Long-acting feline granulocyte macrophage colony-stimulating factor recombinant protein, and cho cell expression method and application thereof

Abstract

The application belongs to the technical field of bioengineering, and discloses a long-acting cat granulocyte macrophage colony-stimulating factor recombinant protein, a CHO cell expression method and application thereof. The cat granulocyte macrophage colony-stimulating factor recombinant protein GM-CSF-Fc(L) provided by the application can exhibit a significant curative effect advantage in the treatment intervention of cat leukopenia. In addition, the GM-CSF-Fc(L) can not only realize the rapid correction of plasma leukocytes, especially neutrophils, of a cat with leukopenia within 24 hours after the treatment intervention, but also can continuously exert a treatment effect, and can significantly improve the half-life period, prolong the treatment time and improve the overall prognosis relative to the traditional GM-CSF.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a long-acting recombinant feline granulocyte-macrophage colony-stimulating factor protein, its CHO cell expression method, and its application. Background Technology

[0002] In clinical practice, cats often experience bone marrow suppression due to viral and parasitic infections, drug side effects (such as chemotherapy), or stress, leading to a significant decrease in white blood cell count and impaired immune function. Some cats develop short-term or persistent immunodeficiency after infection or treatment intervention, with a significant drop in total white blood cell count, affecting the recovery process and even endangering their lives. Currently, clinical practice often uses comprehensive therapies such as broad-spectrum antibiotics, glucocorticoids, and nutritional supplements to control secondary infections, but this may further suppress immunity or lead to drug resistance, failing to effectively promote the rapid reconstruction of the body's own immune function. Therefore, improving the immune function of sick cats, promoting white blood cell production, and enhancing their anti-infection capabilities have become more forward-looking intervention strategies.

[0003] Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a key hematopoietic cell growth factor that stimulates bone marrow stem / progenitor cells to differentiate into neutrophils and monocytes, promoting the generation and activation of immune cells such as neutrophils and macrophages. In human and canine medicine, recombinant GM-CSF has been widely used to treat leukopenia following chemotherapy, promote hematopoietic recovery, and serve as an immune adjuvant. However, systematic studies on feline GM-CSF are currently lacking. Furthermore, due to the strong species specificity of GM-CSF, the bioactivity and safety of heterologous (e.g., human or canine) GM-CSF in cats are difficult to guarantee, and there may be immunogenicity risks, which severely limits its clinical application in cats.

[0004] Therefore, developing an efficient, safe, and easily scalable GM-CSF production technology is key to solving the aforementioned problems. The Chinese hamster ovary (CHO) cell expression system possesses advantages such as well-developed protein glycosylation modification function, accurate post-transcriptional modification, low risk of viral contamination, and low endotoxin production. Furthermore, the CHO cell expression system exhibits strong gene amplification capacity and high exogenous protein expression capacity, enabling efficient and stable expression of recombinant proteins. Its fibroblast characteristics result in almost no secretion of endogenous proteins, greatly simplifying the isolation and purification process of recombinant proteins and reducing production costs and process complexity. Utilizing the CHO cell expression system to produce recombinant GM-CSF holds promise for obtaining products with structures and functions closer to natural proteins and higher safety, demonstrating significant technological advantages and broad market prospects.

[0005] Currently available feline GM-CSF products are mostly conventional injectable or lyophilized powder injections. Due to their short half-life in vivo, frequent injections are required, which can easily lead to adverse reactions and stress at the injection site in pets. Therefore, this invention employs Fc fusion protein technology to achieve long-acting effects. This technology, by fusing the target protein with the Fc fragment of immunoglobulin G (IgG), can significantly prolong its in vivo half-life through an FcRn-mediated circulation mechanism, and improve protein stability and drug-likeness. Although Fc fusion technology is a mature long-acting strategy in human medicine, its application to feline GM-CSF to address the short-acting problem of species-specific proteins is currently unexplored, and its actual efficacy and safety in cats are unknown. Therefore, this invention creatively develops a feline GM-CSF-Fc fusion protein, aiming to provide a long-acting, safe, and highly effective feline leukocyte-boosting drug.

[0006] In summary, to address the efficacy and safety issues of cross-species GM-CSF in feline applications, this invention aims to efficiently prepare recombinant feline GM-CSF-Fc protein with natural activity using a CHO cell expression system, providing a highly efficient, specific, and safe biological product for the clinical treatment of feline leukopenia. Summary of the Invention

[0007] Based on the current problems of high production cost, insufficient safety and short half-life of GM-CSF in cat applications, this invention provides a long-acting feline granulocyte-macrophage colony-stimulating factor mutant GM-CSF-Fc(L), the amino acid sequence of which is shown in SEQ ID NO.7.

[0008] Another object of the present invention is to provide a method for preparing CHO cells of GM-CSF-Fc(L).

[0009] The final objective of this invention is to provide the use of GM-CSF-Fc(L) in the treatment of feline leukopenia.

[0010] To achieve the above objectives, the present invention adopts the following technical measures:

[0011] The applicant linked the original sequence of feline granulocyte-macrophage colony-stimulating factor GM-CSF to the C-terminus with an HRV 3C restriction site, followed by a GGGGS flexible linker peptide. An IgG Fc fragment was then added to enhance its stability and half-life. Furthermore, the F at amino acid terminus 266 of GM-CSF-Fc was replaced with L to further improve its half-life, resulting in the recombinant protein GM-CSF-Fc(L). The amino acid sequence of GM-CSF-Fc(L) is shown in SEQ ID NO.7, and one of the polynucleotide sequences encoding its protein is shown in SEQ ID NO.8.

[0012] The scope of protection of this invention also includes:

[0013] The fusion protein obtained by fusing the recombinant protein shown in SEQ ID NO.7 with a protein tag.

[0014] The gene encoding the recombinant protein shown in SEQ ID NO.7 or the fusion protein described above.

[0015] Expression cassettes, recombinant vectors, recombinant microorganisms, or in vitro recombinant cells containing the above-mentioned coding genes.

[0016] The recombinant cells described above are preferably recombinant CHO cells.

[0017] A method for preparing the recombinant protein shown in SEQ ID NO.7 or the above-described fusion protein, comprising culturing CHO containing an expression cassette or recombinant vector having the above-described encoding gene.

[0018] The application of the recombinant protein shown in SEQ ID NO.7, the above-mentioned fusion protein, the encoding gene, the expression cassette having the above-mentioned encoding gene, the recombinant vector, the recombinant microorganism or the ex vivo recombinant cell in the preparation of long-acting cat granulocyte-macrophage colony-stimulating factor.

[0019] The recombinant protein shown in SEQ ID NO.7, the above-mentioned fusion protein, the encoding gene, the expression cassette having the above-mentioned encoding gene, the recombinant vector, the recombinant microorganism or the ex vivo recombinant cell, and their application in the preparation of a drug for treating feline leukopenia.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] This invention provides for the first time a long-acting feline granulocyte-macrophage colony-stimulating factor mutant, GM-CSF-Fc(L), with low immunogenicity and high safety, offering an effective solution for the treatment of feline leukopenia. GM-CSF-Fc(L) demonstrates good therapeutic effects on feline leukopenia in terms of physiological function and blood routine indicators. Compared to GM-CSF-His, GM-CSF-Fc(L) exhibits a more significant therapeutic effect and longer duration of action in the treatment of feline leukopenia by extending its half-life (P < 0.05). GM-CSF-Fc(L) not only enables rapid recovery of white blood cells in affected cats within 24 hours after treatment intervention, but also maintains therapeutic blood drug concentrations continuously due to its significantly prolonged in vivo half-life, thus providing a sustained and stable therapeutic effect and keeping white blood cell levels within the normal range for a long period.

[0022] The recombinant GM-CSF-Fc(L) provided by this invention can be produced using a CHO cell expression system, and has significant advantages in terms of ease of protein purification, safety, and production cost, providing reliable technical support for large-scale production.

[0023] GM-CSF-Fc(L) demonstrated significant therapeutic advantages in the treatment of feline leukopenia (P<0.05). Furthermore, GM-CSF-Fc(L) not only enabled rapid correction of plasma leukocytes, particularly neutrophils, in cats with leukopenia within 24 hours of treatment intervention, but also exerted a sustained therapeutic effect. Compared to traditional GM-CSF, it significantly increased the half-life, prolonged the treatment duration, and improved overall prognosis. Attached Figure Description

[0024] Figure 1 This is a schematic diagram showing the detection results of SDS-PAGE and Western Blot for different recombinant GM-CSF proteins after purification.

[0025] Note: A: GM-CSF-His; B: GM-CSF-Fc; C: GM-CSF-Fc (YTE); D: GM-CSF-Fc (L).

[0026] Figure 2 A schematic diagram of the weight measurement results for the mouse modeling experiment;

[0027] Note: 0 represents before modeling.

[0028] Figure 3 A schematic diagram of body temperature and weight measurements from a cat modeling experiment.

[0029] Note: A: Schematic diagram of body temperature measurement results in the cat modeling experiment; B: Schematic diagram of weight measurement results in the cat modeling experiment; 0 represents before modeling.

[0030] Figure 4 A schematic diagram showing the weight measurement results of mice in a treatment experiment;

[0031] Note: -1 represents before modeling; 0 represents day 0 of treatment.

[0032] Figure 5 A schematic diagram showing the body temperature and weight measurements of cats in a treatment experiment.

[0033] Note: A: Schematic diagram of body temperature measurement results in the cat treatment experiment; B: Schematic diagram of body weight measurement results in the cat treatment experiment; -1 represents before modeling; 0 represents day 0 of treatment.

[0034] Figure 6 A schematic diagram showing the weight measurement results of the mouse safety experiment;

[0035] Note: 0 represents before treatment.

[0036] Figure 7 A schematic diagram showing the body temperature and weight measurement results of a cat safety experiment.

[0037] Note: A: Schematic diagram of body temperature measurement results in cat safety experiment; B: Schematic diagram of weight measurement results in cat safety experiment; 0 represents before treatment. Detailed Implementation

[0038] The present invention will be further illustrated below through embodiments, which are intended only to provide a better understanding of the research content of the present invention and not to limit the scope of protection of the present invention. Unless otherwise specified, the technical solutions described in this invention are conventional solutions in the art, and the reagents or materials described, unless otherwise specified, are all from commercial sources.

[0039] Example 1: Design and acquisition of four recombinant cat granulocyte-macrophage colony-stimulating factors:

[0040] 1.1 Design of four recombinant feline granulocyte-macrophage colony-stimulating factor nucleotide sequences:

[0041] The gene and amino acid sequences of feline granulocyte-macrophage colony-stimulating factor (GenBank: AAX63391.1) and feline IgG Fc (GenBank accession number: ATI97569.1) were retrieved from NCBI.

[0042] Four amino acid sequences of cat granulocyte-macrophage colony-stimulating factors were designed:

[0043] (1) GM-CSF-His: By linking an HRV 3C restriction site to the C-terminus of the original GM-CSF sequence, followed by a GGGGS flexible linker peptide, a Twin-Strep-tag, and a 10×His tag, a fusion protein obtained by fusing GM-CSF with a protein tag was obtained, which is referred to as GM-CSF-His in this embodiment of the invention. The amino acid sequence of GM-CSF-His is shown in SEQ ID NO.1, and the nucleotide sequence encoding it is shown in SEQ ID NO.2.

[0044] (2) GM-CSF-Fc: By linking an HRV 3C restriction site to the C-terminus of the original GM-CSF sequence, followed by a GGGGS flexible linker peptide, and then adding an IgG Fc fragment to enhance its stability and half-life, a fusion protein obtained by fusing GM-CSF and Fc fragments was obtained, which is referred to as GM-CSF-Fc in this embodiment of the invention. The amino acid sequence of GM-CSF-Fc is shown in SEQ ID NO.3, and the nucleotide sequence encoding it is shown in SEQ ID NO.4.

[0045] (3) GM-CSF-Fc (YTE): By replacing the Y at position 199 of the IgG Fc fragment with S, the T at position 201 with S, and the E at position 203 with T (CN119954968A) in the original GM-CSF-Fc sequence, its half-life was further improved, resulting in the GM-CSF-Fc mutant, which is referred to as GM-CSF-Fc (YTE) in this embodiment of the invention. The amino acid sequence of GM-CSF-Fc (YTE) is shown in SEQ ID NO.5, and the nucleotide sequence encoding it is shown in SEQ ID NO.6.

[0046] (4) GM-CSF-Fc(L): By replacing the F at position 266 of the IgG Fc fragment amino acid in the original GM-CSF-Fc sequence with L to further improve its half-life, the GM-CSF-Fc mutant was obtained, which is referred to as GM-CSF-Fc(L) in this embodiment of the invention. The amino acid sequence of GM-CSF-Fc(L) is shown in SEQ ID NO.7, and the nucleotide sequence encoding it is shown in SEQ ID NO.8.

[0047] The aforementioned recombinant feline granulocyte-macrophage colony-stimulating factor can be directly synthesized commercially or obtained through microbial expression. In this invention, it is obtained through commercial synthesis and CHO eukaryotic expression.

[0048] 1.2 Gene Sequence Synthesis and Plasmid Construction

[0049] DNAman software was used to analyze the restriction endonuclease sites of the target gene. Based on the analysis results and the multiple cloning site on the Pxc17.4 transfer vector, HindIII (5' end) and EcoRI (3' end) restriction sites were introduced at both ends of the target gene sequence to ensure that the selected restriction sites did not appear in the target gene sequence. The identified CHO system codon-optimized target gene sequences SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, and SEQ ID NO.8 were inserted into the Pxc17.4 vector to construct recombinant plasmids, which were named Pxc-17.4-GM-CSF-His, Pxc-17.4-GM-CSF-Fc, Pxc-17.4-GM-CSF-Fc (YTE), and Pxc-17.4-GM-CSF-Fc (L), respectively. These plasmids were used for subsequent transfection of the CHO cell expression system and efficient expression of the recombinant protein.

[0050] 1.3 Expression and purification of GM-CSF-His, GM-CSF-Fc, GM-CSF-Fc (YTE) and GM-CSF-Fc (L) in CHO cells

[0051] 1.3.1 Cell preparation and electroporation equipment treatment: Select CHO suspension cells that have been passaged to the 3rd generation or above and are in good condition as expression host cells. Immerse the electroporation cuvette in 95% ethanol and sterilize it under ultraviolet light for 30 min before use.

[0052] 1.3.2 Cell Collection and Pretreatment: CHO cells were collected and pretreated until they reached the logarithmic growth phase (cell density approximately 3 × 10⁻⁶). 6 When the cell density reaches 100 cells / mL, transfer 20 mL of cell suspension to a sterile centrifuge tube and centrifuge at 1000 rpm for 5 minutes at room temperature, discarding the supernatant. Resuspend the cells in sterile PBS buffer to achieve a density of approximately 1 × 10⁻⁶ cells / mL. 7 Centrifuge repeatedly (1000 rpm, 5 minutes) at a rate of 1000 rpm for 5 minutes, and discard the supernatant.

[0053] 1.3.3 Electroporation: Add 160 μL of electroporation buffer to the cell pellet and gently mix to fully resuspend the cells. Then add 20 μg of the target expression plasmids Pxc-17.4-GM-CSF-His, Pxc-17.4-GM-CSF-Fc, Pxc-17.4-GM-CSF-Fc (YTE), and Pxc-17.4-GM-CSF-Fc (L), respectively, mix well, and transfer to an electroporation cuvette. Electroporation is performed at 200 V and 1000 μs pulses. Each sample is repeated 6 times, with an interval of 1000 ms between each electroporation. After electroporation, the cell suspension is immediately transferred to a sterile shake flask containing 20 mL of culture medium pre-warmed to 37℃ and cultured at 37℃, 5% CO2, and 120 r / min.

[0054] 1.3.4 Large-scale culture and feeding: After the cell state stabilizes, feed the cells at a rate of 0.5 × 10⁻⁶. 6 Cells were seeded at a density of 10 cells / mL into 2 L Erlenmeyer flasks containing 400 mL of culture medium. The vent caps were sealed, and the flasks were incubated on a shaker at 37°C and 5% CO2. Cell density and viability were monitored daily. When the cell density reached 4 × 10⁶ cells / mL, the cell count was increased. 6 Start feeding when the viable cell count reaches 80%; stop the culture when the viable cell count drops to 80%, collect the culture supernatant for further processing.

[0055] 1.3.5 Protein Supernatant Processing and Purification: The culture supernatant was centrifuged to remove cells and debris. The supernatant from the GM-CSF-His group was filtered through a 0.45 μm filter and loaded onto a Ni-NTA affinity chromatography column pre-equilibrated with PBS buffer. After the A280 absorbance returned to baseline, the target protein was eluted with 300 mM imidazole buffer to obtain high-purity recombinant GM-CSF-His protein. The supernatants from the GM-CSF-Fc, GM-CSF-Fc(YTE), and GM-CSF-Fc(L) groups were filtered through a 0.45 μm filter and loaded onto a Protein A affinity chromatography column pre-equilibrated with PBS buffer. After the A280 absorbance returned to baseline, the target protein was eluted with 0.1 M, pH 3.0 sodium acetate buffer to obtain the corresponding high-purity recombinant protein from the eluent.

[0056] 1.3.6 Protein Identification and Concentration Detection: After sterilization filtration through a 0.22 μm filter membrane, the purified proteins were identified and detected using SDS-PAGE and Western Blot. The results showed that the four recombinant GM-CSF proteins exhibited clear banding patterns. The molecular weight of GM-CSF-His was 21 kDa, while the molecular weights of GM-CSF-Fc, GM-CSF-Fc(YTE), and GM-CSF-Fc(L) proteins with the added Fc tag were 55 kDa, consistent with expectations. Figure 1 Furthermore, the concentrations of recombinant proteins GM-CSF-His, GM-CSF-Fc, GM-CSF-Fc (YTE), and GM-CSF-Fc (L) were determined using the BCA method (Thermo Scientific Pierce BCA Protein Quantification Kit). The results showed that approximately 3.81 g of GM-CSF-His protein, 3.66 g of GM-CSF-Fc protein, 3.75 g of GM-CSF-Fc (YTE) protein, and 3.87 g of GM-CSF-Fc (L) protein were obtained per liter of CHO cell supernatant, respectively. These four recombinant proteins were used for subsequent treatment of leukopenia.

[0057] Example 2:

[0058] Establishment of an animal model of leukopenia

[0059] 1. Establishment of a mouse model of leukopenia

[0060] Before the experiment, the animal room was thoroughly disinfected using formaldehyde fumigation combined with potassium permanganate. During the experiment, the ambient temperature in the animal room was controlled at 24±2℃, and the relative humidity was maintained at 40~60%.

[0061] Forty 8-week-old BALB / c mice weighing 22-24 g were randomly divided into four groups (n=10): a 50 mg / kg cyclophosphamide group (CTX-50 group), a 100 mg / kg cyclophosphamide group (CTX-100 group), a 200 mg / kg cyclophosphamide group (CTX-200 group), and a control group. Before the experiment, all mice underwent a 7-day acclimatization period in the animal room with free access to food and water. Cyclophosphamide was dissolved in physiological saline, and 0.2 mL of the corresponding concentration of cyclophosphamide solution was injected intraperitoneally into each group. The control group received an equal volume of physiological saline. This was designated as day 0. Mouse weight was recorded daily after treatment, and anticoagulated blood was collected on days 0, 1, 3, 5, 7, and 10 for complete blood count (CBC) analysis. The results are as follows:

[0062] Compared with the control group, the body weight of mice in each cyclophosphamide treatment group continued to decrease during the experiment. Among them, the body weight of mice in the CTX-50 group and CTX-100 group began to recover on the 5th day after modeling, while the body weight of mice in the CTX-200 group decreased the most significantly and showed no obvious signs of recovery. Figure 2 Blood routine tests showed that at 24 h after cyclophosphamide induction, the total number of peripheral blood leukocytes and the levels of major subsets (neutrophils, lymphocytes, and monocytes) in the medium- and high-dose modeling groups were significantly lower than those in the control group (P < 0.01), and the leukocyte level had dropped below the normal reference range. In contrast, although the low-dose group showed a decreasing trend, its leukocyte level was not statistically different from that of the control group (P > 0.05) and remained within the normal range (Table 1).

[0063] In summary, this experiment successfully established a cyclophosphamide-induced mouse leukopenia model. Compared with the control group, the 100 mg / kg and 200 mg / kg groups significantly reduced peripheral blood leukocyte levels to below the normal physiological range, meeting the criteria for a successful leukopenia model. Compared with the 200 mg / kg group, the leukopenia induced by the 100 mg / kg group was sufficient for the model and more consistent with clinical practice, and the total drug dose was lower. Therefore, 100 mg / kg cyclophosphamide was selected as the modeling dose for mice in subsequent experiments. Furthermore, this experiment found that at 24 hours after modeling, the leukocyte level in the 100 mg / kg group was significantly lower than that in the control group, indicating that the experimental animals were already in the disease progression stage. Therefore, 24 hours after modeling was set as the starting point for intervention treatment to evaluate the therapeutic effect of GM-CSF recombinant protein.

[0064] Table 1. Analysis of blood routine index detection results in the establishment of a mouse leukopenia animal model.

[0065] .

[0066] Note: All data were analyzed using SPSS 22.0 software, and all results are expressed as mean (M) ± standard deviation (SD). t-tests were used to analyze differences between the two groups. A p-value < 0.05 indicated a significant difference; a p-value < 0.01 indicated a highly significant difference; and a p-value > 0.05 indicated no significant difference. Specifically, p1 represents the significant difference between the CTX-50 group and the control group, p2 represents the significant difference between the CTX-100 group and the control group, and p3 represents the significant difference between the CTX-200 group and the control group.

[0067] 2. Establishment of an animal model of feline leukopenia

[0068] Before the experiment, the animal room was strictly disinfected using formaldehyde fumigation and potassium permanganate. During the experiment, the temperature and humidity of the animal room were controlled at approximately 24±2℃ and 40~60%, respectively, and the experimental animals were fed and watered freely.

[0069] Twenty healthy cats aged 8-12 months, weighing approximately 2.5-3.5 kg, were randomly divided into four groups: a low-dose modeling group (CTX-8, 8 mg / kg), a medium-dose modeling group (CTX-16, 16 mg / kg), a high-dose modeling group (CTX-24, 24 mg / kg), and a control group, with five cats in each group. After one week of acclimatization in the animal facility, all cats were anesthetized with Saltoxin® 50 (5-7.5 mg / kg). Subsequently, each modeling group received an intravenous injection of 2.5 mL of cyclophosphamide solution dissolved in physiological saline, while the control group received the same volume of physiological saline intravenously. After modeling, the body temperature and weight of all cats were measured daily, and blood samples were collected periodically to detect complete blood count indicators. The following results were obtained:

[0070] Compared with the control group, the body temperature of cats in each modeling group increased significantly after modeling and fluctuated dramatically during the experiment, and their body weight also showed a significant decreasing trend. There were no significant differences among the modeling groups at different dosages. Figure 3 Blood routine tests showed that, 24 h after cyclophosphamide induction, the total number of peripheral blood leukocytes and their major subsets (neutrophils, lymphocytes, and monocytes) in the medium- and high-dose modeling groups were significantly lower than those in the control group (P < 0.01). In contrast, although the low-dose group showed a decreasing trend, its leukocyte level was not statistically different from that of the control group (P > 0.05) (Table 2).

[0071] Based on the above research, the results of this experiment demonstrate that a feline leukopenia model can be successfully established through cyclophosphamide induction. Compared with the control group, the modeling group showed significant changes in clinical symptoms, physiological function indicators, and blood routine indicators. Furthermore, the total white blood cell count in the medium- and high-dose groups consistently decreased and fell below the normal physiological range, and the model effect was more stable and reliable. Considering drug cost and clinical operability, the medium dose (16 mg / kg) was ultimately determined to be the optimal modeling dose. In addition, this experiment further found that at 24 hours after modeling, significant differences were observed in various indicators between the medium-dose (16 mg / kg) group and the control group, indicating that the experimental animals were already in the disease progression stage at this time. Therefore, 24 hours after modeling was set as the starting point for intervention treatment to evaluate the therapeutic effect of GM-CSF recombinant protein.

[0072] Table 2. Analysis of blood routine indicators in cyclophosphamide-induced leukopenia in cats.

[0073] .

[0074] Note: All data were analyzed using SPSS 22.0 software, and all results are expressed as mean (M) ± standard deviation (SD). t-tests were used to analyze differences between the two groups. A p-value < 0.05 indicated a significant difference; a p-value < 0.01 indicated an extremely significant difference; and a p-value > 0.05 indicated no significant difference. Specifically, p1 represents the significant difference between the CTX-8 group and the control group, p2 represents the significant difference between the CTX-16 group and the control group, and p3 represents the significant difference between the CTX-24 group and the control group.

[0075] Example 3:

[0076] Evaluation of the therapeutic effects of four GM-CSF recombinant proteins prepared in Experiment Example 1 in an animal model of leukopenia

[0077] 1. Evaluation of the therapeutic effects of four GM-CSF recombinant proteins in a mouse model of leukopenia.

[0078] Before the experiment, the animal room was strictly disinfected using formaldehyde fumigation and potassium permanganate. During the experiment, the temperature in the animal room was controlled at 24±2℃, and the relative humidity was controlled at around 40~60%. The experimental animals were fed and watered freely.

[0079] Fifty 8-week-old BALB / c mice weighing 22-24 g were randomly divided into 5 groups: cyclophosphamide model group (control group), GM-CSF-His treatment group (GM-CSF-His group), GM-CSF-Fc treatment group (GM-CSF-Fc group), GM-CSF-Fc(YTE) treatment group (GM-CSF-Fc(YTE) group), and GM-CSF-Fc(L) treatment group (GM-CSF-Fc(L) group), with 10 mice in each group. The modeling method for leukopenia in each group was based on the protocol determined in Section 1 of Example 2 (100 mg / kg cyclophosphamide, single intraperitoneal injection). Mice in each treatment group were injected intraperitoneally with cyclophosphamide 24 h later, and then subcutaneously injected with GM-CSF-His, GM-CSF-Fc, GM-CSF-Fc(YTE), and GM-CSF-Fc(L) proteins (10 μg / mouse), respectively. This was designated as day 0 of treatment. Mouse body weight was recorded daily during the experiment. Anticoagulated blood samples were collected from mice before modeling and on days 0, 1, 3, 5, 7, and 10 of treatment for complete blood count analysis. The following results were obtained:

[0080] Compared with the control group, the body weight of mice in each treatment group gradually recovered after treatment. However, the body weight of mice in the GM-CSF-His group began to decrease on day 6 after treatment, while the body weight of mice in the GM-CSF-Fc, GM-CSF-Fc(YTE), and GM-CSF-Fc(L) groups maintained a steady increase. There were no significant differences among the groups. Figure 4 The results of routine blood tests are shown in Table 3. In the control group, the white blood cell count, including neutrophils, lymphocytes, and monocytes, decreased significantly after cyclophosphamide injection. In the GM-CSF-His group, the peripheral blood white blood cell level rapidly recovered on day 1 after treatment, showing a significant difference compared to the control group (P < 0.01), quickly returning to near-normal levels, indicating that GM-CSF-His can effectively reverse leukopenia. However, the efficacy of the GM-CSF-His group was time-sensitive; from day 5 onwards, the treatment effect gradually weakened, and by day 10, the white blood cell level was no longer significantly different from the control group. Compared to the GM-CSF-His group, the GM-CSF-Fc group with the added Fc tag, as well as the mutant GM-CSF-Fc (YTE) group and the GM-CSF-Fc (L) group, showed a significant increase in white blood cell count, neutrophil count, lymphocyte count and monocyte count from day 1 of treatment, which rapidly returned to normal levels, and all showed stronger and more lasting therapeutic effects within 10 days of the experiment.

[0081] Comprehensive experimental data showed that, compared with the control group, the GM-CSF-His, GM-CSF-Fc, GM-CSF-Fc (YTE), and GM-CSF-Fc (L) groups all exhibited good therapeutic effects in terms of physiological function indicators and leukopenia. Compared with the GM-CSF-His group, the GM-CSF-Fc, GM-CSF-Fc (YTE), and GM-CSF-Fc (L) groups were able to exert a sustained therapeutic effect, which helped restore white blood cell levels. It is worth noting that, compared with the GM-CSF-Fc and GM-CSF-Fc (YTE) groups, the GM-CSF-Fc (L) group had the best therapeutic effect, recovering to normal levels in the shortest time and still exhibiting a high therapeutic effect on the 10th day of treatment.

[0082] Table 3. Analysis of blood routine index test results in the mouse leukopenia treatment experiment.

[0083] .

[0084] Note: All data were analyzed using SPSS 22.0 software, and all results are expressed as mean (M) ± standard deviation (SD). t-tests were used to analyze differences between the two groups. A p-value < 0.05 indicated a significant difference; a p-value < 0.01 indicated a highly significant difference; and a p-value > 0.05 indicated no significant difference. Specifically, p1 represents the significant difference between the GM-CSF-His group and the control group; p2 represents the significant difference between the GM-CSF-Fc group and the control group; p3 represents the significant difference between the GM-CSF-Fc(YTE) group and the control group; and p4 represents the significant difference between the GM-CSF-Fc(L) group and the control group.

[0085] 2. Evaluation of the therapeutic effects of four GM-CSF recombinant proteins in a feline leukopenia animal model.

[0086] Before the experiment, the animal room was strictly disinfected using formaldehyde fumigation and potassium permanganate. During the experiment, the temperature in the animal room was controlled at 24±2℃, and the relative humidity was controlled at around 40~60%. The experimental animals were fed and watered freely.

[0087] Twenty-five healthy cats aged 8-12 months and weighing approximately 2.5-3.5 kg were randomly divided into five groups (n=5 per group): cyclophosphamide model group (control group), GM-CSF-His treatment group (GM-CSF-His group), GM-CSF-Fc treatment group (GM-CSF-Fc group), GM-CSF-Fc(YTE) treatment group (GM-CSF-Fc(YTE) group), and GM-CSF-Fc(L) treatment group (GM-CSF-Fc(L) group). After one week of acclimatization in the animal facility, the experimental cats were anesthetized with Saltocin® 50 anesthetic (5-7.5 mg / kg). The feline leukopenia model in each group was established according to the protocol determined in Section 2 of Example 2 (16 mg / kg cyclophosphamide, single intravenous injection). Cats in each treatment group received an intraperitoneal injection of cyclophosphamide 24 hours later, followed by subcutaneous injections of 10 μg / kg of GM-CSF-His, GM-CSF-Fc, GM-CSF-Fc (YTE), and GM-CSF-Fc (L) proteins, respectively. The control group received the same volume of physiological saline subcutaneously. During treatment, body temperature and weight were measured daily, and injection site reactions were observed and recorded. Anticoagulated blood samples were collected daily before modeling and after treatment for complete blood count (CBC) analysis. The following results were obtained:

[0088] Temperature results showed that, compared with the control group, on the 2nd and 3rd days after treatment, the body temperature of cats in each treatment group was significantly lower than that of the abnormally elevated control group cats (P<0.05), and remained stable without significant fluctuations throughout the treatment period. Figure 5 (A). Weight results showed that the control group cats exhibited a significant decreasing trend in weight, while the weight of cats in each treatment group resumed a stable increasing trend after treatment. No significant differences were observed among the treatment groups (P>0.05). Figure 5(B) Blood routine test results showed that the white blood cell level in the control group decreased rapidly after modeling, consistent with the symptoms of leukopenia. Compared with the control group, the GM-CSF-His group showed a significant effect in promoting white blood cell recovery in the early stage of treatment (days 1 and 3), but the effect was short-lived, and the effect was no longer obvious after day 7. The GM-CSF-Fc group and the mutant GM-CSF-Fc (YTE) and GM-CSF-Fc (L) groups also showed significant advantages in improving hematological indicators, with significant differences from the control group from day 1 (P<0.01), indicating that GM-CSF-Fc (L) can effectively reverse leukopenia (Table 4). Comparing the treatment groups, the therapeutic effect of GM-CSF-His gradually weakened from day 5, and there was no significant difference in white blood cell level between the GM-CSF-His group and the control group by day 10 (P>0.05). The GM-CSF-Fc group, GM-CSF-Fc(YTE) group, and GM-CSF-Fc(L) group all showed stable therapeutic effects within 10 days of the experiment. Among them, the GM-CSF-Fc(L) group had the best therapeutic effect. On the 5th day of treatment, the white blood cell level, especially the neutrophil count, was significantly higher than that of the GM-CSF-Fc group and the GM-CSF-Fc(YTE) group (P < 0.05), and it was the first to recover to the normal range.

[0089] In summary, the results of this embodiment demonstrate that the GM-CSF-Fc(L) recombinant protein prepared in this invention exhibits both significant therapeutic efficacy and good safety in treating a cat model of leukopenia, effectively promoting the recovery of peripheral blood leukocytes. Most importantly, the Fc fragment mutated at the L site not only endows the protein with long-lasting therapeutic properties but also allows it to maintain sustained blood drug concentrations while exhibiting optimal therapeutic effects.

[0090] Table 4. Analysis of blood routine index test results in the feline leukopenia treatment experiment.

[0091] .

[0092] Note: All data were analyzed using SPSS 22.0 software, and all results are expressed as mean (M) ± standard deviation (SD). t-tests were used to analyze differences between the two groups. A p-value < 0.05 indicated a significant difference; a p-value < 0.01 indicated a highly significant difference; and a p-value > 0.05 indicated no significant difference. Specifically, p1 represents the significant difference between the GM-CSF-His group and the control group, p2 represents the significant difference between the GM-CSF-Fc group and the control group, p3 represents the significant difference between the GM-CSF-Fc(YTE) group and the control group, and p4 represents the significant difference between the GM-CSF-Fc(L) group and the control group.

[0093] Example 4:

[0094] Determination of half-life of four GM-CSF recombinant proteins

[0095] Before the experiment, the animal room was strictly disinfected using formaldehyde fumigation and potassium permanganate. During the experiment, the temperature in the animal room was controlled at 24±2℃, and the relative humidity was controlled at around 40~60%. The experimental animals were fed and watered freely.

[0096] Twenty healthy cats aged 8-12 months and weighing approximately 2.5-3.5 kg were randomly divided into four groups (n=5 per group): GM-CSF-His group, GM-CSF-Fc group, GM-CSF-Fc (YTE) group, and GM-CSF-Fc (L) group. After one week of acclimatization in the animal facility, the cats were anesthetized with Sutair® 50 anesthetic (5-7.5 mg / kg). The protein injection method for each group of cats was the same as described in Section 3.2 of Example 3. Blood samples were collected venously at 0, 1, 2, 4, 8, 16, 24, 48, 72, 96, 120, 144, 168, 192, 216, and 240 hours after administration. The blood samples were coagulated at 4°C, centrifuged at 3000 rpm for 5 min, and the serum was separated and stored at -20°C for analysis. The concentration of GM-CSF protein in serum at each time point was determined by a double-antibody sandwich ELISA method. Anti-GM-CSF monoclonal antibody was used as the capture antibody, and HRP-labeled anti-GM-CSF secondary antibody was used as the detection antibody. After the detection was completed, DAS pharmacokinetic software was used for curve fitting and pharmacokinetic parameters were calculated.

[0097] The experiment yielded the following results:

[0098] This study analyzed the significant differences in the half-life of four GM-CSF recombinant proteins. The results showed that the half-life of the GM-CSF-Fc group (50.83±4.92 h) was significantly longer than that of the GM-CSF-His group (7.84±2.51 h) (P<0.01), an increase of approximately 6.5-fold. Further comparison within the Fc fusion protein group revealed that the mutants exhibited a further increase in half-life compared to the basic Fc fusion protein: the half-life of the GM-CSF-Fc(YTE) group (57.71±4.26 h) was significantly longer than that of the GM-CSF-Fc group (P<0.05), an increase of approximately 13.5%; while the half-life of the GM-CSF-Fc(L) group (64.1±2.57 h) showed a highly significant increase compared to the GM-CSF-Fc group (P<0.01), an increase of approximately 21.4%. Notably, the half-life of the GM-CSF-Fc(L) group was also significantly longer than that of the GM-CSF-Fc(YTE) group (P < 0.05), an increase of approximately 11.9%. In conclusion, the GM-CSF-Fc(L) mutant exhibited the longest in vivo circulation time among all groups, demonstrating optimal pharmacokinetic characteristics.

[0099] In summary, this invention employs a protein engineering strategy combining Fc domain fusion and L-site site-directed mutagenesis to significantly improve the pharmacokinetic properties of recombinant feline GM-CSF. Fc fusion is key to prolonging the drug's half-life and achieving an order-of-magnitude increase; further L-site mutation leads to even better pharmacokinetic characteristics. This invention not only significantly prolongs the half-life, reduces dosing frequency, and improves administration convenience and animal welfare, but also enhances therapeutic efficacy by maintaining stable blood drug concentrations, demonstrating significant clinical translational value.

[0100] Table 5. Comparison of half-lives of four GM-CSF recombinant proteins in cats.

[0101] .

[0102] Note: All data were analyzed using SPSS 22.0 software, and all results are expressed as mean (M) ± standard deviation (SD). The t-test was used to analyze differences between the two groups. A p-value < 0.05 indicated a significant difference; a p-value < 0.01 indicated a highly significant difference; and a p-value > 0.05 indicated no significant difference. P1 represents the statistical significance of differences between the GM-CSF-His group and the GM-CSF-Fc group, the GM-CSF-Fc(YTE) group, and the GM-CSF-Fc(L) group. P2 represents the statistical significance of differences between the GM-CSF-Fc group and the GM-CSF-His group, the GM-CSF-Fc(YTE) group, and the GM-CSF-Fc(L) group. P3 represents the statistical significance of differences between the GM-CSF-Fc(YTE) group and the GM-CSF-His group, the GM-CSF-Fc group, and the GM-CSF-Fc(L) group. P4 represents the statistical significance of differences between the GM-CSF-Fc(L) group and the GM-CSF-His group, the GM-CSF-Fc group, and the GM-CSF-Fc(YTE) group. " / " indicates no data.

[0103] Example 5:

[0104] Animal safety studies of GM-CSF-Fc(L)

[0105] 1. Safety experiment in mice

[0106] To verify the safety of the GM-CSF-Fc(L) prepared in this invention in mice, 20 8-week-old BALB / c mice weighing 22-24 g were randomly divided into two groups: a safety inspection group and a control group. Before the experiment, the animal room was strictly disinfected using formaldehyde fumigation and potassium permanganate. During the experiment, the temperature of the animal room was controlled at 24±2℃, and the humidity was controlled at approximately 40-60%. The experimental animals were fed and watered freely.

[0107] Mice in the security check group received a single subcutaneous injection of 5 times the therapeutic dose (50 μg / mouse) of GM-CSF-Fc (L) solution in their necks; mice in the control group received an equal volume of physiological saline solution via the same method. Mouse weight was recorded daily on day 0 (before security check) and after security check, and observed for 14 consecutive days. The following results were obtained:

[0108] Throughout the observation period, the body weight of mice in both the security inspection group and the control group showed the same steady upward trend, and there was no significant difference between the two groups (P>0.05). Figure 6 ).

[0109] The results showed that within 14 days after injection of GM-CSF-Fc(L) at 5 times the therapeutic dose, the weight of mice maintained a steady increase, indicating that GM-CSF-Fc(L) exhibited good safety in mice.

[0110] 2. Cat safety experiment

[0111] To verify the safety of the GM-CSF-Fc(L) prepared in this invention in cats, ten healthy domestic cats aged 8-12 months and weighing 2.5-3.5 kg were randomly divided into two groups: a safety inspection group and a control group. Before the experiment, the animal room was strictly disinfected using formaldehyde fumigation and potassium permanganate. During the experiment, the temperature in the animal room was controlled at 24±2℃, and the humidity was controlled at approximately 40-60%. The experimental animals were fed and watered freely.

[0112] Cats in the security check group received a single subcutaneous injection of 5 times the therapeutic dose (50 μg / kg) of GM-CSF-Fc (L) solution in their necks; cats in the control group received an equal volume of physiological saline solution via the same method. Body temperature, weight, and abnormal behaviors (such as vomiting, diarrhea, lethargy, etc.) were recorded daily on day 0 (before security check) and after security check, for a total of 14 days. The following results were obtained:

[0113] Throughout the observation period, neither the security inspection group nor the control group exhibited any abnormal clinical symptoms such as vomiting or diarrhea; their mental state and body temperature remained normal. Both groups maintained a physiological weight gain trend, with no significant difference between the groups (P > 0.05). Figure 7 ).

[0114] The above results indicate that within 14 days after injection of GM-CSF-Fc(L) at 5 times the therapeutic dose, no significant adverse reactions were observed in the cats, their body temperature remained stable, and their weight gain was stable, demonstrating good safety.

[0115] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A synthetically produced recombinant protein of feline granulocyte-macrophage colony-stimulating factor, the amino acid sequence of which is shown in SEQ ID NO.

7.

2. The fusion protein obtained by fusing the recombinant protein of claim 1 with a protein tag.

3. The gene encoding the recombinant protein of claim 1 or the fusion protein of claim 2.

4. An expression cassette or recombinant vector having the gene encoding as described in claim 3.

5. A recombinant microorganism or an isolated recombinant cell having the encoding gene of claim 3.

6. The recombinant cell according to claim 5, characterized in that: The recombinant cells mentioned are recombinant CHO cells.

7. A method for preparing the recombinant protein of claim 1 or the fusion protein of claim 2, comprising culturing CHO cells having an expression cassette or recombinant vector encoding the gene of claim 3.

8. The use of the recombinant protein of claim 1, the fusion protein of claim 2, the encoding gene of claim 3, the expression cassette or recombinant vector of claim 4, the recombinant microorganism of claim 5, and the recombinant cell of claim 5 or 6 in the preparation of long-acting feline granulocyte-macrophage colony-stimulating factor.

9. The use of the recombinant protein of claim 1, the fusion protein of claim 2, the encoding gene of claim 3, the expression cassette or recombinant vector of claim 4, the recombinant microorganism of claim 5, or the recombinant cell of claim 5 or 6 in the preparation of a medicament for treating feline leukopenia.

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