A prrsv-derived recombinant protein and use thereof

Abstract

This invention provides a recombinant protein comprising: an ORF-6 fragment of the antigenic protein of porcine reproductive and respiratory syndrome virus (PRRSV), the ORF-6 fragment having the amino acid sequence of SEQ ID NO: 1; an ORF-5 fragment of the antigenic protein of PRSV, the ORF-5 fragment having the amino acid sequence of SEQ ID NO: 2; and a linker connecting the C-terminus of the ORF-6 fragment to the N-terminus of the ORF-5 fragment, the linker having the amino acid sequence of SEQ ID NO: 3. This invention also provides the use of this recombinant protein in the preparation of pharmaceutical compositions for promoting macrophage polarization to M1 type or treating diseases associated with macrophage M2 type polarization.

Description

Technical Field

[0001] The present invention relates to a recombinant protein, specifically, to a recombinant protein derived from PRRSV, and the use of the recombinant protein in preparing a pharmaceutical composition for promoting macrophage polarization to M1 type or treating diseases associated with macrophage M2 polarization. Prior Art

[0002] According to the latest statistics from the Ministry of Health and Welfare of the Republic of China (Taiwan) in 2022, cancer has been the leading cause of death in Taiwan for 40 consecutive years. The cancer clock is being accelerated year by year, not only due to environmental damage that has led to an influx of carcinogens in our lives, but also due to advances in health education and medical technology, which have increased life expectancy, reaching the age at which cancer develops. As the elderly population grows, so too does their risk of cancer. However, cancer cells have the ability to release soluble molecules that activate oncogene signaling, facilitating their growth, survival, and metastasis, making cancer difficult to control and completely cure. Therefore, the development of effective cancer treatments is an urgent task.

[0003] Cancer treatments are rapidly evolving. In addition to the long-standing surgeries of surgery, chemotherapy, and radiotherapy, various molecular or cellular immunotherapies have emerged in recent years. These include CAR-T cell therapy (chimeric antigen receptor T cell), DC-CIK cell immunotherapy (coculture of dendritic cell-cytokine induced killer cells), immune checkpoint inhibitors, autologous immune cell activation immunotherapies, bispecific antibodies, and monoclonal antibodies.

[0004]

[0005] Among these novel treatments, DC (dendritic cell) therapy offers high specificity and low side effects. It can deliver tumor antigen signals to T lymphocytes, enabling T cells to accurately and effectively attack cancer cells. However, the tumor antigens required for this therapy are difficult to obtain, and due to the rapid mutation of cancer cells, using a single antigen system is ineffective, thus compromising efficacy.

[0006] Another new treatment, CAR-T cell therapy, involves genetically engineering a monoclonal antibody that can recognize cancer cells directly onto T cells in vitro, creating a chimeric antigen receptor (CAR). This allows these T cells to recognize and attack cancer cells. Once the expanded CAR-T cells are injected into the body, they will attack and eliminate the tumor cells upon recognizing antigens on their surface, without the need for MHC or co-stimulatory ligands. This therapy is highly specific and potent, and has been successfully used in clinical trials to cure B-cell acute lymphoblastic leukemia (by recognizing the CD19 protein on B cells, a specific target for leukemia). However, clinical trials primarily target solid tumors such as gliomas, breast cancer, lung cancer, and colorectal cancer. Solid tumors have an extremely complex tumor microenvironment, and CAR-T cells are less effective in treating these tumors, which lack specific markers. Furthermore, CAR-T therapy is associated with significant side effects. The induction of an immune storm caused by the release of excessive cytokines can be fatal, and there is also the potential risk of off-target effects. In addition, CAR-T therapy requires customized therapeutic cells for each patient, so the cost of treatment is very high. [Prior Art Literature] [Patent Document]

[0007] none Summary of the Invention

[0008] [Problems to be solved by the invention]

[0009] Macrophages are the white blood cells in the immune system responsible for engulfing and presenting invading pathogen antigens and attacking cancer cells. They are crucial mediators of the innate and adaptive immune systems and a major cell population in the tumor microenvironment. Signals within the microenvironment activate and polarize macrophages into distinct phenotypes: M1 (classically activated) and M2 (alternatively activated). Classically activated M1 macrophages are induced by lipopolysaccharide (LPS) and interferon-γ (IFN-γ) and produce high levels of proinflammatory cytokines such as interleukin-1 (IL-1), IL-6, IL-23, and tumor necrosis factor-α (TNF-α). M2 macrophages, alternatively activated by IL-4, IL-10, and IL-13, possess tissue repair and anti-inflammatory capabilities. Within the tumor microenvironment, they can promote tumor progression and suppress anti-tumor immune responses.

[0010] Cancer cells typically release various signaling factors that adapt them to the tumor microenvironment, where immune cells thrive. For example, specific factors can trigger the polarization of M1 macrophages, which initiate inflammation and phagocytose cancer cells, into M2 tumor-associated macrophages (TAMs), which promote tumor growth and suppress immune responses. TAMs promote tumor angiogenesis by secreting vascular endothelial growth factor (VEGF). VEGF also increases the expression of programmed death ligand 1 (PD-L1) on TAMs and cancer cells through autocrine and paracrine effects. These cell-surface proteins bind to the programmed death protein 1 (PD-1) receptor on T cells, B cells, and natural killer cells, leading to apoptosis of immune cells and ultimately suppressing the cancer patient's immune system. TAMs can also secrete metalloproteinases to destroy the extracellular matrix (ECM), promoting cancer cell metastasis. Furthermore, TAMs can induce the transformation of infiltrating macrophages from M1 to M2 by secreting the cytokine IL-10. For these reasons, inhibiting the polarization of macrophages to M2 tumor-associated macrophages is currently considered a promising immunotherapy for cancer cells.

[0011] Therefore, the present invention aims to provide a recombinant protein derived from PRRSV. Compared to the aforementioned prior art, this recombinant protein is easier to produce and possesses a superior ability to promote macrophage polarization to the M1 phenotype. Therefore, it can be further applied in clinical anticancer pharmaceutical compositions to effectively inhibit cancer cell growth and induce apoptosis. Furthermore, because its mechanism targets the influence of individual macrophage polarization patterns, it can avoid the generation of an immune storm. Furthermore, compared to DC dendritic cell therapy, the recombinant protein of the present invention is not designed to recognize tumor cell surface antigens in order to identify and eliminate cancer cells. Therefore, even if the target cancer cells mutate, the recombinant protein of the present invention still maintains its anticancer efficacy. [Technical means]

[0012] After extensive research on this topic, the inventors discovered that a recombinant protein derived from porcine reproductive and respiratory syndrome virus (PRRSV) can stimulate macrophage polarization to the M1 phenotype, activating M1 macrophages to phagocytose cancer cells and inhibiting the formation of M2 tumor-associated macrophages, preventing the continued proliferation and migration of cancer cells. This discovery suggests that the protein could be used as an anticancer drug. Importantly, PRRSV is a porcine virus that causes blue ear disease in commercial animal markets and does not infect humans. Currently, two distinct serotypes of PRRSV are prevalent: the North American and European serotypes. Porcine alveolar macrophages are its host cells. The CD163 receptor on their membranes binds to the M-GP5 glycoprotein complex on the PRRS virus envelope, making the virus host-specific. However, compared to the prior art, the PRRSV recombinant protein provided by the present invention also exhibits cross-reactivity against mouse, rat, and human cells, surpassing the host cell specificity of the PRRS virus strain.

[0013] Therefore, in order to achieve the above-mentioned objectives, the present invention provides the following technical means.

[0014] In one aspect, the present invention provides a recombinant protein characterized in that it comprises: an ORF-6 fragment of an antigenic protein of porcine reproductive and respiratory syndrome virus, wherein the ORF-6 fragment of the antigenic protein has an amino acid sequence of SEQ ID NO: 1; A porcine reproductive and respiratory syndrome virus antigen protein ORF-5 fragment, the antigen protein ORF-5 fragment having the amino acid sequence of SEQ ID NO: 2; A linker connects the C-terminus of the antigen protein ORF-6 fragment and the N-terminus of the antigen protein ORF-5 fragment, and the linker has the amino acid sequence of SEQ ID NO: 3.

[0015] In some embodiments, the recombinant protein has the amino acid sequence of SEQ ID NO: 4.

[0016] In some embodiments, the recombinant protein is produced by a baculovirus expression vector system.

[0017] In one embodiment, the present invention provides a use of the recombinant protein as described above for preparing a drug, wherein the drug is used to promote macrophage polarization to the M1 type.

[0018] In one aspect, the present invention provides a use of the recombinant protein as described above for preparing a medicament, wherein the medicament is used to treat a disease associated with macrophage M2 polarization.

[0019] In one aspect, the present invention provides a use as described above, wherein the disease associated with M2 macrophage polarization is cancer. In another aspect, the disease associated with M2 macrophage polarization is a solid tumor, including but not limited to glioma, breast cancer, lung cancer, colorectal cancer, etc.

[0020] In one embodiment, the present invention provides a use of the recombinant protein as described above for preparing a drug, wherein the drug is used to promote the expression of at least one of the following biomarkers in macrophages: CXCL10, CCR7, IL-1β, CCL2, Cox2, CD80. [Effects of the Invention]

[0021] The present invention provides a recombinant protein derived from PRRSV that can be used to promote the polarization of human macrophages into M1 rather than M2 tumor-associated macrophages (TAMs). This recombinant protein significantly and effectively increases the mRNA expression of M1 macrophage biomarkers CXCL10, CCR7, IL-1β, CCL2, Cox2, and CD80. Furthermore, the recombinant protein of the present invention exhibits excellent tumor cell growth inhibition. Therefore, this recombinant protein can also be used to prepare a drug for treating conditions associated with M2 macrophage polarization, such as cancer, including solid tumors such as gliomas. Simple diagram description

[0022] [Figure 1] Figure 1A shows the linear model of the genomic structure of the recombinant protein ORF-6L5 (A3); Figure 1B shows the linear model of the genomic structure of the recombinant protein G6L10T (A1). [Figure 2] Figure 2A shows an amino acid sequence alignment of ORF-6 of recombinant proteins A1 and A3; Figure 2B shows an amino acid sequence alignment of ORF-5 of recombinant proteins A1 and A3. The C-termini of the recombinant proteins are located outside the ORF-6 and ORF-5 sequences. The C-terminus of A1 (also known as g6Ld10T) is located at the last amino group of the His-Tag (HHHHH), while the C-terminus of A3 (also known as ORF-6L5) is located after the His-Tag (HHHHH). [ FIG. 3 ] shows the mRNA expression levels of M1 macrophage biomarkers in each treatment group of porcine alveolar macrophages (PAMs) in Example 1. [Figure 4] shows the mRNA expression levels of M2 macrophage biomarkers in each treatment group of porcine alveolar macrophages in Example 2. [ FIG. 5 ] shows the mRNA expression levels of M1 macrophage biomarkers in each treatment group of human THP-1-derived macrophages in Example 3. [ FIG. 6 ] shows the mRNA expression levels of M2 macrophage biomarkers in each treatment group of human THP-1-derived macrophages in Example 4. Figure 7 shows the results of the MTT assay of rat C6 glioma cell line treated with different concentrations of recombinant protein A3 in Example 5 (Figure 7A), the half-inhibitory concentration (IC50) obtained based on the survival rate (Figure 7B), and the cell growth status under a light microscope (Figure 7C). [ FIG. 8 ] shows the mRNA expression levels of M1 macrophage biomarkers in each treatment group of porcine alveolar macrophages in Comparative Example 1. [ FIG. 9 ] shows the mRNA expression levels of M2 macrophage biomarkers in each treatment group of porcine alveolar macrophages in Comparative Example 2. [ Figure 10 ] shows the results of MTT assay of rat C6 glioma cell line cell survival rate analysis ( Figure 10A ), the half-inhibitory concentration (IC 50 ) obtained based on the survival rate ( Figure 10B ), and the cell growth status under a light microscope ( Figure 10C ) when recombinant protein A1 was administered at different concentrations. Implementation Method

[0023] The following discloses an embodiment of the present invention. It should be noted that the present invention is not limited to the embodiment entirely including the following description. The following description is only used to illustrate the details of the present invention and the effects of its implementation.

[0024] The definitions of terms used in this invention are based on currently accepted definitions in the biomedical field, and appropriate explanations are provided herein. These definitions apply throughout this specification, unless otherwise specified in the specification based on specific circumstances.

[0025] The term "protein" in the present invention refers to an organic polymer composed of two or more amino acid monomers and / or their analogs, including, for example, full-length proteins or fragments of full-length proteins.

[0026] The term "amino acid" in this invention refers to any of the 20 naturally occurring amino acids, including synthetic amino acids with non-natural side chains, and also includes dextrorotatory (D) and levorotatory (L) optical isomers.

[0027] The term "recombinant protein" in the present invention refers to a functional fragment of a full-length protein, or a chimeric protein containing functional fragments from different sources.

[0028] As used herein, the terms "link," "connect," and "link" refer to the connection or binding of two or more proteins by bonding or non-bonding interactions, including direct or indirect connections. In some embodiments, the connection is indirect, for example, via a linker.

[0029] The term "treatment" as used herein refers to methods for achieving beneficial or desired clinical outcomes in a patient. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: alleviation of symptoms, reduction in disease severity, failure to progress in the disease state, prevention of disease spread, prevention of disease occurrence or recurrence, slowing of disease progression, improvement in the disease state, and remission.

[0030] In the embodiments described below, baculovirus is selected as the expression vector system to produce the recombinant protein of the present invention, which further illustrates the embodiments of the present invention, but the present invention is not limited to this embodiment.

[0031] Construction of recombinant protein: The coding sequence of the desired PRRSV gene is cloned into the restriction enzyme cutting site of the baculovirus transfer plasmid to form a transfer vector. This transfer vector and baculovirus are co-transfected into Hi-5 insect cells to form a recombinant baculovirus containing PRRSV antigens. Recombinant protein 3 (A3) was produced using a baculovirus expression vector. The recombinant protein A3 was constructed as follows: a partial amino acid sequence (amino acids 31-168) of ORF-5 of the glycoprotein GP5 of PRRSV was combined with a partial amino acid sequence (amino acids 1-174) of ORF-6 of the M protein of PRRSV. A linker was used to connect the C-terminus of the ORF-6 fragment of the antigenic protein to the N-terminus of the ORF-5 fragment of the antigenic protein. Subsequently, a hexahistidine tag was attached to the C-terminus of the ORF-5 fragment, as shown in Figure 1A. The recombinant protein 1 (A1) was produced using a baculovirus expression vector. The recombinant protein A1 is constructed as follows: it contains the PRRSV structural protein, a partial amino acid sequence of ORF-6 of the M protein (amino acids 1-175), a partial amino acid sequence of ORF-5 of the glycoprotein GP5 (amino acids 11-201), the T cell antigenic determinant T1 fragment (amino acids 117-131), and the T cell antigenic determinant T2 fragment (amino acids 149-163). A hexahistidine tag is attached to the C-terminus of the T2 fragment. The amino acid sequence of the recombinant protein A1 from the N-terminus to the C-terminus is: N-terminus - structural protein (SP) - ORF-6 - ORF-5 - T1 - T2 - (His) 6 - C-terminus. The detailed structure is shown in Figure 1B.

[0032] Furthermore, although both recombinant protein A1 and recombinant protein A3 contain the ORF-5 and ORF-6 fragments of PRRSV, their sequences differ slightly because recombinant protein A1 utilizes the mainland PRRSV strain, while recombinant protein A3 utilizes the Taiwan PRRSV strain. A further amino acid sequence alignment is shown in Figure 2.

[0033] The amino acid sequences (SEQ ID NOs: 1-4) and the corresponding nucleotide sequences (SEQ ID NOs: 39-42) used in the A3 plasmid construction are shown in Table 1 below.

[0034] Table 1

[0035] The amino acid sequences (SEQ ID NOs: 43-45) and the corresponding nucleotide sequences (SEQ ID NOs: 46-48) used in the A1 plasmid construction are shown in Table 2 below.

[0036] Table 2

[0037] Collection of porcine alveolar macrophages: First, pigs were euthanized and the trachea was ligated to prevent complete alveolar collapse. Subsequently, the heart and lungs were removed from the thoracic cavity. Alveolar macrophages were collected from fresh lungs under aseptic procedures. The lungs were washed intratracheally two to four times with phosphate-buffered saline (PBS), and the washes containing PAMs were centrifuged at 800 x g for 10 minutes. The collected PAMs were cultured in 12-well plates using complete RPMI-1640 medium (Corning, Manassas, OH, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA) at 37°C in a humidified atmosphere of 5% carbon dioxide.

[0038] In the following examples, various primers used in quantitative real-time polymerase chain reaction (qPCR) to analyze cytokine expression in porcine alveolar macrophages (PAMs) are shown in Table 3. The target gene names, primer sequences, and sequence numbers are listed below. The qPCR amplification step was set at 95°C for 3 minutes, followed by 40 denaturation cycles at 95°C for 3 seconds, and a final binding temperature of 60°C for 20 seconds. The collected mRNA expression data were compared using the 2-ΔΔCT method for relative quantification.

[0039] Table 3

[0040] In the following examples, various primers were used in quantitative real-time polymerase chain reaction (qPCR) to analyze cytokine expression in human leukocyte monocytic cells (THP-1). The target gene names, primer sequences, and sequence numbers are shown in Table 4. The qPCR amplification step was set at 95°C for 3 minutes, followed by 40 denaturation cycles at 95°C for 3 seconds, and a final binding temperature of 60°C for 20 seconds. The collected mRNA expression data were compared using the 2-ΔΔCT method for relative quantification.

[0041] Table 4 [Example]

[0042] Example 1: Effect of recombinant protein A3 on promoting the polarization of porcine alveolar macrophages to M1 type

[0043] Pigs (n = 4) that had not been administered recombinant protein A3 were euthanized. Porcine alveolar macrophages (PAMs) were collected as previously described and divided into seven groups. The cells were stimulated with LPS, IL-4, baculovirus expression vectors, PRRSV at infection doses (MOIs) of 0.01 and 0.05, and recombinant protein A3. PAMs treated with PBS served as a control. The cells were cultured in 12-well plates using RPMI-1640 medium supplemented with 5% fetal bovine serum for 24 hours (37°C, 5% CO₂). Total RNA was then extracted and subjected to real-time polymerase chain reaction (qPCR) using the primers listed in Table 3 to quantify proinflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-12) and β-actin. The instrument used was a Rotor-Gene Q 5plex (Qiagen, USA). The expression levels of cytokine genes were normalized based on the expression level of β-actin gene and expressed as n-fold increase or decrease relative to the PBS control group.

[0044] Figure 3 shows the expression of pro-inflammatory cytokines. Compared to other groups, porcine alveolar macrophages stimulated with recombinant protein A3 or LPS showed significantly higher mRNA levels of TNF-α, IL-1β, IL-6, and IL-12. Recombinant protein A3, like LPS, effectively stimulates macrophage polarization to the M1 phenotype. In Figure 3, "*" indicates a significant difference compared to the control group (p < 0.05), "ϕ" indicates a significant difference compared to the 0.01 MOI PRRSV-treated group (p < 0.05), and "ψ" indicates a significant difference compared to the 0.05 MOI PRRSV-treated group (p < 0.05).

[0045] Example 2: Effect of recombinant protein A3 on the polarization of porcine alveolar macrophages to M2 type

[0046] Following the experimental procedures of Example 1, porcine alveolar macrophages were collected and divided into seven groups. The cells were stimulated with LPS, IL-4, baculovirus expression vectors, PRRSV at infection doses (MOI) of 0.01 and 0.05, and recombinant protein A3. Cells treated with PBS served as a control group. Cells were cultured in 12-well plates using RPMI-1640 medium supplemented with 5% fetal bovine serum for 24 hours (37°C, 5% CO₂). Total RNA was then extracted and subjected to real-time polymerase chain reaction (qPCR). The primers listed in Table 3 were used to quantify M2 macrophage biomarkers (Arg1 and Chil-3) and β-actin using a Rotor-Gene Q 5plex (Qiagen, USA). Cytokine gene expression levels were normalized to β-actin expression and expressed as n-fold increase or decrease relative to the PBS control group.

[0047] The experimental results are shown in Figure 4. Compared to the control group, there was no significant difference in the mRNA expression of Arg1 and Chil-3 in porcine alveolar macrophages stimulated with recombinant protein A3. However, IL-4 and 0.05 MOI of PRRSV increased the expression of Arg1 and Chil-3. This result indicates that recombinant protein A3 does not induce macrophage polarization to the M2 phenotype. In Figure 4, "*" indicates a significant difference compared to the control group (p < 0.05), "ϕ" indicates a significant difference compared to the 0.01 MOI of PRRSV treatment group (p < 0.05), and "ψ" indicates a significant difference compared to 0.05 MOI of PRRSV treatment (p < 0.05).

[0048] Example 3: Effect of recombinant protein A3 on promoting polarization of human THP-1-derived macrophages to M1 type

[0049] The purpose of this example was to further confirm the efficacy of recombinant protein A3 in promoting M1 polarization of human THP-1 cell-derived macrophages. Human monocytic leukocytes (THP-1) were treated with 100 nM phorbol myristate acetat (PMA) for 48 hours to polarize into M0 macrophages. These M0 cells were then divided into seven groups. Group 1 included PBS-treated cells. The M0 cells were then stimulated with LPS, IL-4, vehicle, recombinant protein A3 (2 μg / ml), and recombinant protein A1 (2 μg / ml). THP-1 cells that had not been treated with PMA and had been treated with PBS served as control group 2. Cells were cultured in 12-well plates using RPMI-1640 medium supplemented with 5% fetal bovine serum for 24 hours (37°C, 5% CO₂). Total RNA was then extracted and subjected to real-time polymerase chain reaction (qPCR). Several classic macrophage M1 biomarkers (CXCL10, CCR7, IL-1β, CCL2, Cox2, and CD80) and β-actin were quantified by qPCR using the primers listed in Table 4. The instrument was a Rotor-Gene Q 5plex (Qiagen, USA). Cytokine gene expression levels were normalized to β-actin gene expression and expressed as n-fold increase or decrease relative to the PBS control group. Experimental data are determined by mean ± SEM, sample number n=7-10, "a" indicates a significant difference compared with control group 1 (p<0.05); "b" indicates a significant difference compared with the LPS group (p<0.05); "c" indicates a significant difference compared with the IL-4 added group (p<0.05); "d" indicates a significant difference compared with the recombinant protein A3 added group (p<0.05); "e" indicates a significant difference compared with control group 2 (p<0.05).

[0050] The experimental results, shown in Figure 5, show that compared to the control group, human macrophages stimulated with recombinant protein A3 or LPS exhibited significantly higher mRNA expression levels of CXCL10, CCR7, IL-1β, CCL2, Cox2, and CD80, further confirming that recombinant protein A3, like LPS, can effectively stimulate M1 polarization in human macrophages. In contrast, while recombinant protein A1 effectively promoted M1 polarization in porcine alveolar macrophages in the comparative example described below, it failed to successfully induce M1 polarization in human macrophages when administered to them. This suggests that the effect of recombinant protein A1 is related to host cell specificity.

[0051] Example 4: Effect of recombinant protein A3 on polarization of human macrophages to M2 type

[0052] We further evaluated the effect of recombinant protein A3 on the polarization of human macrophages to the M2 type when administered to macrophages derived from human THP-1 cell line.

[0053] Human monocytic leukocytes (THP-1) were treated with 100 nM phorbol myristate acetat (PMA) for 48 hours to polarize them into macrophages (M0). Subsequently, the M0 cells were divided into seven groups. PBS-treated cells served as control group 1. Human M0 cells were stimulated with LPS, IL-4, vehicle, recombinant protein A3 (2 μg / ml), and recombinant protein A1 (2 μg / ml). THP-1 cells that were not treated with PMA or treated with PBS served as control group 2. The cells were cultured in 12-well plates in RPMI-1640 medium supplemented with 5% fetal bovine serum. The treated cells were incubated for 24 hours (37°C, 5% CO2).

[0054] Next, total RNA was extracted from these samples and subjected to real-time polymerase chain reaction (qPCR). Quantification of several macrophage M2 biomarkers (CD206, CD209, and CCL17) and β-actin was performed using the primers listed in Table 4. The instrument was a Rotor-Gene Q 5plex (Qiagen, USA). Cytokine gene expression levels were normalized to β-actin gene expression and expressed as an n-fold increase or decrease relative to the PBS control group. Experimental data are determined by mean ± SEM, sample number n=7-10, "a" indicates a significant difference compared with control group 1 (p<0.05); "b" indicates a significant difference compared with the LPS group (p<0.05); "c" indicates a significant difference compared with the IL-4 supplemented group (p<0.05); "d" indicates a significant difference compared with the recombinant protein A3 supplemented group (p<0.05); "e" indicates a significant difference compared with control group 2 (p<0.05).

[0055] The experimental results are shown in Figure 6. Compared with the control group, there was no significant difference in the mRNA expression levels of CD206, CD209, and CCL17 in human macrophages stimulated by recombinant protein A3, while IL-4 increased the expression levels of these markers. This result indicates that recombinant protein A3 does not induce macrophage polarization to the M2 type.

[0056] Example 5: Effect of recombinant protein A3 on inhibiting glioma cells

[0057] Examples 1-4 demonstrated that recombinant protein A3 effectively promotes macrophage polarization to the M1 phenotype, while also preventing macrophage polarization to the M2 phenotype, regardless of whether administered to porcine or human macrophages. To further confirm the ability of recombinant protein A3 to effectively induce apoptosis and inhibit cancer cell growth, the MTT assay was used to analyze the cell viability of rat C6 glioma cells treated with recombinant protein A3.

[0058] First, rat C6 glioma cells (n=7) were treated with various concentrations of recombinant protein A3 and cultured for 72 hours. Eight experimental groups were divided into the control group, groups treated with recombinant protein A3 at concentrations of 0.312 µg / ml, 0.625 µg / ml, 1.25 µg / ml, 2.5 µg / ml, 5 µg / ml, and 10 µg / ml, and a group treated with 7 µM cisplatin. MTT formazan absorbance was measured at a wavelength of 570 nm. Cell viability analysis using the MTT assay is shown in Figures 7A-7C. As shown in the bar graph in Figure 7A, the cell viability of C6 glioma cells exposed to different concentrations of recombinant protein A3 decreased with increasing concentrations. Figure 7B shows the half-inhibitory concentration (IC50) of recombinant protein A3 against rat C6 glioma cells, calculated based on MTT assay results. The IC50 of recombinant protein A3 is 9.419 µg / ml. Figures 7A and 7B show that higher concentrations of recombinant protein A3 significantly reduce the survival rate of C6 glioma cells. Furthermore, the cell survival rate (Figure 7A) demonstrates that A3 concentrations below 2.5 µg / ml do not cause cytotoxicity. The aforementioned macrophage polarization assay was also conducted based on the safety concentration standard shown in Figure 7A, indicating that macrophage polarization is not affected by drug cytotoxicity.

[0059] The growth of C6 glioma cells after administration of different concentrations of recombinant protein A3 was observed under an optical microscope at 200x magnification 24, 48, and 72 hours later. The results, shown in Figure 7C, clearly show that after 72 hours of administration of recombinant protein A3 at a concentration of 10 µg / ml, the glioma cells not only stopped growing and dividing, but even underwent apoptosis compared to the control group.

[0060] Therefore, as shown in Figures 7A-7C, administration of a certain concentration of recombinant protein A3 to C6 glioma cells significantly reduced the survival rate of glioma cells. This result confirms that recombinant protein A3 does have the excellent ability to eliminate cancer cells in solid tumors such as gliomas.

[0061] In this experiment, cell viability was defined as the ratio (percentage) of the absorbance values ​​of C6 glioma cells treated with recombinant protein A3 relative to those of untreated C6 glioma cells. The vehicle + 10% FBS + DMEM-treated group served as the negative control, and the cisplatin-treated group served as the positive control. Data in Figure 7A are presented as mean ± SEM. Significant differences compared to the negative control are indicated by "*" (P < 0.05), "**" (P < 0.005), and "***" (P < 0.0005). These data are based on seven replicate experiments (n = 7).

[0062] Comparative Example 1: Effect of Recombinant Protein A1 on Promoting the Polarization of Porcine Alveolar Macrophages to M1 Type

[0063] Following the same experimental procedures as described in Example 1, pigs (n = 4) that had not been administered recombinant protein A1 were euthanized. Porcine alveolar macrophages (PAMs) were collected as described above and divided into five groups. The cells were stimulated with LPS, IL-4, PRRSV virus, or recombinant protein A1, respectively. PAMs treated with PBS served as a control. The cells were cultured in 12-well plates in RPMI-1640 medium supplemented with 5% fetal bovine serum for 24 hours (37°C, 5% CO₂). Total RNA was then extracted and subjected to real-time polymerase chain reaction (qPCR) using the primers listed in Table 3 to quantify proinflammatory cytokines (TNF-α, IL-6, and IL-12) and β-actin. The assay was performed using a Rotor-Gene Q 5plex (Qiagen, USA). The expression levels of cytokine genes were normalized based on the expression level of β-actin gene and expressed as n-fold increase or decrease relative to the PBS control group.

[0064] The results of pro-inflammatory cytokine expression are shown in Figure 8. Compared with other groups, porcine alveolar macrophages stimulated with recombinant protein A1 and LPS had significantly higher mRNA expression levels of TNF-α, IL-6, and IL-12, indicating that recombinant protein A1 can indeed effectively stimulate macrophage polarization to the M1 type.

[0065] Comparative Example 2: Effect of Recombinant Protein A1 on the Polarization of Porcine Alveolar Macrophages to M2 Type

[0066] Following the same experimental procedures as in Comparative Example 1, porcine alveolar macrophages were collected and divided into five groups. The cells were stimulated with LPS, IL-4, PRRSV virus, and recombinant protein A1, respectively. A PBS-treated group served as a control. The cells were cultured in 12-well plates using RPMI-1640 medium supplemented with 5% fetal bovine serum for 24 hours (37°C, 5% CO₂). Total RNA was then extracted and subjected to real-time polymerase chain reaction (qPCR). Expression of the M2 macrophage biomarker (Arg1) and β-actin was quantified by qPCR using the primers listed in Table 3. The instrument was a Rotor-Gene Q 5plex (Qiagen, USA). Cytokine gene expression levels were normalized to β-actin expression and expressed as n-fold increase or decrease relative to the PBS control group.

[0067] The experimental results are shown in Figure 9. After stimulation with recombinant protein A1, there was no significant difference in the expression of Arg1 mRNA in porcine alveolar macrophages compared with the control group, while IL-4 increased the expression of Arg1. This result indicates that recombinant protein A1 does not induce macrophage polarization to the M2 type.

[0068] Comparative Example 3: Effect of Recombinant Protein A1 on Inhibiting Glioma Cells

[0069] The purpose of this study was to evaluate whether recombinant protein A1 has the ability to induce apoptosis and inhibit the growth of cancer cells like recombinant protein A3. The cell survival rate of rat C6 glioma cell line treated with recombinant protein A1 was analyzed by MTT assay.

[0070] The experimental procedures were the same as in Example 5. First, rat C6 glioma cells (n = 7) were treated with various concentrations of recombinant protein A1 and cultured for 72 hours. Nine experimental groups were divided into the following: a control group, 10% FBS + DMEM, groups treated with recombinant protein A1 at concentrations of 0.312 µg / ml, 0.625 µg / ml, 1.25 µg / ml, 2.5 µg / ml, 5 µg / ml, and 10 µg / ml, and a group treated with 7 µM cisplatin. MTT formazan absorbance was measured at a wavelength of 570 nm. Cell viability was analyzed using an MTT assay, and the results are shown in Figures 10A-10C. As shown in the bar graph in Figure 10A, there was no significant difference in cell viability among C6 glioma cells exposed to different concentrations of recombinant protein A1, and no significant difference was observed compared to the control group. Figure 10B shows the half-inhibitory concentration (IC50) of recombinant protein A1 against rat C6 glioma cells, calculated from MTT assay results. The IC50 of recombinant protein A1 reached as high as 3958 µg / ml. Figures 10A and 10B show that even at a concentration of 10 µg / ml, recombinant protein A1 was unable to reduce the survival rate of C6 glioma cells.

[0071] The growth of C6 glioma cells was observed under an optical microscope at 200x magnification 24, 48, and 72 hours after administration of different concentrations of recombinant protein A1. As shown in Figure 10C , even after administration of a high concentration of 10 µg / ml of recombinant protein A1 for 72 hours, there was no significant difference in cell growth compared to the control group, indicating continued cell proliferation and no apoptosis.

[0072] Therefore, as shown in Figures 10A-10C , even when high concentrations of recombinant protein A1 were administered to C6 glioma cells, their survival rate was not affected. This result confirms that recombinant protein A1, unlike recombinant protein A3, does not possess the ability to eliminate cancer cells or affect their growth.

[0073] As shown in the results of the above examples and comparative examples, the present invention provides a recombinant protein derived from PRRSV and its uses. This recombinant protein can be used to promote the polarization of human macrophages to the M1 phenotype, significantly and effectively increasing the expression of M1 macrophage biomarkers CXCL10, CCR7, IL-1β, CCL2, Cox2, and CD80. The recombinant protein of the present invention has excellent tumor cell growth inhibition capabilities and can therefore be used to prepare a medicament. This medicament can be used to treat conditions associated with M2 macrophage polarization, such as cancer, including solid tumors such as gliomas.

[0074] none

[0075] TW202411240A_111133934_SEQL.xml

Claims

1. A recombinant protein, characterized in that it comprises: an ORF-6 fragment of an antigenic protein of porcine reproductive and respiratory syndrome virus (PRRSV), the ORF-6 fragment having the amino acid sequence of SEQ ID NO: 1; an ORF-5 fragment of an antigenic protein of PRSV, the ORF-5 fragment having the amino acid sequence of SEQ ID NO: 2; and a linker connecting the C-terminus of the ORF-6 fragment to the N-terminus of the ORF-5 fragment, the linker having the amino acid sequence of SEQ ID NO:

3.

2. The recombinant protein as described in claim 1, wherein, The recombinant protein has the amino acid sequence of SEQ ID NO:

4.

3. The recombinant protein as described in claims 1 to 2, wherein, This recombinant protein was produced using a baculovirus expression vector system.

4. Use of the recombinant protein according to any one of claims 1 to 3 for the preparation of a medicament, characterized in that the medicament is used to promote macrophage polarization to the M1 type.

5. Use of the recombinant protein according to any one of claims 1 to 3 for the preparation of a medicament, characterized in that the medicament is for the treatment of glioma.

6. Use of the recombinant protein of any one of claims 1 to 3 for the preparation of a medicament, characterized in that the medicament is used to promote the expression of at least one of the following biomarkers of macrophages: CXCL10, CCR7, IL-1β, CCL2, Cox2, CD80.

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