAACGCA (SEQ ID NO: 7)

PCR was performed under the following conditions: 98°C 1 min, 30 cycles (98°C 10 sec, 63°C 10 sec, 72°C 2 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 RG211740 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.

The pFUSE-hlgG4e-Fc2 vector (InvivoGen) was used to clone the target sequences. It allows one to obtain sequences fused with the Fc domain of lgG4 and study their properties. The Bglll restriction site (PDGFRA_R) was included in one of the primers. After amplification and restriction, it was possible to obtain a DNA fragment with one sticky (Bglll) site and one blunt site. The vector was treated with EcoRV and Bglll restriction enzymes (Thermo), and the PCR product was treated with Bglll restriction enzyme (Thermo) for 1 hour at 37°C, then purified using the GeneJet PCR purification kit (Fermentas) and ligated using 1 IU of ligase (Thermo) according to the manufacturer's protocol. The ligase mixture E. coli XL1-Blue competent cells were transformed and plated on LB medium containing zeocin.

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 FRAT2 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.

PROMF2: GCCTGACCCTGCTTGCTCAACT (SEQ ID NO: 8)

FRAT2: CGAGGCACTGCTCACTTCCAAGA (SEQ ID NO: 9)

FC: CTCACGTCCACCACCACGCA (SEQ ID NO: 10)

The resulting constructed plasmid (expression vector) containing the target nucleotide sequences (SEQ ID NO: 5) is shown in Fig. 1.

Example 2. Production of a hybrid protein

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

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 (Infors, 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).

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 kcells/ml. Electroporation was performed according to the protocol: 3 pulses of 1130 V with a duration of 20 ms.

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. Protein production was analyzed using standard SDS-PAGE gel electrophoresis. The secreted fusion protein was visualized using rabbit antibodies against the human Fc domain (Jackson Immunoresearch, USA). The target protein PDGFRa-hFc in the culture fluid was also determined by bilayer interferometry using Protein A biosensors and the Octet K2 system (Fortebio, Pall, USA). Binding of the Fc fragment of the fusion protein to Protein A immobilized on the sensor was assessed. When culturing these pools in the fed-batch mode, the production of fusion polypeptides PDGFRa-hFc and their secretion in the culture fluid were confirmed.

Thus, as a result of the conducted studies, a nucleotide sequence encoding the fusion protein PDGFRa-hFc and being the expression basis for its production was developed, and a fusion protein PDGFRa-hFc was obtained, containing functional domains of the extracellular part of PDGFRa fused with the constant part (Fc-domain) of human immunoglobulin IgG4, capable of effectively blocking the tyrosine kinase activity of PDGFR pathways, which can be used in the therapy of oncological and fibrotic diseases. This invention has a number of improved properties compared to analogues, and therefore expands the range of available candidates for the treatment of fibrotic and oncological diseases, in the pathogenesis of which PDGFR signaling pathways are involved.

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 - PDGFRa antagonist, represented by the amino acid sequence SEQ ID NO: 3, comprising a fragment of the soluble extracellular portion of the human PDGFRa protein and the Fc fragment of human IgG4, attached to the fragment of the PDGFRa protein from the C-terminus via the 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. (IT).