Bifunctional fusion protein as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to a bifunctional fusion protein as well as a preparation method and application thereof. The bifunctional fusion protein comprises (1) an antibody fragment capable of being specifically combined with a human FcRn receptor, and (2) at least one amino acid mutated IL-2 structural domain, and (3) a connecting peptide for connecting the antibody fragment and the IL-2 structural domain to form a continuously expressed fusion protein, wherein the amino acid sequence of the connecting peptide has a flexible peptide chain with 1-30 amino acid residues. Through unique molecular design, the bifunctional fusion protein solves the key problems of insufficient specificity, limited curative effect and the like during treatment of autoimmune diseases in an existing treatment method, can more accurately regulate and control immune response, reduces side effects of medicines in clinical use, and particularly shows good prospects in treatment of immune related diseases such as SLE (systemic lupus erythematosus) and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a bifunctional fusion protein and a preparation method and application thereof. BACKGROUND

[0002] Lupus nephritis (LN) is an immune complex nephritis caused by systemic lupus erythematosus (SLE) involving the kidney, which seriously affects the survival rate and quality of life of patients. The pathogenesis of lupus nephritis includes immune complex deposition, inflammatory cell recruitment and activation, and excessive activation of the complement system. IgG (immunoglobulin G) immune complex is one of the main pathogenic factors, which deposits in the glomerulus, leading to the recruitment of inflammatory cells and inducing inflammatory reactions. In SLE patients, anti-dsDNA antibodies form immune complexes with IgG and deposit in the glomerulus, inducing local inflammatory reactions. At the same time, after the combination of complement C1q and IgG immune complex, the complement system is activated to form a membrane attack complex, leading to damage to glomerular epithelial cells and renal tubular epithelial cells. Therefore, IgG plays a crucial role in the pathogenesis of lupus nephritis, and IgG is directly involved in the inflammatory response and tissue damage of the kidney, and the treatment effect of LN can be enhanced by inhibiting the production of IgG.

[0003] There are autoreactive B cells in the body of SLE patients, which cause damage to normal tissues by producing a large number of autoantibodies. The ASC subpopulation is considered to be the final product of pathogenic B cells, which has the ability to rapidly produce IgG antibodies. The ASC subpopulation includes memory B cells and plasma cells. The rapid proliferation and diversification of the ASC subpopulation make them the main source of IgG antibodies. The ASC subpopulation gradually differentiates into long-lived plasma cells (LLPCs), leading to long-term antibody secretion, especially anti-nuclear antibodies (ANA) and anti-dsDNA antibodies. The autoantibodies present in SLE patients are mainly released by long-lived plasma cells, and these IgG antibodies can activate the complement system by forming immune complexes with antigens, triggering systemic inflammatory reactions.

[0004] In the prior art, the treatment of autoimmune diseases (such as systemic lupus erythematosus) mainly adopts the following methods: 1. Traditional immunosuppressants: such as glucocorticoids, cyclophosphamide, mycophenolate mofetil, etc. These drugs exert their effects by inhibiting the function of the immune system in a broad spectrum, but are prone to cause serious adverse reactions, such as increased risk of infection, osteoporosis, metabolic disorders, cardiovascular complications, etc.; and the therapeutic window is narrow, and the balance between efficacy and toxicity is difficult to control, and the disease is prone to relapse after stopping the drug, requiring long-term maintenance therapy.

[0005] 2. B cell targeting therapy: Anti-CD20 monoclonal antibodies such as Rituximab, mainly target CD20 positive B cells, but have limited effect on differentiated plasma cells (which do not express CD20) and are almost ineffective on long-lived plasma cells, leading to overall B cell depletion, which can affect normal anti-infective immune function, and the therapeutic effect has a lag, usually requiring several months to observe a significant decrease in autoantibody levels.

[0006] 3. Conventional IL-2 (interleukin-2) therapy: Natural IL-2 has been used as an immunomodulatory factor for experimental treatment of certain autoimmune diseases, but due to its ability to activate both effector T cells and regulatory T cells, it has a dual effect in autoimmune disease treatment, which can exacerbate immune imbalance in some patients, causing infection or recurrence and exacerbation of certain diseases; IL2 can activate CD25 + B cells, leading to increased IgG release and further promoting humoral immune response; moreover, this therapy lacks targeting specificity, has a narrow therapeutic window and a short half-life, requires frequent administration, and has poor patient compliance.

[0007] 4. BAFF / APRIL inhibitors: such as Belimumab, which works by inhibiting B cell survival factors, but has limited effect on long-lived plasma cells, has a slow therapeutic response, and shows limited improvement in immune complex-mediated diseases, and some patients may develop drug resistance.

[0008] 5. Proteasome inhibitors: such as Bortezomib, which can target and kill plasma cells, but lacks specificity and is prone to serious adverse reactions, and the disease is prone to relapse after discontinuation.

[0009] 6. Conventional FcRn (neonatal Fc receptor) modulators: There are monoclonal antibodies against FcRn in the prior art (such as Efgartigimod), which promote IgG degradation by blocking the binding of FcRn to IgG. However, these drugs lack specificity in targeting plasma cells and cannot distinguish between pathogenic IgG and protective IgG, which can affect the body's defense against infection; and only promote the clearance of IgG, do not directly inhibit the production of antibodies by plasma cells, and cannot solve the problem of autoantibody production from the source, and require continuous drug use to maintain therapeutic effect, and the level of autoantibodies rises rapidly after drug discontinuation.

[0010] 7. Unmodified IL-2 fusion proteins: Some studies have explored the fusion of IL-2 with other proteins, but lack the ability to specifically target plasma cells, can simultaneously activate multiple immune cells, leading to an imbalance of immune response, the biological half-life of such IL-2 fusion proteins is poor, frequent administration is required, and there is insufficient data on in vivo stability and safety.

[0011] In summary, the prior art is mainly limited to a single mechanism of action, either inhibiting B cell / plasma cell production or promoting IgG clearance, and has common shortcomings such as lack of specificity, multiple adverse reactions, and poor durability of efficacy. SUMMARY

[0012] The technical problem to be solved by the present application is to provide a bifunctional fusion protein, a preparation method and application thereof, which can specifically target and inhibit the release of IgG by plasma cells and can be used for the treatment of systemic lupus erythematosus.

[0013] To solve the above technical problems, the technical scheme adopted by the present application is as follows: a bifunctional fusion protein, comprising: (1) an antibody fragment capable of specifically binding to human FcRn receptor, and (2) at least one amino acid mutated IL-2 domain; (3) a connecting peptide for connecting the antibody fragment and the IL-2 domain to form a continuous expression fusion protein, the amino acid sequence of the connecting peptide has a flexible peptide chain of 1-30 amino acid residues.

[0014] Another technical scheme adopted by the present application is a preparation method of the bifunctional fusion protein, comprising the following steps: S1: constructing an expression vector of a nucleic acid encoding the bifunctional fusion protein; S2: transfecting the expression vector into mammalian cells; S3: sequentially performing expression, separation and purification to obtain the bifunctional fusion protein.

[0015] Still another technical scheme adopted by the present application is the use of the bifunctional fusion protein in the preparation of a drug, wherein the drug comprises the bifunctional fusion protein and a pharmaceutically acceptable carrier or excipient.

[0016] The beneficial effects of the present application are that the bifunctional fusion protein of the present application plays a role through at least one of the following matrices: (a) binding to the FcRn receptor to reduce the recycling of IgG through lysosomes, promote IgG degradation; (b) activating Treg cells to enhance peripheral tolerance; (c) inhibiting the survival and antibody secretion of plasma cells; (d) reducing the production of age-associated B cells (ABCs). Through the unique molecular design of the bifunctional fusion protein, the key problems such as insufficient specificity and limited efficacy in the prior art in the treatment of autoimmune diseases are solved, the immune response can be more accurately regulated, the side effects of the drug in clinical use are reduced, and especially in the treatment of immune-related diseases such as SLE, good prospects are shown. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 It is a molecular weight electrophoresis analysis chart of the bifunctional fusion protein in the embodiment of the present application; Figure 2 It is a detection chart generated after the bifunctional fusion protein in the embodiment of the present application is analyzed by a high performance liquid chromatograph (HPLC); Figure 3 It is an HE staining chart of the kidney pathology of MRL / Lpr mice treated with IL-2 wild type (WT-IL-2), IL-2 mutant (mIL-2), Anti-FcRn, different concentrations of Anti-FcRn-IL-2 bifunctional fusion protein (Anti-FcRn-IL-2) in the embodiment of the present application; Figure 4 It is a histological score of the lesion degree of glomerulus, renal interstitium and renal blood vessels of MRL / Lpr mice treated with WT-IL-2, mIL-2, Anti-FcRn, different concentrations of Anti-FcRn-IL-2 in the embodiment of the present application; Figure 5 It is the change of IgG level in the plasma of MRL / Lpr mice treated with WT-IL-2, mIL-2, Anti-FcRn, different concentrations of Anti-FcRn-IL-2 in the embodiment of the present application; Figure 6 It is the change of anti-dsDNA antibody level in the plasma of MRL / Lpr mice treated with WT-IL-2, mIL-2, Anti-FcRn, different concentrations of Anti-FcRn-IL-2 in the embodiment of the present application; Figure 7The stability of WT-IL-2, mIL-2 and Anti-FcRn-IL-2 in healthy human plasma in in vitro experiments in this embodiment of the invention; Figure 8 This invention relates to the stability of WT-IL-2, mIL-2, and Anti-FcRn-IL-2 in patient plasma during in vitro experiments in this embodiment of the invention. Figure 9 This is a schematic diagram of the protein thermal drift experiment in an embodiment of the present invention; Figure 10 These are melting temperature curves of WT-IL-2, mIL-2, Anti-FcRn, and Anti-FcRn-IL-2 at different pH values ​​in embodiments of the present invention. Figure 11 These are the melting peak curves of WT-IL-2, mIL-2, Anti-FcRn, and Anti-FcRn-IL-2 at pH=7 in the embodiments of the present invention; Figure 12 This illustrates the changes in plasma IL-2 concentration in C57BL / 6 mice after subcutaneous injection of WT-IL-2, mIL-2, and Anti-FcRn-IL-2 in an embodiment of the present invention.

[0018] Figure 13 This illustrates the changes in spleen follicular helper T cells (Tfh) in MRL / Lpr mice after treatment with different concentrations of Anti-FcRn-IL-2 in this embodiment of the invention. Figure 14 This invention relates to CD19 in MRL / Lpr mice after treatment with different concentrations of Anti-FcRn-IL-2 in embodiments of the present invention. + IgG + Changes in cells; Figure 15 The proportion of p-Stat5 in conventional T cells (Tcon) of C57B6L mice after treatment with different concentrations of WT-IL-2, mIL-2, Anti-FcRn, and Anti-FcRn-IL-2 in this embodiment of the invention. Detailed Implementation

[0019] To explain in detail the technical content, objectives, and effects of the present invention, the following description is provided in conjunction with the embodiments and accompanying drawings.

[0020] A bifunctional fusion protein comprising: (1) an antibody fragment that specifically binds to a human FcRn receptor; and (2) an IL-2 domain with at least one amino acid mutation; and (3) a linker peptide for linking the antibody fragment to the IL-2 domain to form a continuously expressed fusion protein, the linker peptide having a flexible peptide chain having an amino acid sequence of 1 to 30 amino acid residues.

[0021] From the above description, the beneficial effects of the present application are as follows: the prior art is mainly limited to a single mechanism, lacking a treatment scheme with both inhibiting B cell / plasma cell generation and promoting IgG clearance, and lacking the ability to specifically target plasma cells. The present application provides a grafted FcRn monoclonal antibody and an IL-2 fusion protein with targeted inhibition of plasma cell IgG release, which is composed of three functional modules: 1) a monoclonal antibody part against FcRn receptor, which can block the interaction between FcRn and IgG, promote the clearance of autoantibodies; 2) a modified IL-2 domain, which has the effect of selectively regulating immune function and stronger IgG inhibition ability; 3) a fusion module of FcRn coupled IL-2, which enhances the inhibition of plasma cells. The present application has the dual immunoregulatory effects of clearing circulating IgG and inhibiting plasma cells, and has the following technical advantages: 1. Dual mechanism: by blocking the interaction between FcRn and IgG to promote the rapid clearance of autoantibodies, and directly inhibiting the production of new autoantibodies by the IL-2 domain, the dual therapeutic effect of "clearing existing antibodies" and "inhibiting the production of new antibodies" is achieved, which synergistically acts from the source and clearance.

[0022] 2. Safety of protein drug therapy: the FcRn-IL-2 fusion protein of the present application can specifically target plasma cells, selectively inhibit conventional T cells and the expression of various inflammatory factors, thereby reducing immunotoxicity and preventing tissue damage; by balancing the activity of T cells, B cells and plasma cells, precise immune regulation is achieved, which significantly reduces the systemic immunosuppressive effect compared to traditional immunosuppressants, and reduces the risk of infection and other systemic adverse reactions. 3. Synergistic regulation of multiple immune subpopulations: the fusion protein of the present application exhibits balanced regulation of multiple immune cell subpopulations, including inhibition of T cell exhaustion, maintenance of Treg activation characteristics, inhibition of B cell subpopulation activation, and effective inhibition of plasma cell subpopulation, achieving comprehensive and precise immune regulation.

[0023] 4. Specific protection of the kidney: the fusion protein significantly enhances the protective effect on lupus nephritis, and preclinical studies have confirmed that it can effectively inhibit the deposition of IgG and C3 in kidney tissue, significantly improve the kidney pathological score, and provide a more effective treatment option for patients with lupus nephritis.

[0024] 5. Synergistic effect: the innovative combination of FcRn monoclonal antibody part and IL-2 functional domain produces a synergistic effect, specifically enhancing the inhibition of plasma cells, while improving the efficiency of autoantibody clearance, significantly enhancing the therapeutic effect on autoimmune diseases, and showing more significant efficacy in animal models than using FcRn inhibitors or IL-2 treatment alone.

[0025] 6. Stability enhancement: The optimized fusion protein structure significantly improves the stability of the molecule, increases the drug concentration in the blood in vivo, reduces the frequency of administration, and improves patient compliance and quality of life.

[0026] 7. Precise immune regulation: The specially modified IL-2 structure can selectively regulate immune response, inhibit pathological immune activity while maintaining the body's defense against infection, and achieve balanced regulation of the immune system.

[0027] 8. Broadening of therapeutic window: The optimized structure increases the safety interval between effective dose and toxic dose of the drug, expanding the therapeutic window for clinical application. 9. Immune function balance maintenance: The modified IL-2 structure can selectively regulate immune response, inhibit pathological immune activity while maintaining normal immune defense function, reducing the risk of infection in conventional immunosuppressive therapy. 10. Effective inhibition of long-lived plasma cells: In vitro and in vivo, it shows inhibition of plasma cell survival and effectively reduces the secretion of IgG class autoantibodies; overcomes the limitations of existing B cell targeted therapies that cannot effectively eliminate long-lived plasma cells, and can specifically intervene in the main source of continuous production of autoantibodies. 11. Enhanced durability of disease control: Preclinical studies show that the fusion protein can achieve more durable disease activity control, reduce disease recurrence, and improve long-term prognosis. 12. Simplification of production process: Compared with producing two protein drugs separately, the design of the fusion protein simplifies the production process, reduces production costs, and improves drug accessibility. 13. Compared with natural IL-2 or IL-2-Fc fusion protein, it has longer in vivo half-life, stronger plasma stability and higher selective immune regulation ability.

[0028] Through the above innovative mechanisms, the technical solution of the present application successfully overcomes various major shortcomings in the prior art, providing a new choice for the treatment of autoimmune diseases, especially systemic lupus erythematosus.

[0029] Unlike the prior art, WO2023217288 discloses a new fusion protein comprising an IL-2 part and an FC part, which is significantly different from the present application in terms of the synthetic composition of the fusion protein, the mechanism of prolonging the half-life of the drug, the disease spectrum targeted, and the different donor-recipient relationship between the Fc region variant and FcRn. Chinese invention patent CN105814080A discloses a protein combining an anti-FcRn antibody with an IL-2 effector molecule, which is significantly different from the present application in terms of the design focus of the protein, the need for specific improvement of the anti-FcRn antibody, and the lack of modification of the IL-2 effector molecule.

[0030] Further, the IL-2 domain comprises at least one amino acid mutation: D20A, D20E, T51S, N88R, F42A, R38A, Y45A, N103R, D109A, Q74R, E62A, K48A, C125S, S8P and I5T.

[0031] Further, the IL-2 domain retains the binding ability to CD25 after mutation.

[0032] As can be seen from the above description, Treg cells express CD25, CD122 and CD132 on the surface, which constitute a complete IL-2 receptor complex. Tcon only expresses CD122 and CD132; although B cells express CD25, they rarely express CD122 and CD132 on the surface. Treg cells can inhibit immune activation by regulating the immune system, thereby preventing excessive inflammatory response, while the activation of Tcon can trigger stronger inflammatory and immunotoxic response. Therefore, the present application designs and develops a biased IL-2 that reduces the affinity to CD122 and CD132 while retaining the activation characteristics of CD25, thereby reducing the activation of Tcon while ensuring the activation stability of Treg; the modified IL-2 domain can selectively regulate immune response, reducing the risk of infection in conventional immunosuppressive therapy. The IL-2 domain is obtained by genetic engineering methods such as point mutation, combinatorial mutation, and regional saturation mutation, and can be further fused with FcRn antibody or Fc segment to construct an expressible fusion protein form.

[0033] The functions achieved by mutation include: (1) reducing the affinity to IL-2Rβ chain (CD122) and / or γ chain (CD132), thereby reducing the activation effect on Tcon or NK cells; (2) retaining the binding ability to IL-2Rα chain (CD25), which has less effect on the activation of Treg cells; (3) improve the solubility, expression amount, expression efficiency, conformational stability and in vivo half-life of IL-2 protein; (4) optimize signal flux, reduce Tcon selective phosphorylation STAT5 signaling, and reduce the activation effect on Tcon cells by adjusting the intensity of IL-2 signaling; (5) weaken the misactivation potential of interleukin-2 receptor-expressing incomplete B cells or Teff cells (effector T cells).

[0034] Further, the antibody fragment is a single-chain variable fragment that specifically binds to human FcRn or a single-domain antibody that specifically binds to human FcRn.

[0035] Further, the antibody fragment is of murine origin, humanized or synthetic origin.

[0036] Further, the bifunctional fusion protein is expressed by a mammalian cell expression system, the coding nucleic acid of the fusion protein comprises a promoter and regulatory element required for mammalian expression, and the functional protein can be obtained by purification.

[0037] Another technical solution adopted by the present application is: the preparation method of the bifunctional fusion protein described above, comprising the following steps: S1: constructing an expression vector of the coding nucleic acid of the bifunctional fusion protein; S2: transfecting the expression vector into mammalian cells; S3: obtaining the bifunctional fusion protein after sequentially performing expression, separation and purification.

[0038] Further, the expression vector is suitable for expressing the fusion protein in CHO cells or HEK293 cells.

[0039] Still another technical solution adopted by the present application is: the application of the bifunctional fusion protein described above in the preparation of a drug, wherein the drug comprises the bifunctional fusion protein and a pharmaceutically acceptable carrier or excipient.

[0040] Further, the drug is an injection, a freeze-dried powder injection or a pre-filled injection.

[0041] Further, the drug is used for treating autoimmune diseases, immunoglobulin-mediated diseases, antibody-related allergic reactions, vaccine adjuvant immunomodulation, chronic inflammatory diseases and for inducing immune tolerance for transplantation.

[0042] From the above description, the anti-FcRn-IL-2 bifunctional fusion protein of the application grafts the biased IL-2 and the FcRn monoclonal antibody together, effectively avoids the short half-life of traditional IL-2 and the disadvantage that it may affect other immune cells, reduces the level of IgG in the blood by reducing pathogenic IgG recycling through lysosomes through the FcRn antibody. It can reduce the frequency of drug administration of patients and increase the drug compliance of patients. Through the optimized biased IL-2, it targets the change of IL-2 receptor subunit, which can increase the safety and effectiveness of IL-2, and may have better efficacy for patients with more combined underlying diseases or combined with other autoimmune diseases.

[0043] Further, the drug is used for treating systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, myasthenia gravis, autoimmune thrombocytopenia, IgG4-related diseases, autoimmune hepatitis, inflammatory dermatomyositis, Sjogren's syndrome and glomerular immune complex deposition diseases.

[0044] From the above description, the anti-FcRn-IL-2 bifunctional fusion protein of the application can be applied to systemic lupus erythematosus nephritis, treatment of rheumatoid arthritis, myasthenia gravis, autoimmune thrombocytopenia and other autoimmune antibody-mediated diseases, and has a broad clinical application prospect.

[0045] The anti-FcRn-IL-2 bifunctional fusion protein significantly enhances the protective effect on lupus nephritis, can effectively inhibit the deposition of IgG and C3 in the kidney tissue, and significantly improves the kidney pathological score, thereby providing a more effective treatment option for patients with lupus nephritis.

[0046] Embodiment 1 of the application is a preparation method of the bifunctional fusion protein, and the steps are as follows: 1. Plasmid construction 1.1 Primer design: design Anti-FcRn scFv and IL-2 target fragment primers, the IL-2 target fragment primer contains a D20A mutation, which retains the binding ability with CD25 after mutation; a homologous recombination region sequence is added at the 5' end of the primer, which is used for recombination with the pcDNA3.4 vector.

[0047] 1.2 Target gene PCR amplification: according to the primer design, the Anti-FcRn-IL-2 fusion gene is amplified.

[0048] PCR system (25 μL): PrimeSTAR Max DNA Polymerase 12.5 μL, ddH2O 9.5 μL, forward primer (10 μM) 1 μL, reverse primer (10 μM) 1 μL, template DNA 1 μL; PCR procedure: 98℃ 5min→(98℃ 20s→60℃ 15s→72℃ 30s)×30→72℃ 5min→4℃ keep.

[0049] 1.3 Linearization of vector: pcDNA3.4 vector was digested by Fast Digest EcoRV.

[0050] Digest system (200μL): pcDNA3.4 plasmid 20μg, 10×Fast Digest Buffer 20μL, FastDigest EcoRV: 10μL, ddH2O to 200μL; 37℃ for 30min.

[0051] 1.4 DNA fragment recovery: PCR products and digested vectors were subjected to agarose gel electrophoresis and gel recovery purification 1.5 Homologous recombination and transformation: Hieff Clone Plus One Step Cloning Kit was used.

[0052] Reaction system (6μL): linearized vector 20~50 ng, Anti-FcRn-IL-2 insert at a molar ratio of 1:3, 2×Hieff Clone Enzyme Premix 3μL, ddH2O to 6μL; 37℃ for 30min, and then transformed into DH5α competent cells. 1.6 Transformation and positive clone identification: single colonies were picked for PCR verification and sequencing alignment to confirm the correct sequence of Anti-FcRn-IL-2 fusion gene.

[0053] 2, Protein expression 2.1 Cell preparation: ExpiCHO cells were cultured to (3~4)×10 6 cells / mL the day before transfection.

[0054] 2.2 Transfection: the cell density was adjusted to 6×10 6 cells / mL on the day of transfection, with a viability of >98%; Anti-FcRn-IL-2 expression plasmid DNA and ExpiFectamine CHO were diluted with OptiPRO SFM; after dilution, incubate at room temperature for 4 minutes, add cell suspension, and cultivate at 37℃ with 8% CO 2 .

[0055] 2.3 Feed: add ExpiCHO Enhancer and Feed 20h after transfection, and cultivate at 32℃ with 5% CO 2 ; feed again on the fifth day, and continue to cultivate until the cell viability is 70%, then harvest the supernatant.

[0056] 3. Protein purification 3.1 System preparation: The column was packed with 2 mL Protein A packing material and washed with DI water and CIP buffer.

[0057] 3.2 Equilibration: Equilibrated with 15 CV Binding Buffer, flow rate controlled at 4 min retention time.

[0058] 3.3 Loading: The cell supernatant was centrifuged at 13,000 x g for 20 min and filtered with 0.22 μm before loading into the Protein A column.

[0059] 3.4 Washing: Washed with 10 CV, 15 CV and 20 CV Wash Buffer I / II in sequence.

[0060] 3.5 Elution: Eluted the Anti-FcRn-IL-2 fusion protein with 15 CV Elution Buffer, and collected 5 mL per tube.

[0061] 3.6 End: The concentration was detected by micro-spectrophotometer, and the sample was concentrated by ultrafiltration and then aliquoted and stored in the freezer.

[0062] Example 2 of the present application is: structure verification of the bifunctional fusion protein in Example 1 1. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel) electrophoresis analysis of molecular weight: the molecular weight of the target protein was determined by comparison with standard molecular weight marker (marker), to verify whether the fusion protein was expressed in the expected size, to detect whether there were degradation products or aggregates, to evaluate the integrity and uniformity of the sample, and the analysis results are shown in Table 1 and Figure 1 From Table 1 and Figure 1 It can be seen that the fusion protein prepared in Example 1 is expressed in the expected size.

[0063] Table 1

[0064] 2. HPLC verification of purity: detection of main peak purity (usually required ≥ 95%), types and contents of related impurities, and proportion of aggregates and degradation products, to ensure that the chemical purity meets the standard, and the detection results are shown in Table 2 and Figure 2 From Table 2 and Figure 2 It can be seen that the fusion protein prepared in Example 1 has high purity.

[0065] Chromatographic column configuration: XBridge BEH200Å SEC chromatographic column, particle size 3.5 μm, size 7.8 x 300 mm, specific for molecular exclusion chromatography separation, suitable for analysis of protein molecular weight range.

[0066] Mobile phase system: 0.15M phosphate buffer + sodium chloride system, pH adjusted to 6.0, to provide a stable ionic strength environment to avoid non-specific interactions between proteins and stationary phases.

[0067] Detection parameters: The flow rate was set to 0.8 mL / min, the sample concentration was 0.5 mg / ml, the injection volume was 20 μL, and the ultraviolet detection wavelength was 280 nm for protein quantitative analysis. This combination of conditions ensures good separation and detection sensitivity, suitable for purity verification and aggregate analysis of bifunctional fusion proteins.

[0068] Table 2

[0069] Example three of the present application is: the effect verification of the bifunctional fusion protein in example 1.

[0070] 1. Lesion degree test: 1.1 Divide the MRL / Lpr mice into Control group, WT-IL-2 group, Anti-FcRn group, Anti-FcRn-IL-2 (0.5 mg / kg) group, Anti-FcRn-IL-2 (2 mg / kg) group, Anti-FcRn-IL-2 (4 mg / kg) group (n=5~6 per group), subcutaneously inject the drugs every 3 days until the 18th day, and collect the kidneys of the mice on the 30th day for paraffin section, HE pathological staining, and the results are shown in Figure 3 , Figure 4 The lesion degree histological score of glomerulus, renal interstitium and renal blood vessels, the score adopts semi-quantitative grading of 1-4 grades, and the specific score is shown in Table 3. From Figure 3 and Figure 4 It can be seen that compared with the Control group, WT-IL-2 group and Anti-FcRn group, the pathological damage of the kidneys of the Anti-FcRn-IL-2 treatment group is obviously reduced, which is manifested as the reduction of inflammatory cell infiltration around the glomerulus, renal interstitium and around the blood vessels, and with the increase of the concentration, the improvement of the kidney pathology is more obvious.

[0071] Table 3

[0072] 1.2 Collect the peripheral blood of MRL / Lpr mice (Control group, WT-IL-2 group, Anti-FcRn group, different concentrations of Anti-FcRn-IL-2 group) by the way of internal canthus vein on the 0th day, 6th day, 12th day, 18th day and 30th day, and detect the concentration change of IgG and the absorbance (OD value) change of dsDNA in the plasma by the method of Elisa, and the results are shown in Figure 5 andFigure 6 As shown in Figure 5 and Figure 6 It can be seen that Anti-FcRn-IL-2 can effectively reduce the levels of IgG and dsDNA in the peripheral blood of MRL / Lpr mice, and the more the concentration increases, the more obvious the reduction of Anti-FcRn-IL-2 on IgG and dsDNA.

[0073] 1.3 Add WT-IL-2, mIL-2 and Anti-FcRn-IL-2 (all at a concentration of 100 ng / ml) to the plasma of healthy people, incubate in a cell incubator for 24 hours, and detect the IL-2 levels in the plasma at different time points by Elisa method. Divide the IL-2 levels measured at each time point by the baseline IL-2 level to calculate the percentage decrease of IL-2, and the results are shown in Figure 7 The plasma stability of the drug in patients was detected, and the results are shown in Figure 8 As shown in Figure 7 and Figure 8 Compared with WT-IL-2, the percentage decrease of IL-2 levels in mIL-2 and Anti-FcRn-IL-2 groups is smaller, indicating that the plasma stability of the two is stronger.

[0074] 2. Protein thermal shift experiment, the experimental steps are as follows: S1: Resuspend WT-IL-2, mIL-2, Anti-FcRn and Anti-FcRn-IL-2 in deionized water to a concentration of 1 mg / ml to obtain a protein solution; S2: Add 2.5ul of 5000X SYPRO concentrate to 250ul of deionized water to prepare a 50X SYPRO working solution; S3: Add 10ul of protein solution to each well of the 96-well plate.

[0075] S4: Add 2.5ul of 50X SYPRO working solution to each well of the 96-well plate.

[0076] S5: Construct a melt curve with a pH value interval of 3.5~9.0 at an interval of 0.5, and distribute 12.5ul of each pH value buffer to the corresponding duplicate wells of the 96-well plate, so that the final concentration of SYPRO in each well is 5x.

[0077] S6: Repeat steps S1~S5 to create a buffer and salt concentration range for each different pH buffer.

[0078] The protein thermal shift experiment formula is shown in Table 4, and the program settings are as follows: use a full-automatic medical PCR analyzer (model SLAN-96S) to set the program, and the specific program is shown in Figure 9Melting curve: range from 10.0 to 95.0 °C, increment of 0.5 °C, duration of 10 seconds and collection of fluorescence signal. Sample volume was 25 ul. Channel selection: HEX channel.

[0079] Table 4

[0080] The stability of WT-IL-2, mIL-2, Anti-FcRn, Anti-FcRn-IL-2 was observed between pH 3.5 to 9.0. The melting temperature (Tm) of the drugs at different pH values was recorded, and the results are shown in Figure 10 . It can be seen from Figure 10 that compared with WT-IL-2, mIL-2, Anti-FcRn, Anti-FcRn-IL-2 has a higher melting temperature and better protein stability. The melting peak values of the above four drugs as a function of temperature at pH = 7.0 were recorded, and the melting peak value curve was plotted using Graphpad Prism 10.0, and the results are shown in Figure 11 . It can be seen from Figure 11 that at pH = 7, mIL-2, Anti-FcRn, Anti-FcRn-IL-2 have a higher Tm than WT-IL-2, and their stability is better.

[0081] 3. WT-IL-2, mIL-2 and Anti-FcRn-IL-2 were subcutaneously injected into C57BL / 6 mice at a concentration of 1 mg / kg (n = 4 in each group), and the peripheral blood of mice at different time points (0 h, 2 h, 5 h, 8 h, 12 h, 24 h, 48 h, 72 h, 96 h) was collected by the method of collecting blood from the inner canthus. The IL-2 level in the peripheral blood at different time points was detected by ELISA, and the results are shown in Figure 12 . It can be seen from Figure 12 that the peak time and peak concentration of Anti-FcRn-IL-2 are higher than those of WT-IL-2 and mIL-2, and its absorption is better.

[0082] 4. MRL / Lpr mice were subcutaneously injected with different concentrations of Anti-FcRn-IL-2, and the specific implementation method is shown in the disease degree test. After 1 month, the spleen of MRL / Lpr mice was collected, a single cell suspension of the spleen was prepared, and the proportion changes of Tfh, CD19 + IgG + cells in the spleen of mice were detected by flow cytometry, and the results are shown in Figure 13 and Figure 14 . It can be seen from Figure 13 that when the concentration of Anti-FcRn-IL-2 is 4 mg / kg, it can effectively reduce the proportion of Tfh in the spleen. It can be seen from Figure 14It was found that three different concentrations of Anti-FcRn-IL-2 could reduce CD19 levels in the spleen of MRL / Lpr mice. + IgG + The proportion of cells.

[0083] 5. Different concentrations of WT-IL-2, mIL-2, Anti-FcRn, and Anti-FcRn-IL-2 (0.0001 nM, 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1000 nM) were added to single-cell suspensions of spleen cells from wild-type C57BL / 6 mice. After culturing in a cell culture incubator for 30 min, the proportion of p-STAT5 in Tcon cells was detected by flow cytometry. The results are shown in the figure below. Figure 15 .because Figure 15 It can be seen that the EC50 of Anti-FcRn-IL-2 is higher than that of WT-IL-2 and mIL-2, indicating that it is less likely to stimulate Tcon cells, and Anti-FcRnIL-2 has better selectivity.

[0084] In summary, the bifunctional fusion protein, its preparation method, and its applications provided by this invention significantly enhance the clearance effect of IgG compared to FcRn or IL-2 alone, suggesting a synergistic effect between CD25 and FcRn on IgG cell circulation. Furthermore, the construction of this novel fusion protein is expected to enhance its targeting effect on plasma cells in vivo, providing a more effective option for the treatment of autoimmune diseases. This invention has the following advantages: 1. Addressing the non-specificity of traditional immunosuppressants: The FcRn-IL-2 fusion protein of this invention achieves precise immune regulation by specifically targeting plasma cells, avoiding the severe adverse reactions caused by broad-spectrum suppression of the entire immune system. The FcRn monoclonal antibody only blocks specific pathways without affecting other immune functions, thereby significantly reducing the risk of infection.

[0085] 2. Overcoming the limitation of B-cell targeted therapy being ineffective against plasma cells: Existing B-cell targeted therapies (such as anti-CD20 monoclonal antibodies) cannot act on differentiated plasma cells, while the IL-2 domain of the fusion protein of this invention is specially modified to specifically target and inhibit plasma cell function, including long-lived plasma cells, thereby cutting off the continuous production of autoantibodies from the source.

[0086] 3. Overcoming the shortcomings of conventional IL-2 therapy in bidirectional regulation: Traditional IL-2 therapy has a dose-dependent bidirectional regulatory effect. This invention eliminates the activation effect on effector T cells by specially modifying the IL-2 domain, while retaining the inhibitory function on plasma cells, thus avoiding immune response imbalance.

[0087] 4. Solve the problem of limited effect of BAFF / APRIL inhibitors on long-lived plasma cells: Unlike BAFF / APRIL inhibitors that only act on B cell survival and differentiation, the fusion protein can directly act on already formed plasma cells, significantly shortening the treatment response time and improving the effect on immune complex-mediated disease manifestations.

[0088] 5. Avoid non-specific toxicity of proteasome inhibitors: Proteasome inhibitors have serious adverse reactions due to non-specific mechanisms. The fusion protein specifically targets plasma cells, significantly reducing the impact on other cell types, significantly improving safety, and allowing long-term medication.

[0089] 6. Improve the single mechanism of conventional FcRn modulators that only promote IgG clearance: Existing FcRn modulators only promote IgG clearance and do not directly inhibit antibody production by plasma cells. The present application achieves the dual effect of promoting pathological IgG clearance and inhibiting new antibody production through the FcRn-IL-2 fusion module, solving the problem of rapid rebound of autoantibody levels after drug withdrawal.

[0090] 7. Increased drug stability and enhanced effect: Unmodified IL-2 fusion proteins lack specificity in targeting plasma cells and have a short half-life. The present application enhances the stability of the molecule by optimizing the molecular structure, increases the blood concentration of the drug in the body, and increases the targeting of the kidneys, increasing the effect on lupus nephritis, reducing the frequency of drug administration, and improving patient compliance.

[0091] 8. Solve the limitations of single-action mechanism therapy: Existing therapies are often limited to single-action mechanisms, making it difficult to meet the treatment needs of complex autoimmune diseases. The fusion protein achieves a complete treatment strategy of "treating the symptoms" (clearing existing autoantibodies) and "treating the root cause" (inhibiting the production of new autoantibodies) through a synergistic mechanism of action, thereby achieving a more optimal therapeutic effect and more durable disease control.

[0092] The above only describes the embodiments of the present application and does not limit the patent scope of the present application. Any equivalent transformation or direct or indirect application in related technical fields using the content of the present application specification and drawings is also included in the patent protection scope of the present application.

Claims

1. A bifunctional fusion protein, characterized in that, The bifunctional fusion protein comprises: (1) an antibody fragment capable of specifically binding to a human FcRn receptor, and (2) an IL-2 domain with at least one amino acid mutation; and (3) a connecting peptide for connecting the antibody fragment and the IL-2 domain to form a continuously expressed fusion protein, wherein the amino acid sequence of the connecting peptide has a flexible peptide chain of 1-30 amino acid residues.

2. The bifunctional fusion protein of claim 1, wherein, The IL-2 domain comprises at least one amino acid mutation selected from the group consisting of D20A, D20E, T51S, N88R, F42A, R38A, Y45A, N103R, D109A, Q74R, E62A, K48A, C125S, S8P and I5T.

3. The bifunctional fusion protein of claim 2, wherein, The IL-2 domain retains the binding ability to CD25 after the mutation.

4. The bifunctional fusion protein of claim 1, wherein, The antibody fragment is a single-chain variable fragment capable of specifically binding to a human FcRn or a single-domain antibody capable of specifically binding to a human FcRn.

5. The bifunctional fusion protein of claim 1, wherein, The bifunctional fusion protein is expressed by a mammalian cell expression system, and the nucleic acid encoding the fusion protein comprises a promoter and regulatory elements required for mammalian expression, and the functional protein can be obtained by purification.

6. A method of producing a bifunctional fusion protein according to any one of claims 1 to 5, wherein The method comprises the following steps: S1: constructing an expression vector of a nucleic acid encoding the bifunctional fusion protein; S2: transfecting the expression vector into mammalian cells; S3: obtaining the bifunctional fusion protein after sequentially performing expression, separation and purification.

7. Use of the bifunctional fusion protein according to any one of claims 1 to 5 for the manufacture of a medicament, characterized in that, The drug comprises the bifunctional fusion protein and a pharmaceutically acceptable carrier or excipient.

8. Use according to claim 7, characterized in that, The drug is an injection, a lyophilized powder injection or a pre-filled injection.

9. Use according to claim 7, characterized in that, The drug is used for treating autoimmune diseases, immunoglobulin-mediated diseases, antibody-related allergic reactions, vaccine adjuvant immunomodulation, chronic inflammatory diseases and for inducing immune tolerance for transplantation.

10. Use according to claim 7, characterized in that, The drug is used for treating systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, myasthenia gravis, autoimmune thrombocytopenia, IgG4-related diseases, autoimmune hepatitis, inflammatory dermatomyositis, Sjogren's syndrome and glomerular immune complex deposition diseases.

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