A recombinant human coagulation factor ix-fc variant fusion protein and uses thereof

Abstract

The application provides a recombinant human coagulation factor IX-Fc variant fusion protein and application thereof, and relates to the field of biological medicines.The fusion protein of the application is a dimeric molecule composed of coagulation factor IX, a cleavable linker and a human IgG1 Fc variant fragment.Compared with monomers or wild-type dimers, the fusion protein of the application has significantly improved affinity to FcRn and molar ratio activity of long-acting coagulation factor IX;at the same time, the problem that the pharmacokinetics of dimeric Fc fusion proteins is poorer than that of monomeric Fc fusion proteins is overcome, the half-life of the recombinant human coagulation factor IX is effectively improved, and the stability thereof is improved.Therefore, the recombinant human coagulation factor IX-Fc variant fusion protein of the application has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a recombinant human coagulation factor IX-Fc variant fusion protein and its uses. Background Technology

[0002] Human clotting factor IX (hFIX) is a glycoprotein with a molecular weight of approximately 55 kDa. It belongs to the vitamin K-dependent clotting factors and exists in the blood as a serine protease protease, playing a crucial role in the intrinsic coagulation pathway. hFIX is synthesized in the human liver and requires various post-translational modifications to become a fully active mature protein, including N-linked and O-linked glycosylation, cleavage of the signal peptide and leader peptide, and vitamin K-dependent γ-carboxylation of the N-terminal GLA region. hFIX deficiency or dysfunction leads to hemophilia B, an X-linked recessive bleeding disorder. The incidence of hemophilia B in males is approximately 1 in 30,000, while it is extremely rare in females. Clinical manifestations of hemophilia B include prolonged post-traumatic bleeding and recurrent spontaneous soft tissue and joint bleeding, which can be life-threatening in severe cases. Currently, clinical treatment for hemophilia B mainly relies on regular transfusions of fresh plasma or concentrated human clotting factor IX preparations to maintain effective FIX activity levels in the body. Because hemophilia B patients require lifelong prophylactic replacement therapy, frequent intravenous injections not only impose a heavy treatment burden on patients but also severely impact their quality of life. Therefore, the development of long-acting recombinant human factor IX (rhFIX) formulations with extended half-lives has significant clinical implications and enormous market demand.

[0003] Currently, there are three main long-acting rhFIX products approved for marketing internationally: Alprolix® (recombinant human coagulation factor IX-Fc fusion protein, Sanofi SA), Idelvion® (recombinant human coagulation factor IX-albumin fusion protein, CSL Behring), and Rebinyn® (glycosylated recombinant human coagulation factor IX, Novo Nordisk). However, none of these products are available in China. Alprolix® is a monomeric long-acting rhFIX formulation constructed using Fc fusion protein technology. It significantly prolongs the in vivo half-life of rhFIX by covalently linking a single-molecule hFIX to the Fc domain of IgG1 and utilizing the pH-dependent binding of Fc to the neonatal Fc receptor (FcRn). Through the FcRn-mediated recycling pathway, the fusion protein is prevented from being degraded by lysosomes. However, while the Fc fusion strategy leads to a prolonged half-life, it also results in a significant decrease in the specific activity of coagulation factor IX. Studies have shown that the molar activity (IU / nmol) of Alprolix® is only about 50% of that of recombinant FIX, which limits its full clinical efficacy to some extent. Furthermore, Alprolix® does not use a cleavable linker; after coagulation factor IX is activated, it remains fused to its Fc domain. This non-cleavable design prevents activated FIXa from participating in the coagulation cascade in its natural form, potentially affecting the full expression of its procoagulant activity and posing certain safety risks.

[0004] More notably, according to research published in *Blood* by Peters et al. and related disclosures in the monograph *Therapeutic Fc-Fusion Proteins*, the monomeric recombinant human coagulation factor IX-Fc / Fc fusion protein exhibits significantly superior pharmacokinetic (PK) performance compared to the dimer form. However, in actual production, the use of monomers inevitably results in the formation of a certain proportion of dimer recombinant human coagulation factor IX-Fc fusion protein. This significantly increases the difficulty and cost of downstream purification processes, posing a severe challenge to industrial-scale production. On the other hand, the Fc domain of natural IgG1 can bind with high affinity to Staphylococcal protein A (SPA). While this property can be used for affinity chromatography purification of Protein A, it also causes SPA to interfere with the application of wild-type IgG1 Fc-based fusion proteins in anti-Staphylococcal infection treatment and other applications, limiting the further application of Fc variants in the fields of antimicrobial antibodies and antimicrobial peptide fusions.

[0005] Idelvion® employs a strategy of fusing recombinant human coagulation factor IX with albumin, and by introducing a cleavable linker peptide derived from the endogenous activating peptide of natural FIX, it achieves automatic cleavage and release of the albumin moiety upon FIX activation, offering certain advantages in safety design. However, albumin fusion proteins have a large molecular weight, and their half-life extension mechanism also relies on FcRn-mediated recycling, resulting in significant differences in manufacturing processes and quality control standards compared to Fc fusion proteins. Rebinyn®, on the other hand, utilizes glycosyl PEGylation technology, covalently linking 40 kDa branched-chain PEG molecules to the FIX-activating peptide region to reduce renal clearance, thereby extending the half-life. However, PEG modification may pose potential immunogenicity risks, and its preparation process is relatively complex.

[0006] Structurally, the Fc domain binds to FcRn via the CH2-CH3 region in a pH-dependent manner, which is the core mechanism for the long half-life of IgG and Fc fusion proteins. Simultaneously, the CH2-CH3 interface of Fc is also the main binding site for SPA, with histidine 435 (H435) and tyrosine 436 (Y436) residues playing crucial roles in the interaction between SPA and Fc. Studies have shown that mutating H435 and Y436 of IgG1 Fc to arginine and phenylalanine (H435R / Y436F) respectively, corresponding to IgG3, can essentially eliminate the binding ability of Fc to SPA. However, the H435 residue also participates in the interaction between Fc and FcRn; simply introducing the H435R mutation significantly weakens the FcRn binding affinity, leading to a significant shortening of the in vivo half-life of the fusion protein. Therefore, how to eliminate SPA binding while maintaining or even enhancing FcRn-mediated recycling efficiency is a key challenge in Fc variant design.

[0007] In summary, existing long-acting recombinant human coagulation factor IX formulations still have the following technical problems that urgently need to be solved: (1) The production of monomeric Fc fusion protein is accompanied by dimer impurities, making purification difficult: Although the monomeric FIX-Fc fusion protein is superior to the dimer in terms of pharmacokinetics, dimers are inevitably generated during the production process, resulting in complex purification processes, high costs, and low yields, which is not conducive to industrial production.

[0008] (2) The pharmacokinetic properties of the dimer Fc fusion protein are inferior to those of the monomer: Although the natural dimer FIX-Fc fusion protein is easier to purify by Protein A affinity chromatography and has better structural stability, its in vivo half-life and pharmacokinetic parameters are significantly worse than those of the monomer structure, which limits its long-term effect.

[0009] (3) Fc fusion leads to a significant decrease in the specific activity of FIX: Existing Fc fusion strategies generally lead to a significant decrease in the molar specific activity of FIX (e.g., Alprolix® is only about 50% of recombinant FIX), which affects the therapeutic effect per unit dose.

[0010] (4) Safety risks caused by non-cleavable linkers: Some existing Fc fusion proteins do not use cleavable linker peptides. After FIX activation, they remain fused with Fc, which may affect the native conformation and activity of FIXa, posing potential safety risks.

[0011] (5) The binding of wild-type Fc to SPA limits its application expansion: The high affinity binding of wild-type IgG1 Fc to SPA may not only be interfered with by SPA in anti-infective therapy, but also limit the application of Fc variants in the fields of antimicrobial antibodies, antimicrobial peptide fusions, etc.

[0012] Therefore, there is an urgent need in this field to develop a novel recombinant human coagulation factor IX-Fc fusion protein that is easy to produce and purify, has excellent pharmacokinetic performance and high specific activity, and at the same time takes into account safety and application expansion. Summary of the Invention

[0013] The purpose of this invention is to provide a recombinant human coagulation factor IX-Fc variant fusion protein and its uses.

[0014] The present invention provides a human IgG1 Fc variant fragment, the amino acid sequence of which is shown in SEQ ID NO.4.

[0015] The human IgG1 Fc variant fragment in this application contains four mutations: Y349C, S354C, T394C, and Y407C.

[0016] The present invention also provides the use of the aforementioned human IgG1 Fc variant fragment in the preparation of fusion proteins; preferably, the fusion protein is a recombinant human coagulation factor IX-Fc variant fusion protein.

[0017] The present invention also provides the use of the aforementioned human IgG1 Fc variant fragment in the preparation of fusion protein drugs, wherein the fusion protein drugs are fusion proteins comprising an antimicrobial antibody or antimicrobial peptide and the human IgG1 Fc variant fragment; preferably, the drugs are drugs for treating or preventing Staphylococcus aureus infection.

[0018] The present invention also provides a recombinant human coagulation factor IX-Fc variant fusion protein, which is composed of coagulation factor IX, a cleavable linker and the aforementioned human IgG1 Fc variant fragment; the cleavable linker is located between coagulation factor IX and the human IgG1 Fc variant fragment; the fusion protein forms a dimer molecule through disulfide bonds between the human IgG1 Fc variant fragments.

[0019] Furthermore, The coagulation factor IX is human coagulation factor IX; The amino acid sequence of the cleavable linker includes the intrinsic activation site of human coagulation factor IX: SVSQTSKLTRAETVFPDVD or SVSQTSKLTRAEAVFPDVD. Preferably, The amino acid sequence of the human coagulation factor IX is shown in SEQ ID NO. 5; The amino acid sequence of the cleavable linker is shown in SEQ ID NO.3; More preferably, the amino acid sequence of the recombinant human coagulation factor IX-Fc variant fusion protein is shown in SEQ ID NO.2.

[0020] The present invention also provides a gene fragment encoding the aforementioned recombinant human coagulation factor IX-Fc variant fusion protein.

[0021] The present invention also provides a recombinant plasmid comprising the aforementioned gene fragment.

[0022] The present invention also provides an expression system comprising the aforementioned gene fragment for expressing the aforementioned recombinant human coagulation factor IX-Fc variant fusion protein, wherein the expression system is a mammalian cell expression system, a prokaryotic expression system, a yeast expression system, a transgenic animal mammary gland bioreactor expression system, or a baculovirus-insect cell expression system; preferably, the expression system is Vero cells, CHO cells, or HEK293 cells.

[0023] The present invention also provides a method for preparing the aforementioned recombinant human coagulation factor IX-Fc variant fusion protein, comprising the following steps: (1) Transfect the aforementioned recombinant plasmid into host cells using cationic liposome transfection; (2) Culture the host cells to obtain a stable cell line; (3) The cell culture supernatant was obtained by culturing the cell line and then purified to obtain the recombinant human coagulation factor IX-Fc variant fusion protein.

[0024] The present invention also provides the use of the aforementioned recombinant human coagulation factor IX-Fc variant fusion protein, the aforementioned gene fragment, the aforementioned recombinant plasmid, or the aforementioned expression system in the preparation of a drug for treating hemophilia.

[0025] The cleavable linker described in this invention contains the natural activation sequence of human coagulation factor IX. As coagulation factor IX is activated, it exerts its biological function. Simultaneously, coagulation factor IX is cleaved from the human IgG1 Fc fragment, without affecting the coagulation function and normal metabolism of the activated coagulation factor IX, thus avoiding the risks of drug-induced thrombosis. Compared with the prior art, the present invention has the following beneficial effects: This invention provides a recombinant human coagulation factor IX-Fc variant fusion protein. Compared with the monomer or wild-type dimer, this fusion protein exhibits significantly improved affinity for FcRn and longer-acting factor IX molar activity. Simultaneously, it overcomes the pharmacokinetic inferiority of the dimer Fc fusion protein compared to the monomeric Fc fusion protein, effectively increasing the half-life and stability of recombinant human coagulation factor IX. Therefore, the recombinant human coagulation factor IX-Fc variant fusion protein of this invention has promising application prospects.

[0026] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0027] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0028] Figure 1 The FIX (%) concentration-time curves in the plasma of SD rats in each group are shown (n=6, Mean ± SD).

[0029] Figure 2 The figure shows the stability results of each sample in a room temperature solution. The initial FIX activity on day 0 is defined as 100%. Detailed Implementation

[0030] Unless otherwise specified, the raw materials and equipment used in the specific embodiments of the present invention are all known products and were obtained by purchasing commercially available products.

[0031] Example 1: Stable cell line of recombinant human coagulation factor IX-Fc / Fc fusion protein monomer CHO (DG44) A stable cell line of CHO (DG44) recombinant human coagulation factor IX-Fc / Fc fusion protein monomer was constructed according to the method disclosed in patent application number 201810259116.5, entitled "A Feed-Based Fermentation Method for Recombinant Human Coagulation Factor IX Active Molecule". The specific method is as follows: 1. Materials, plasmids, and strains The eukaryotic expression plasmid pRH was constructed by Chengdu Rongsheng Pharmaceutical Co., Ltd.; the dihydrofolate reductase (DHFR) deficient Chinese hamster ovary cell line (CHO-DG44) was purchased from Invitrogen; and Escherichia coli TOP10 was preserved by Chengdu Rongsheng Pharmaceutical Co., Ltd.

[0032] 1.2 Main Reagents Lipofectamine 2000 cationic liposome transfection reagent was purchased from Invitrogen; PERMAb serum-free medium was purchased from HyClone; fetal bovine serum (FBS) was purchased from PAA; restriction endonucleases and various modifying enzymes were purchased from TaKaRa; 1 kb DNA marker was purchased from NEB; protein marker, APS, TEMED, and polyacrylamide concentrate were purchased from Bio-Rad; methotrexate (MTX), hypoxanthine, and thymidine (HT) were purchased from Sigma; DNA fragment gel extraction kit and plasmid extraction kit were purchased from QIAGEN.

[0033] 1.3 Cell resuscitation The CHO-DG44 cell line was revived from liquid nitrogen in 30 mL of serum-free PERMAb medium and cultured in suspension at 37 °C, 5% CO2, and 100 rpm in a shaker to prepare for cell transfection.

[0034] 1.4 Construction of recombinant plasmids The human FIX-Fc gene and Fc fragment gene were synthesized by Suzhou Genewise Biotech Co., Ltd. The human FIX-Fc fusion gene and Fc fragment gene were cloned into the eukaryotic expression plasmid pRH using the BssHII / XbaI restriction enzyme sites. Expression was controlled by the CMV promoter, and the expression vector contained the DHFR gene as a selection marker gene. The correctness of the recombinant expression plasmid construction was verified by double enzyme digestion and sequencing analysis.

[0035] 1.5 CHO cell transfection The host cells used in the experiment were CHO-DG44 cells. Recombinant plasmids for transfection were prepared, and transfection was performed using cationic liposome transfection. One day before transfection, CHO-DG44 cells in logarithmic growth phase were cultured in 6-well plates in antibiotic-free PERMAb medium (2 mL / well), supplemented with 10% (v / v) FBS and HT. Transfection was performed when cell confluence reached 90%. 4 μg of the constructed recombinant expression plasmid and 10 μL of cationic liposome transfection reagent (Lipofectamine 2000) were gently mixed in 200 μL of antibiotic-free medium and allowed to stand for 5 minutes. The plasmid and liposome transfection reagent mixture was then added to the cell wells, gently shaken, and incubated at 37 ℃ and 5% CO2 for 6 h. After 6 h, the medium was replaced with fresh PERMAb medium, supplemented with 10% FBS and HT, and cultured at 37 ℃ for another 24 h. Cells were digested with 0.25% trypsin, seeded into 96-well plates, and cultured at 37 °C using a HT-free selection culture medium. The culture medium was changed every 3 days until clones appeared. The activity of coagulation factor IX in the culture supernatant of each clone was measured.

[0036] 1.6 Screening and Expression of Engineered Cell Lines Positive cell clones were sequentially transferred from 96-well plates to 24-well plates, T25 square flasks, and shake flasks for stepwise selection and scale-up culture. The selected cell lines were subjected to stepwise pressurization with methotrexate (MTX), and several rounds of subclonal selection were performed using the limiting dilution method. The activity of human FIX in the supernatant was monitored during the selection process. The high-expression clones obtained in the final selection were identified by SDS-PAGE, confirming that the expression product was recombinant human coagulation factor IX-Fc fusion protein.

[0037] Using the methods described above, a stable cell line of recombinant human coagulation factor IX-Fc / Fc fusion protein monomer CHO (DG44) was successfully constructed.

[0038] Example 2: Construction of stable cell lines of recombinant human coagulation factor IX-Fc fusion protein dimer and recombinant human coagulation factor IX-Fc variant fusion protein dimer CHO (DG44) The following recombinant plasmid was constructed according to the method disclosed in the patent application number 201810259116.5, entitled "A Feed-Based Fermentation Method for Recombinant Human Coagulation Factor IX Active Molecules": 1. Contains recombinant human clotting factor IX-L-Fc Recombinant plasmid of fusion protein gene pRH-IX-L-Fc ; 2. Contains recombinant human clotting factor IX-L-Fc Recombinant plasmid of variant fusion protein gene pRH-IX-L- FcY349C / S354C / T394C / Y407C ; 3. Individuals with deleted transmembrane domainsFurin Recombinant plasmid of protease gene pRH-Furin .

[0039] All of the above genes were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0040] According to the method disclosed in the patent application number 201810259116.5, entitled "A Feed Fermentation Method for Recombinant Human Coagulation Factor IX Active Molecule", recombinant plasmids "1" and "3" and recombinant plasmids "2" and "3" are co-transfected into CHO (DG44) to obtain stable cell lines of recombinant human coagulation factor IX-Fc fusion protein and recombinant human coagulation factor IX-Fc variant fusion protein.

[0041] Among them, recombinant human coagulation factor IX-Fc Fusion protein is composed of pRH-IX-L-Fc The recombinant plasmid was integrated into the CHO (DG44) genome. CMV The amino acid sequence expressed under promoter control is shown in SEQ ID NO.1 below: MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFP CGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVS GWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLTSVSQTSKLTRAETVFPDVDGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Recombinant human coagulation factor IX-Fc Variant fusion protein is composed of pRH-IX-L-FcY349C / S354C / T394C / Y407C The recombinant plasmid was integrated into the CHO (DG44) genome. CMV The amino acid sequence expressed under promoter control is shown in SEQ ID NO.2 below: MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFP CGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVS GWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLTSVSQTSKLTRAETVFPDVDGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPCRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTCPPVLDSDGSFFLCSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK The linker amino acid sequence used by the above fusion protein between recombinant human coagulation factor IX and human IgG1 Fc fragment is shown in SEQ ID NO.3: SVSQTSKLTRAETVFPDVDGS Recombinant human coagulation factor IX-Fc The amino acid sequence of the human IgG1 Fc fragment in the variant fusion protein is shown in SEQ ID NO. 4: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPCRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTCPPVLDSDGSFFLCSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Recombinant human coagulation factor IX-Fc The amino acid sequence of recombinant human coagulation factor IX in the variant fusion protein is shown in SEQ ID NO. 5: MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSY ECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGG EDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIF LKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT People who delete transmembrane domains Furin The protease gene is integrated into the CHO (DG44) genome and expressed under the control of the CMV promoter. It is used for the precursor processing of recombinant human coagulation factor IX. The amino acid sequence is shown in SEQ ID NO.6 below: MELRPWLLWVVAATGTLVLLAADAQGQKVFTNTWAVRIPGGPAVANSVARKHGFLNLGQIFGDYYHFWHRGVTKRSLSPHRPRHSRLQREPQVQWLEQQVAKRRTKRDVYQEPTDPKFPQQWYLSGVTQRDLNVKAAWAQGYTG HGIVVSILDDGIEKNHPDLAGNYDPGASFDVNDQDPDPQPRYTQMNDNRHGTRCAGEVAAVANNGVCGVGVAYNARIGGVRMLDGEVTDAVEARSLGLNPNHIHIYSASWGPEDDGKTVDGPARLAEEAFFRGVSQGRGGLGSI FVWASGGNGREHDSCNCDGYTNSIYTLSISSATQFGNVPWYSEACSSTLATTYSSGNQNEKQIVTTDLRQKCTESHTGTSASAPLAAGIIALTLEANKNLTWRDMQHLVVQTSKPAHLNANDWATNGVGRKVSHSYGYGLLDAG AMVALAQNWTTVAPQRKCIIDILTEPKDIGKRLEVRKTVTACLGEPNHITRLEHAQARLTLSYNRRGDLAIHLVSPMGTRSTLLAARPHDYSADGFNDWAFMTTHSWDEDPSGEWVLEIENTSEANNYGTLTKFTLVLYGTAPEG Example 3: Expression of recombinant human coagulation factor IX-Fc / Fc fusion protein monomer, recombinant human coagulation factor IX-Fc fusion protein dimer, and recombinant human coagulation factor IX-Fc variant fusion protein dimer. AM02 culture medium, AF02, and AFS01 feed reagent were purchased from Shanghai Aopumai Biotechnology Co., Ltd.

[0042] 1. Cell resuscitation and passage: Recombinant human coagulation factor IX-Fc / Fc fusion protein monomer, recombinant human coagulation factor IX-Fc fusion protein dimer, and recombinant human coagulation factor IX-Fc variant fusion protein dimer cell lines were resuscitated from liquid nitrogen in 30 ml AM02 medium and cultured in suspension at 37°C and 110 rpm on a 5% CO2 shaker. The cells were passaged every 3 days.

[0043] 2. Fed-Batch culture: The cells were seeded at a density of 500,000 / ml into AM02 medium with an initial culture volume of 120 ml and 10 μg / ml VK1 and 125 nM MTX. The cells were cultured at 37.0℃ and 110 rpm on a 5% CO2 shaker.

[0044] 3. Fed culture: Starting from day 3, add feed culture medium according to the percentage of the initial culture volume in Table 1. On day 3, adjust the calcium ion concentration in the culture medium in the shake flask to 2mM and the magnesium ion concentration to 1.5mM. After day 6, cool down to 33.0℃ for culture.

[0045] Table 1. Feeding Strategy Example 4: Purification of recombinant human coagulation factor IX-Fc / Fc fusion protein monomer The cell culture supernatant containing the recombinant human coagulation factor IX-Fc / Fc fusion protein monomer obtained in Example 3 was replaced with a Cytiva Vivaspin 20 (30 kDa) ultrafiltration tube using a buffer system of (20 mM L-His, 20 mM CaCl2, 150-300 mM NaCl, 0.02% Tween-80). The supernatant was then clarified using a Sartorius Sartolab P20 plus (0.2 µm) filter and loaded into protein A affinity medium to capture the fusion protein. The supernatant was washed with 5 column volumes of equilibration buffer (20 mM L-His, 20 mM CaCl2, 150-300 mM NaCl, 0.02% Tween-80) and 10 column volumes of washing buffer (20 mM L-His, 20 mM CaCl2, 1000 mM NaCl, 0.02% Tween-80). Finally, the product was eluted with 0.1 M (pH 2.7-3.5) glycine-hydrochloric acid buffer, and then immediately neutralized with 1 M Tris (pH 9.0) to obtain the purified recombinant human coagulation factor IX-Fc / Fc fusion protein monomer.

[0046] Example 5: Purification of recombinant human coagulation factor IX-Fc fusion protein dimer and recombinant human coagulation factor IX-Fc variant fusion protein dimer The cell culture supernatants containing the recombinant human coagulation factor IX-Fc fusion protein dimer and the recombinant human coagulation factor IX-Fc variant fusion protein dimer obtained in Example 3 were exchanged using a Cytiva Vivaspin 20 (30 kDa) ultrafiltration tube with a buffer system of (20-50 mM Tris, 1-5 mM EDTA, 100-150 mM NaCl, pH 7.0-8.0). The supernatants were then clarified using a Sartorius Sartolab P20 plus (0.2 µm) filter and loaded onto anion exchange chromatography medium to capture the fusion protein. The cells were washed with 5 column volumes of washing buffer (20-50 mM Tris, 1-5 mM EDTA, 100-250 mM NaCl, pH 7.0-8.0). Finally, the protein was eluted with elution buffer (20-50 mM Tris, 5-50 mM CaCl2, 0-150 mM NaCl, pH 7.0-8.0) to obtain purified recombinant human coagulation factor IX-Fc fusion protein dimer and recombinant human coagulation factor IX-Fc variant fusion protein dimer.

[0047] The following specific experimental examples demonstrate the beneficial effects of the present invention.

[0048] Example 1: Affinity of recombinant human coagulation factor IX-Fc / Fc fusion protein monomer, recombinant human coagulation factor IX-Fc fusion protein dimer, and recombinant human coagulation factor IX-Fc variant fusion protein dimer to FcRn Following the methods in Examples 4 and 5, recombinant human coagulation factor IX-Fc / Fc fusion protein monomers, recombinant human coagulation factor IX-Fc fusion protein dimers, and recombinant human coagulation factor IX-Fc variant fusion protein dimers were obtained. The affinity of the purified recombinant human coagulation factor IX-Fc / Fc fusion protein monomers, recombinant human coagulation factor IX-Fc fusion protein dimers, and recombinant human coagulation factor IX-Fc variant fusion protein dimers for FcRn was detected using a Biacore T200 (Cytiva) instrument. Specifically, 0.5 μg / ml human FcRn protein (Sino Biologicals) was immobilized on a CM5 chip via amino-coupling at a coupling level of 211.5 RU. The above samples were sequentially injected into the chip channels at concentration gradients of 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.625 nM, and 7.8125 nM for binding, with a binding time of 120 s, immediately followed by injection of running buffer (pH 6.0). The flow cell was dissociated using 1×PBS-P for 360 s, and then regenerated using 1×PBS-P buffer at pH 7.4 for 40 s. Blank injections of flow cell and run buffer in each cycle were used as double deductions for resonance units. Data were processed using Biacore T200 evaluation software, and the results are shown in Table 2.

[0049] Table 2. Affinity test results with FcRn The affinity of recombinant human coagulation factor IX-Fc variant fusion protein dimer for FcRn is higher than that of recombinant human coagulation factor IX-Fc / Fc fusion protein monomer for FcRn, and is also higher than that of recombinant human coagulation factor IX-Fc fusion protein dimer for FcRn, being 2 times and 2.4 times higher, respectively.

[0050] Experimental Example 2: Affinity of Recombinant Human Coagulation Factor IX-Fc / Fc Fusion Protein Monomer, Recombinant Human Coagulation Factor IX-Fc Fusion Protein Dimer, and Recombinant Human Coagulation Factor IX-Fc Variant Fusion Protein Dimer with Protein A Following the methods in Examples 4 and 5, recombinant human coagulation factor IX-Fc / Fc fusion protein monomers, recombinant human coagulation factor IX-Fc fusion protein dimers, and recombinant human coagulation factor IX-Fc variant fusion protein dimers were obtained. The affinity of the purified recombinant human coagulation factor IX-Fc / Fc fusion protein monomers, recombinant human coagulation factor IX-Fc fusion protein dimers, and recombinant human coagulation factor IX-Fc variant fusion protein dimers for Protein A was detected by a Biacore T200 instrument, i.e., 1 μg / ml Protein A ligands were immobilized onto the CM5 chip via amino coupling at a coupling level of 231.4 RU. The sample was sequentially injected into the chip channels at concentration gradients of 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.625 nM, and 7.8125 nM for binding, with a binding time of 120 s. Dissociation was then performed by injecting running buffer (1×HBS-EP) for 360 s, followed by regeneration using glycine hydrochloride (pH 2.0) for 30 s. Blank injections of the flow cell and running buffer in each cycle were used for double deduction of resonance units. Data were processed using Biacore T200 evaluation software, and the results are shown in Table 3.

[0051] Table 3. Affinity test results with Protein A Note: N / A indicates that the recombinant human coagulation factor IX-Fc variant fusion protein dimer did not bind to the Protein A ligand and could not be fitted, therefore no specific value is given.

[0052] Both the monomeric and dimer Fc fragments of recombinant human coagulation factor IX-Fc / Fc fusion protein can bind normally to Protein A, while the variant dimer of recombinant human coagulation factor IX-Fc fusion protein loses its ability to bind to Protein A. This indicates that this product is not interfered with by staphylococcal protein A (SPA) in cases of Staphylococcus aureus and other infections. Furthermore, the Fc variant described in this invention has promising applications and value in the fields of antimicrobial antibodies and antimicrobial peptide fusions.

[0053] Experimental Example 3: Molar ratio of recombinant human coagulation factor IX-Fc / Fc fusion protein monomer, recombinant human coagulation factor IX-Fc fusion protein dimer, and recombinant human coagulation factor IX-Fc variant fusion protein dimer. The recombinant human coagulation factor IX-Fc / Fc fusion protein monomer molar ratio activity data were obtained from the literature "Prolonged activity of factor IX as a monomeric Fc fusion protein" (Robert T. Peters et al., Blood, 2010, 115(10): 2057-2064).

[0054] The recombinant human coagulation factor IX-Fc fusion protein dimer and the recombinant human coagulation factor IX-Fc variant fusion protein dimer were obtained by purification in Example 5.

[0055] Recombinant coagulation factor IX activity was determined using the BIOPHEN™ FIX(6) kit (HYPHEN BioMed). Protein concentration was determined using NanoDrop OneC (Thermo Scientific). The results are shown in Table 4.

[0056] Table 4. Molar ratios of different long-acting coagulation factors IX The molar specific activity of the recombinant human coagulation factor IX-Fc variant fusion protein dimer, after one-step chromatography purification, was 2.1 times that of the recombinant human coagulation factor IX-Fc fusion protein dimer. This was also significantly higher than the molar specific activity of the unpurified recombinant human coagulation factor IX-Fc / Fc fusion protein monomer and the monomer after three-step chromatography purification, being 2.4 times and 1.7 times that of the monomer before and after three-step chromatography, respectively. These results indicate that the recombinant human coagulation factor IX-Fc variant fusion protein dimer significantly enhanced the molar biological activity of recombinant human coagulation factor IX, achieving higher catalytic efficiency / specific activity while maintaining the dimer structure.

[0057] Experiment 4: Half-life of blood-derived coagulation factor IX, recombinant human coagulation factor IX-Fc / Fc fusion protein monomer, and recombinant human coagulation factor IX-Fc variant fusion protein dimer in SD rats Blood-derived coagulation factor IX was prepared by Chengdu Rongsheng Pharmaceutical Co., Ltd. Recombinant human coagulation factor IX-Fc / Fc fusion protein monomers and recombinant human coagulation factor IX-Fc variant fusion protein dimers were purified in Examples 4 and 5.

[0058] Eighteen 6-8 week old SD rats (half male and half female) were randomly divided into three groups of six each. Grouping and drug administration information are shown in Table 5.

[0059] Table 5. Grouping and Dosing Information Whole blood samples (≥100 μL / time point) were collected from all animals at 0.25 h, 4 h, 8 h, 24 h, 48 h, 72 h, 96 h, and 168 h after drug administration. Plasma was separated from the citrate-anticoagulated whole blood and frozen at ultra-low temperature (< -65℃) for coagulation factor IX activity assay (BIOPHEN™ FIX(6) kit, HYPHEN BioMed) and PK parameter calculation (Winnolin 8.5). The results are shown in Table 6.

[0060] Table 6. PK parameters of FIX (%) concentration in plasma of SD rats in each group (n=6, Mean ± SD) The concentration-time curves of FIX (%) in plasma of SD rats in each group are shown below. Figure 1 The recombinant human coagulation factor IX-Fc variant fusion protein dimer significantly increased the half-life of recombinant human coagulation factor IX in SD rats (43.13 hours and 11.04 hours, respectively), approximately 3.9 times that of the blood-derived coagulation factor IX. Furthermore, the recombinant human coagulation factor IX-Fc variant fusion protein dimer achieved a half-life comparable to that of the recombinant human coagulation factor IX-Fc / Fc fusion protein monomer, thus achieving an unexpectedly prolonged half-life while maintaining long-acting efficacy.

[0061] Experiment 5: Room Temperature Stability Study of Recombinant Human Coagulation Factor IX-Fc Variant Fusion Protein Dimer The stability of recombinant human coagulation factor IX-Fc / Fc fusion protein monomers, recombinant human coagulation factor IX-Fc fusion protein dimers, and recombinant human coagulation factor IX-Fc variant fusion protein dimers, as well as blood-derived coagulation factor IX (prepared by Chengdu Rongsheng Pharmaceutical Co., Ltd.), prepared in solutions at room temperature, was investigated. Purified samples were replaced with 1×PBS using Cytiva Vivaspin 20 (30kDa) ultrafiltration tubes. Blood-derived coagulation factor IX lyophilized powder was reconstituted with sterile water. The initial FIX activity of each group of samples was adjusted to 10-20 IU / mL, and the samples were stored at room temperature for 1, 2, 3, 7, 14, 21, and 28 days, with FIX activity measured at each time point. Each time samples were collected, equal aliquots were frozen at <-65 °C for subsequent analysis.

[0062] The results are as follows Figure 2As shown, with prolonged storage time in room temperature solution, the activities of recombinant human coagulation factor IX-Fc / Fc fusion protein monomers, recombinant human coagulation factor IX-Fc fusion protein dimers, and blood-derived coagulation factor IX all showed a decreasing trend, while the activity of recombinant human coagulation factor IX-Fc variant fusion protein dimers remained relatively stable throughout the storage period. Among them, blood-derived coagulation factor IX showed the fastest decline, with only 4.34% of its initial activity remaining by day 14. The recombinant human coagulation factor IX-Fc / Fc fusion protein monomer showed only 3.3% of its initial activity by day 21. By day 28, the residual activities of recombinant human coagulation factor IX-Fc / Fc fusion protein monomers, recombinant human coagulation factor IX-Fc fusion protein dimers, and recombinant human coagulation factor IX-Fc variant fusion protein dimers were 0.88%, 7.72%, and 93.82% of their initial activities, respectively.

[0063] The above results indicate that the recombinant human coagulation factor IX-Fc variant fusion protein dimer structure prepared in this invention has the potential to be developed into liquid formulations or lyophilized formulations that can be stored at room temperature. Furthermore, its excellent stability provides favorable conditions for subcutaneous administration. Currently, recombinant FIX products are all lyophilized formulations, requiring storage at 2–8 °C and administration via intravenous injection.

[0064] In summary, this invention provides a recombinant human coagulation factor IX-Fc variant fusion protein. Compared with the monomer or wild-type dimer, this fusion protein exhibits significantly improved affinity for FcRn and longer-acting factor IX molar activity. Simultaneously, it overcomes the pharmacokinetic inferiority of the dimer Fc fusion protein compared to the monomeric Fc fusion protein, effectively increasing the half-life and stability of recombinant human coagulation factor IX. Therefore, the recombinant human coagulation factor IX-Fc variant fusion protein of this invention has promising application prospects.

Claims

1. A human IgG1 Fc variant fragment, characterized in that: The amino acid sequence of the human IgG1 Fc variant fragment is shown in SEQ ID NO.

4.

2. Use of the human IgG1 Fc variant fragment of claim 1 in the preparation of a fusion protein; preferably, the fusion protein is a recombinant human coagulation factor IX-Fc variant fusion protein.

3. The use of the human IgG1 Fc variant fragment of claim 1 in the preparation of a fusion protein drug, wherein the fusion protein drug is a fusion protein comprising an antimicrobial antibody or antimicrobial peptide and the human IgG1 Fc variant fragment; preferably, the drug is a drug for treating or preventing Staphylococcus aureus infection.

4. A recombinant human coagulation factor IX-Fc variant fusion protein, characterized in that: It is composed of coagulation factor IX, a cleavable linker, and the human IgG1 Fc variant fragment as described in claim 1; the cleavable linker is located between coagulation factor IX and the human IgG1 Fc variant fragment; the fusion protein forms a dimer molecule through disulfide bonds between the human IgG1 Fc variant fragments.

5. The recombinant human coagulation factor IX-Fc variant fusion protein according to claim 4, characterized in that: The coagulation factor IX is human coagulation factor IX; The amino acid sequence of the cleavable linker includes the intrinsic activation site of human coagulation factor IX: SVSQTSKLTRAETVFPDVD or SVSQTSKLTRAEAVFPDVD. Preferably, The amino acid sequence of the human coagulation factor IX is shown in SEQ ID NO. 5; The amino acid sequence of the cleavable linker is shown in SEQ ID NO.3; More preferably, the amino acid sequence of the recombinant human coagulation factor IX-Fc variant fusion protein is shown in SEQ ID NO.

2.

6. A gene fragment encoding the recombinant human coagulation factor IX-Fc variant fusion protein of claim 4 or 5.

7. A recombinant plasmid, characterized in that: The recombinant plasmid comprises the gene fragment as described in claim 6.

8. An expression system comprising the gene fragment of claim 6 and the recombinant human coagulation factor IX-Fc variant fusion protein of claim 4 or 5, wherein the expression system is a mammalian cell expression system, a prokaryotic expression system, a yeast expression system, a transgenic animal mammary gland bioreactor expression system, or a baculovirus-insect cell expression system; preferably, the expression system is Vero cells, CHO cells, or HEK293 cells.

9. A method for preparing the recombinant human coagulation factor IX-Fc variant fusion protein according to claim 4 or 5, characterized in that: Includes the following steps: (1) Transfect the recombinant plasmid of claim 7 into host cells by cationic liposome transfection; (2) Culture the host cells to obtain a stable cell line; (3) The cell culture supernatant was obtained by culturing the cell line and then purified to obtain the recombinant human coagulation factor IX-Fc variant fusion protein.

10. Use of the recombinant human coagulation factor IX-Fc variant fusion protein of claim 4 or 5, the gene fragment of claim 6, the recombinant plasmid of claim 7, or the expression system of claim 8 in the preparation of a medicament for treating hemophilia.

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