Fc fusion protein of a growth factor and interferon and application thereof

Abstract

This invention discloses a growth factor and interferon Fc fusion protein and its applications. It belongs to the field of fusion protein technology. The fusion protein provided by this invention consists of two heavy-like chains linked by disulfide bonds; it sequentially comprises a keratinocyte growth factor functional region, a human immunoglobulin G1 Fc segment, and an interferon functional region, and its amino acid sequence is shown in SEQ ID NO.1. This fusion protein integrates mucosal epithelial repair function and local antiviral / immunomodulatory function into a single molecule, which can synergistically promote the re-epithelialization of damaged mucosa after radiotherapy and chemotherapy, while inducing epithelial cells to enter an antiviral state and rebuilding the local immune barrier. Compared with existing monotherapy or combination therapy, this invention has a dual synergistic effect, which can effectively block the vicious cycle of "damage-infection-inflammation-damage", and is suitable for preparing drugs to treat severe mucositis and related symptoms after radiotherapy and chemotherapy in cancer patients.

Description

Technical Field

[0001] This invention relates to the field of fusion protein technology, and more specifically to an Fc fusion protein of growth factor and interferon and its applications. Background Technology

[0002] Mucositis following radiotherapy and chemotherapy in cancer patients is one of the most common and severely impactful treatment-related complications affecting quality of life. Literature reports that the incidence of severe oral mucositis can reach 70%–80% in high-risk populations. The pathological mechanism of this process is extremely complex, involving multiple stages and interactions of various cells and molecules. Sonis's five-stage model summarizes the development of mucositis as follows: initiation stage (radiotherapy and chemotherapy cause DNA breaks and reactive oxygen species production), primary damage (activation of inflammatory pathways and release of pro-inflammatory cytokines), signal amplification (positive feedback loop of pro-inflammatory factors leads to amplification of apoptosis signals), ulceration (disruption of epithelial integrity, bacterial colonization exacerbates tissue damage), and healing stage (epithelial migration, proliferation, and differentiation). Radiotherapy-induced NF-κB activation releases large amounts of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF. These inflammatory mediators not only directly damage tissues but also further amplify apoptosis signals through positive feedback loops, leading to a continuously worsening inflammatory response. However, current clinical treatment strategies for mucositis caused by radiotherapy and chemotherapy mainly focus on prevention, symptomatic pain relief, nutritional support, and prevention of secondary infections, lacking effective drugs that can fundamentally break the vicious cycle of "damage-inflammation-infection-damage".

[0003] Keratinocyte growth factor (KGF) is a paracrine growth factor expressed in mesenchymal cells and acting on epithelial cells, promoting proliferation, differentiation, and migration of epithelial cells in various tissues. Palifermin (brand name Kepivance®) is the first and currently the only recombinant human keratinocyte growth factor approved by the US FDA for the prevention of severe oral mucositis, primarily used in patients with hematologic malignancies who have undergone autologous hematopoietic stem cell transplantation. Clinical trials in head and neck cancer patients showed that palifermin reduced the incidence of severe oral mucositis from 67%–69% to 51% and 54%, respectively. Preclinical studies have also shown that KGF stimulates cell proliferation and differentiation by activating FGFR2b receptors on the surface of epithelial cells, upregulating cytoprotective mechanisms, thereby thickening and strengthening the mucosal barrier and resisting cytotoxic damage from radiotherapy and chemotherapy. Nevertheless, the clinical application of KGF-based drugs has significant limitations. First, the benefit effect of parivmin is highly context-dependent, and no significant efficacy has been observed in patients receiving allogeneic stem cell transplantation or those receiving high-dose melphalan pretreatment. Second, its clinical research and application in patients with solid tumors is severely limited due to the theoretical risk of stimulating the growth of non-hematologic malignancies expressing KGF receptors. More importantly, KGF primarily focuses on the physical repair of epithelial structures and lacks direct intervention in local infections and excessive inflammatory responses induced by radiotherapy and chemotherapy, factors that, according to clinical data, are crucial pathological links hindering mucosal healing.

[0004] Type III interferon (IFN-λ, also known as λ interferon or IL-28 / 29) is a novel family of interferons discovered in recent years. A significant characteristic of type III interferon compared to other types is that its receptor, IFNLR1, is preferentially expressed on the surface of epithelial lineage cells and specific immune cell populations. This expression profile allows IFN-λ to specifically act on mucosal epithelial cells—the primary target and gateway for viral invasion—inducing epithelial cells into an antiviral state, upregulating the expression of interferon-stimulating genes, and inhibiting viral replication and spread locally in the mucosa. Studies have shown that IFN-λ plays an irreplaceable antiviral protective role on the mucosal barrier surface, and its function cannot be replaced by type I interferon. Simultaneously, type III interferon also has important immunomodulatory functions, regulating the immune response while inducing antiviral activity and reducing excessive inflammatory damage. It is noteworthy that cancer patients are often in an immunosuppressed state and are highly susceptible to opportunistic viral infections (such as herpes simplex virus, Epstein-Barr virus, and cytomegalovirus). A prospective cohort study showed that in head and neck cancer patients receiving radiotherapy, the frequency of EBV oral shedding was significantly higher than that of other viruses, and it was positively correlated with grade ≥2 oral mucositis, especially during the period three weeks after radiotherapy when the severity of mucositis significantly increased. Other studies have also found that herpes simplex virus infection is common in chemotherapy patients and can manifest as severe lesions, requiring urgent antiviral intervention. Therefore, for patients undergoing radiotherapy and chemotherapy, damaged mucosa not only needs physical re-epithelial repair but also the reconstruction of the local anti-infection barrier.

[0005] Although KGF has a clear efficacy in promoting mucosal epithelial repair, and type III interferon also shows unique advantages in local antiviral protection and immunomodulation, there is currently no drug in clinical practice that integrates mucosal repair function with local antiviral infection and immunomodulatory function into a single molecule. Existing monotherapy (such as using KGF alone) cannot simultaneously solve the vicious cycle of "damage-inflammation-infection-damage" that occurs in the mucosa after radiotherapy and chemotherapy. KGF can promote epithelial regeneration, but it cannot address viral reactivation and secondary infections that occur under immunosuppression after radiotherapy and chemotherapy; while type III interferon can establish a local antiviral state, it does not have the function of directly repairing damaged epithelial structures.

[0006] Therefore, how to break the vicious cycle of "damage-inflammation-infection-damage" is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides an Fc fusion protein of growth factor and interferon and its applications, achieving dual synergistic effects through a single molecule, aiming to overcome the limitations of current cancer treatments in treating mucosal ulcers. Specifically, the present invention employs a dual mechanism of "structural repair + immune protection": keratinocyte growth factor is responsible for physical repair, and interferon is responsible for biological defense, both working together to maintain mucosal homeostasis. This fusion protein is suitable for the prevention and treatment of severe mucositis caused by radiotherapy and chemotherapy, while protecting patients from opportunistic viral infections.

[0008] To solve the above-mentioned technical problems, this application adopts the following technical solution:

[0009] The primary objective of this application is to provide: an Fc fusion protein of growth factor and interferon, wherein the fusion protein is a homodimer formed by two heavy-like chains linked by disulfide bonds; The heavy chain-like structure refers to a polypeptide chain that mimics the Fc heavy chain structure of an antibody, which includes: a keratinocyte growth factor functional region sequence, an Fc segment of human immunoglobulin G1, and an interferon functional region sequence.

[0010] As a preferred technical solution, the amino acid sequence of the keratinocyte growth factor functional region is shown in SEQ ID NO.2: SEQ ID NO.2.

[0011] The amino acid sequence of the Fc region of the human immunoglobulin G1 is shown in SEQ ID NO.3: AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGAS, SEQ ID NO.3.

[0012] The amino acid sequence of the interferon functional region is shown in SEQ ID NO.4: SEQ ID NO.4

[0013] As a preferred technical solution, the keratinocyte growth factor functional region sequence in the heavy chain is connected to the Fc region of human immunoglobulin G1 via a flexible linker peptide 1; the Fc region of human immunoglobulin G1 is connected to the interferon functional region sequence via a flexible linker peptide 2. The amino acid sequence of the flexible linker peptide 1 is shown in SEQ ID NO. 5; the amino acid sequence of the flexible linker peptide 2 is shown in SEQ ID NO. 6. GGGGSGGGGSGGGGS, SEQ ID NO.5; GSSGGSSGTSGSSGGSSGTS, SEQ ID NO. 6.

[0014] As a preferred technical solution, the N-terminus-C-terminus of the fusion protein are sequentially: keratinocyte growth factor functional region - flexible linker peptide 1 - Fc segment of human immunoglobulin G1 - flexible linker peptide 2 - interferon functional region. The heavy chain-like amino acid sequence is shown in SEQ ID NO.1: , SEQ ID NO.1.

[0015] Another object of this application is to provide: a nucleic acid molecule encoding the said fusion protein.

[0016] Another object of this application is to provide: an expression vector or host cell for the said nucleic acid molecule.

[0017] Another object of this application is to provide a method for preparing the fusion protein, comprising culturing the host cells under suitable expression conditions and recovering the fusion protein from the culture.

[0018] Another object of this application is to provide the use of the fusion protein described herein or the fusion protein prepared by the method in the preparation of a medicament for treating severe mucositis that occurs after radiotherapy and chemotherapy in cancer patients.

[0019] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: (1) Promote epithelial reconstruction: Keratinocyte growth factor can specifically stimulate the proliferation, differentiation and migration of mucosal epithelial cells, accelerate the re-epithelialization of damaged mucosa, and thus repair the ulcer surface.

[0020] (2) Optimize repair quality: Mucosal damage caused by radiotherapy and chemotherapy is often accompanied by inflammatory storms. Type III interferon has immunomodulatory effects, which can moderately regulate the local microenvironment, reduce excessive inflammatory damage, and prevent "secondary damage".

[0021] (3) Resisting infectious interference: Damaged mucosa is easily colonized by bacteria or fungi, which can seriously hinder healing. Type III interferon can stimulate the antiviral / antibacterial state of local epithelial cells and prevent healing delay caused by infection. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0023] Figure 1 Here is a schematic diagram of the structure of the fusion protein 119 of the present invention.

[0024] Figure 2 The image shows the enzyme digestion identification results of the fusion protein expression plasmid in Example 1 of this invention; where the left side is a schematic diagram of the nucleic acid marker bands; lane 1 is the marker; lane 2 is the plasmid pcDNA3.4-119 band; lane 3 is the XbaI and EcoRV double enzyme digestion band of plasmid pcDNA3.4-119.

[0025] Figure 3 Figure 1 shows the expression and purification results of the fusion protein prepared in Example 1 of this invention; where A represents the elution volume and OD. 280 The corresponding diagram for pH is shown in Figure B. B shows the SDS-PAGE results, with the left side showing a schematic diagram of the protein ladder. Lane 1 is the protein ladder band, and lane 2 is the denatured and reduced sample band of fusion protein 119.

[0026] Figure 4 Figure 2 shows the results of the fusion protein inhibiting HeLa cell proliferation in Example 2 of this invention.

[0027] Figure 5 Figure A shows the results of transcription of antiviral-related genes in HepG2 cells stimulated by the fusion protein in Example 3 of this invention; Figure A is a bar chart of the mean optical density of RT-PCR, and the optical density is derived from Figure B; Figure B is a nucleic acid gel image of the RT-PCR product.

[0028] Figure 6 Figure 4 shows the results of the fusion protein inducing the migration of epithelial-like cells A2780 in Example 4 of this invention. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] In the examples, all raw materials not mentioned are commercially available, and all experimental methods not mentioned are conventional experimental methods, which will not be described in detail here.

[0031] Example 1 Plasmid construction, protein expression and purification (the structural diagram is shown in Figure 1) Figure 1 (As shown) (1) The aforementioned SEQ ID No.1 (number 119) sequence was synthesized by gene (with restriction endonuclease XbaI and EcoRV restriction sites added to both ends of the sequence), and then constructed into the pcDNA3.4 plasmid expressing the fusion protein using the pcDNA3.4-TOPOTA cloning kit (purchased from Invitrogen (Shanghai) Trading Co., Ltd.), thus constructing pcDNA3.4-119.

[0032] (2) The pcDNA3.4-119 plasmid from the previous step was transformed into Escherichia coli TOP10 (purchased from Beijing Qingke Biotechnology Co., Ltd.), amplified by shaking on LB medium, extracted using a plasmid large-scale extraction kit (Beijing Jumei Biotechnology Co., Ltd.), and then digested with restriction endonucleases XbaI and EcoRV. The results were then analyzed by gel electrophoresis. See below for details. Figure 2 From left to right, lane 1 is the marker band, lane 2 is the plasmid pcDNA3.4-119 band, and lane 3 is the XbaI and EcoRV double digestion band of plasmid pcDNA3.4-119. The upper side of the double digestion band is the linearized band of the empty plasmid, and the lower side is the target band position, which is consistent with the designed size and position.

[0033] (3) The expression plasmid was transfected into 293F cells using PEI transfection reagent for protein expression.

[0034] (4) 120 h after transfection, the supernatant was harvested, centrifuged and filtered. The protein A affinity column (purchased from Cytiva) was treated with 5 column volumes of equilibration buffer (5.6 mM NaH2PO4, 14.4 mM Na2HPO4, 0.15 M NaCl, pH 7.2). The supernatant was loaded onto the column. After washing, contaminating proteins with buffer (5.6 mM NaH2PO4, 14.4 mM Na2HPO4, 0.5 M NaCl, pH 7.2) to baseline. Proteins were then eluted with 50 mM citrate / sodium citrate buffer (pH 3.2), and samples above 80 mAu were collected. The pH was adjusted to 7.0 with 1 M Tris-Cl (pH 8.0), concentrated in a concentration tube, filtered for sterilization, and stored at 4℃. 3 μg of the sample was then used for SDS-PAGE staining. Results are shown below. Figure 3 : Figure 3 A in the diagram is a chromatography chromatogram, showing the correspondence between elution volume and OD280 and pH. Figure 3 Image B shows the SDS-PAGE results, where lane 1 is the protein ladder plot and lane 2 is the 119 denatured / reduced sample band. The protein band positions are consistent with the designed size and position (after expression in cells, 119 forms a Y-shaped homodimer structure, becoming a protein, which then forms a monomer under denaturing / reducing conditions). Figure 3 (As shown in section B). The protein obtained in this step is fusion protein 119.

[0035] Example 2 HeLa proliferation inhibition experiment (1) Cell plating HeLa cells in logarithmic growth phase were digested, resuspended in culture medium, and their density was adjusted to 5 × 10⁻⁶. 4 Add 100 μL of resuspended cell suspension to each well of a 96-well plate (i.e., 5000 cells / well; add PBS to the blank group and surrounding wells to prevent evaporation), and incubate overnight at 37°C with 5% CO2.

[0036] (2) Processing Fusion protein 119 and interferon IFNα2a (purchased from 3SBio Co., Ltd.) were serially diluted with serum-free medium to three concentrations (0.1, 1, 10 μg / mL). The original liquid in the well plate was aspirated, and 100 μL of protein dilution was added to each well (as shown in Table 1 below; serum-free medium was added to the blank group and the control group). The plates were incubated at 37°C for 72 h.

[0037] Table 1. Sample Layout for 96-well Plate

[0038] 1. In the table, 119-10 μg / mL and 119-10 represent adding 10 μg / mL of 119 diluent; 119-1 μg / mL and 119-1 represent adding 1 μg / mL of 119 diluent; 119-0.1 μg / mL and 119-0.1 represent adding 0.1 μg / mL of 119 diluent. 2. In the table, IFNα2a-10 μg / mL and IFNα2a-10 represent the addition of 10 μg / mL of IFNα2a diluent; IFNα2a-1 μg / mL and IFNα2a-1 represent the addition of 1 μg / mL of IFNα2a diluent; IFNα2a-0.1 μg / mL and IFNα2a-0.1 represent the addition of 0.1 μg / mL of IFNα2a diluent.

[0039] (3) CCK8 detection Add 10 μL of CCK-8 reagent to each well (protect from light), incubate at 37°C for 1 h in the dark, and measure the absorbance (OD) at 450 nm using a microplate reader. 450 ).

[0040] The results are as follows Figure 4 As shown, except for the lowest concentration, the other two concentrations of 119 showed significant differences compared to the DMEM control group (p < 0.001); the three concentrations of the IFNα2a positive control group showed significant differences compared to the DMEM control group (p values ​​were 0.003, < 0.001, and < 0.001, respectively). Interferon is known to have anti-tumor activity and can inhibit the proliferation of cervical cancer HeLa cells. The results indicate that 119 can significantly inhibit HeLa cell proliferation and exhibits interferon activity.

[0041] Example 3 HepG2 induction experiment (1) Cell plating HepG2 cells in logarithmic growth phase were digested, cell counting was performed using trypan blue, and the density was adjusted to 23. 10^5 cells / mL were seeded into plates, and 1 mL of cell suspension was added to each well of a 12-well plate. The plates were then incubated overnight at 37°C with 5% CO2.

[0042] (2) Cell treatment The fusion protein 119 and IFNα2a were diluted to 1 μg / mL with serum-free medium. The original liquid in the well plate was discarded, and 1.5 mL of dilution solution was added to each well (as shown in Table 2 below; serum-free medium was added to the control group). The mixture was incubated at 37°C for 1 h.

[0043] Table 2. Layout of 12-well plate samples

[0044] (3) RNA was extracted, reverse transcribed, and then RT-PCR was performed to detect the transcription levels of mRNA of Mxa, Oas, and Gapdh (internal reference gene). The primer sequences are shown in Table 3 below.

[0045] Table 3 RT-PCR Primer Sequences

[0046] The results are as follows Figure 5 As shown, compared with the control group, 119 can induce upregulation of MXA and OAS gene mRNA transcription levels. The results indicate that 119 can effectively induce the transcriptional expression of antiviral-related genes in HepG2 cells.

[0047] Example 4 Cell migration experiment (1) Cell preparation A2780 cells in logarithmic growth phase were digested, resuspended in serum-free medium, counted, and their density adjusted to 2. 10^5 cells / mL, 6-well plate, 2.5 mL per well, i.e. 500,000 cells / well, incubate overnight at 37°C with 5% CO2.

[0048] (2) Scratches: Mark the bottom of the 6-well plate with a ruler. Using the tip of a 200 μL sterile pipette, draw a straight line vertically and at a constant speed along the ruler mark on the cells. Wash twice with PBS to remove detached cells.

[0049] (3) Cell treatment Dilute 119 to working concentration (100, 1000 ng / mL) with serum-free medium, and add 2.5 mL of diluent to each well (control wells are treated with antibody-free medium). Table 4. Sample Layout for 6-well Plates

[0050] (4) Image acquisition: Under an inverted microscope, the fixed locations of each scratch were marked and photographed at predetermined time points (0, 24, 48, 72 h).

[0051] The results are as follows Figure 6 As shown, both concentrations of 119 enhanced cell migration levels after scratch wounds, and after 48 h and 72 h of culture, the cell migration levels within the scratch wounds were superior to those in the control group. Keratinocyte growth factor (KGF) is known to induce epithelial-like cell migration, and 119 possesses KGF function.

[0052] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0053] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An Fc fusion protein of growth factor and interferon, characterized in that, The fusion protein is a homodimer composed of two heavy-like chains linked by disulfide bonds; The heavy chain-like structure refers to a polypeptide chain that mimics the Fc heavy chain structure of an antibody, which includes: a keratinocyte growth factor functional region sequence, an Fc segment of human immunoglobulin G1, and an interferon functional region sequence.

2. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the functional region of the keratinocyte growth factor is shown in SEQ ID NO.2; The amino acid sequence of the Fc region of the human immunoglobulin G1 is shown in SEQ ID NO.3; The amino acid sequence of the interferon functional region is shown in SEQ ID NO.

4.

3. The fusion protein according to claim 1, characterized in that, The keratinocyte growth factor functional region sequence in the heavy chain is linked to the Fc region of human immunoglobulin G1 via a flexible linker peptide 1; the Fc region of human immunoglobulin G1 is linked to the interferon functional region sequence via a flexible linker peptide 2. The amino acid sequence of the flexible linker peptide 1 is shown in SEQ ID NO.5; the amino acid sequence of the flexible linker peptide 2 is shown in SEQ ID NO.

6.

4. The fusion protein according to claim 1, characterized in that, The N-terminus-C-terminus of the fusion protein are, in sequence: keratinocyte growth factor functional region - flexible linker peptide 1 - Fc segment of human immunoglobulin G1 - flexible linker peptide 2 - interferon functional region. The heavy chain-like amino acid sequence is shown in SEQ ID NO.

1.

5. A nucleic acid molecule encoding the fusion protein of any one of claims 1-4.

6. An expression vector or host cell comprising the nucleic acid molecule of claim 5.

7. A method for preparing the fusion protein according to any one of claims 1-4, comprising culturing the host cell as described in claim 6 under conditions suitable for expression, and recovering the fusion protein from the culture.

8. The use of the fusion protein according to any one of claims 1-4 or the fusion protein prepared by the method of claim 7 in the preparation of a medicament for treating severe mucositis in cancer patients after radiotherapy and chemotherapy.

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