Glycosylated fusion protein, nucleic acid molecule, expression vector, host cell and use thereof

Abstract

Provided in the present application are a glycosylated fusion protein, a nucleic acid molecule, an expression vector, a host cell and the use thereof. A first aspect of the present application provides the glycosylated fusion protein, comprising a murine Fc variant and a hepatitis B virus antigen, wherein the murine Fc variant is obtained by performing amino acid mutation and non-mammalian glycosylation modification on a murine Fc fragment, the murine Fc variant comprises at least one of alanine at position 223, alanine at position 228, alanine at position 230, leucine at position 330, and glutamic acid at position 332, the glycosylation modification does not comprise sialic acid modification, and the positions are numbered according to the EU numbering system. The fusion protein can bind to DC cells and activate DC cells, and thus promote the proliferation and activation of specific T cells.

Description

Glycosyl-modified fusion proteins, nucleic acid molecules, expression vectors, host cells, and applications

[0001] This application claims priority to Chinese Patent Application No. 202411894669X, filed on December 20, 2024, entitled "Glycosyl-modified fusion protein, nucleic acid molecule, expression vector, host cell and application", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application relates to a glycosylated fusion protein, nucleic acid molecule, expression vector, host cell, and application, and relates to the field of biopharmaceutical technology. Background Technology

[0003] Human hepatitis B virus (HBV) is a member of a family of DNA viruses that primarily infect the liver. HBV mainly infects hepatocytes and can cause both acute and chronic liver disease, leading to cirrhosis and liver cancer. Approximately 90% of adults infected with HBV clear the infection, while the remaining 10% become chronic carriers, who are highly likely to develop cirrhosis and hepatocellular carcinoma (Block, 2016). The World Health Organization estimates that over 2 billion people are infected with HBV, of whom 350 million are chronically infected. Prophylactic vaccines based on HBV surface antigen (HBsAg) are highly effective in providing protective immunity against HBV infection. These vaccines are developed from HBsAg purified from the plasma of chronic HBV carriers and produced using recombinant DNA technology and synthetic peptides (see U.S. Patents 4,599,230 and 4,599,231). These vaccines are highly effective in preventing infection but in eradicating established chronic infection.

[0004] When a healthy host (human or animal) encounters an antigen (such as a protein derived from bacteria, viruses, and / or parasites), the host typically initiates an immune response. This immune response can be humoral or cellular. In a humoral response, B cells produce antibodies in response to antigenic stimulation and secrete them into the blood and / or lymph. The antibodies then neutralize the antigen (e.g., a virus) by specifically binding to the antigen on their surface, are phagocytosed by phagocytes, and / or killed by complement-mediated mechanisms. A cellular response is characterized by the ability to select and increase specific helper cells and cytotoxic T lymphocytes to directly clear cells containing antigens. However, in many individuals, the immune system does not respond to certain antigens. When antigens do not stimulate the production of specific antibodies and / or cytotoxic T cells, the immune system is unable to prevent the resulting disease. Therefore, an infectious agent (e.g., a virus) can establish a chronic infection, and the host's immune system can develop antigen tolerance to the virus. The most common cases of chronic viral infection include hepatitis B, hepatitis C, human immunodeficiency virus (HIV), and herpes simplex virus (HSV).

[0005] In chronic infection, viruses can evade the host's immune system. Viral antigens are recognized as "self" substances within the body and are therefore not recognized by antigen-presenting cells. The way antigen-presenting cells process antigens depends on their location (Steinman et al., 1999). Exogenous antigens are processed in the APC endosome, and the resulting peptide fragments mix with major histocompatibility complex (MHC) class II molecules and are presented on the cell surface. This complex is presented to CD4+ T cells and stimulates the production of CD4+ T helper cells. Cytokines secreted by the helper cells then stimulate B cells to produce antibodies against the exogenous antigen (humoral response). Immunization using antigens typically generates an antibody response through this endosome antigen processing pathway. On the other hand, intracellular antigens are processed in the proteasome, and the resulting peptide fragments form a complex with MHC class I molecules and are presented on the APC surface. After this complex binds to the co-receptor CD8 molecule, the antigen is presented to CD8+ T cells, triggering a cytotoxic T cell (CTL) immune response that kills the host cells carrying the antigen.

[0006] In patients with chronic viral infections, viral antigens are produced within host cells due to active viral replication, and these secreted antigens circulate in bodily fluids. For example, virions, HBV surface antigen, and core antigen (e antigen) can be detected in the blood of chronic HBV carriers. Effective therapeutic vaccines may be able to induce CTL responses against intracellular antigens or antigens delivered to appropriate cellular compartments, thereby activating the MHC class I processing pathway. CTL-inducible therapeutic vaccines can be processed via the proteasome pathway and presented via the MHC class I pathway (Larsson et al., 2001). This can be achieved either by producing antigens within host cells or by delivering the vaccine to appropriate cellular compartments for processing and presentation to elicit a cellular response. Several methods for intracellular antigen delivery have been documented in the literature.

[0007] Dendritic cells (DCs), derived from blood monocytes, can be immunomodulators that stimulate naïve T-cell responses due to their ability to act as professional antigen-presenting cells (Steinman et al., 1999; Banchereau and Steinman, 1998). This unique property of dendritic cells in capturing, processing, presenting antigens, and stimulating naïve T cells makes them a crucial tool for developing therapeutic vaccines (Laupeze et al., 1999). Antigen targeting of dendritic cells is an important step in antigen presentation, and the presence of several receptors on dendritic cells targeting the Fc region of monoclonal antibodies has been used for this purpose (Regnault et al., 1999). Examples of this approach include ovarian cancer Mab-B43.13 antibodies, anti-PSA antibodies, and anti-HBV antibody-antigen complexes (Wen et al., 1999). Studies have shown that cancer immunotherapy using dendritic cells loaded with tumor-associated antigens generates tumor-specific immune responses and anti-tumor activity (Campton et al., 2000; Fong and Engleman, 2000). Satisfactory results were obtained in in vivo clinical trials using dendritic cells sensitized with tumor antigens (Tarte and Klein, 1999). These studies clearly demonstrate the efficacy of using dendritic cells to generate an immune response against cancer antigens.

[0008] In addition, previous studies have shown that chimeric antigens (fusion of Fc antibody fragments and HBV antigens) can effectively elicit antigen-specific T-cell and B-cell-mediated immune responses against HBV and can also break tolerance to HBV antigens under chronic conditions (George et al., US Patents 8007805B2, 8025873B2, 8029803B2 and 8465745B2, Chinese Patent 200480022747.1, Indian Patent 225281, Japanese Patent 5200201, Korean Patent 10-1327719, New Zealand Patent 545048, Singapore Patent 119567, South African Patent 2006-0804, Australian Patent 2012200998, effective European Patents; German, French and British Patent 1664270; Ma et al., 2020; George et al., 2020). Compared to reports on the modification of the Fc fragment of human immunoglobulins, there are relatively few reports on the modification of the Fc fragment of mouse immunoglobulins. We believe it is necessary to conduct research on the engineering modification of the Fc fragment of mouse immunoglobulins to determine whether the engineering modification can increase the presentation effect. Summary of the Invention

[0009] This application provides a glycosylated fusion protein comprising a murine Fc variant and a hepatitis B virus antigen, wherein the murine Fc variant is obtained by modifying a murine IgG1 Fc fragment. This fusion protein can bind to dendritic cells (DCs), activate DCs, and promote the proliferation and activation of specific T cells.

[0010] This application also provides a nucleic acid molecule encoding the above-mentioned fusion protein, as well as an expression vector and a host cell including the nucleic acid molecule.

[0011] This application also provides the use of the above-mentioned fusion protein in the preparation of a drug for treating hepatitis B virus.

[0012] The first aspect of this application provides a glycosylated fusion protein comprising a murine Fc variant and a hepatitis B virus antigen, wherein the fusion protein binds to dendritic cells (DCs), activates DCs, and promotes the proliferation and activation of specific T cells;

[0013] The mouse-derived Fc variant is obtained by amino acid mutation and glycosylation modification of a mouse-derived Fc fragment;

[0014] The mouse-derived Fc variant includes at least one of alanine at position 223, alanine at position 228, alanine at position 230, leucine at position 330, and glutamic acid at position 332.

[0015] In the glycosylation modification, the glycosyl group is derived from at least one of mannose, N-acetylglucosamine, and fucose;

[0016] In the glycosylation modification, the sugar chain structure formed by the glycosyl linkage is selected from at least one of high-mannose type, oligomannose type, and fucose type;

[0017] The non-mammalian glycosylation modifications do not include sialic acid modification;

[0018] The amino acid sites were numbered according to the EU numbering system.

[0019] In this application, the above-mentioned mouse Fc variant is obtained by modifying the wild-type Fc fragment shown in SEQ ID NO:1 with amino acid mutation and non-mammalian glycosylation.

[0020] Furthermore, the wild-type mouse Fc segment shown in SEQ ID NO:1 is the Fc segment of mouse IgG1. Except for the Fc segment of IgG1, the Fc segments of other mouse IgGs are applicable to this application. For example, when the Fc segment of mouse IgG2 is used as a basis, the Fc variant obtained after amino acid mutation and non-mammalian glycosylation modification has similar effects of binding to DC cells, activating DC cells, and promoting the proliferation and activation of specific T cells.

[0021] In some specific embodiments, the amino acid sequence of the mouse Fc variant is shown in SEQ ID NO:2 or SEQ ID NO:3.

[0022] The amino acid sequence shown in SEQ ID NO:2 consists of 232 amino acid residues. The mutation sites are determined according to the EU numbering system. The alanine residues at positions 223 and 228, and position 230, are shown as underlined in the following sequence:

[0023] The alanine at positions 223 and 228, and positions 230, 330, and 332 in SEQ ID NO:3 are as shown by the underlined positions in the following sequence:

[0024] The difference between non-mammalian glycosylation and mammalian glycosylation lies in the presence or absence of sialic acid modification and the content of mannose. In the technical solution provided in this application, glycosylation is primarily N-glycosylation, with the glycosyl groups derived from at least one of mannose, N-acetylglucosamine, and fucose. The glycan chain structure formed by the glycosyl linkages is selected from one or more of high-mannose, oligomannose, and fucose types. The high-mannose type consists of GlcNAc and mannose, containing 5 to 9 mannose molecules; oligomannose refers to less than 5 mannose molecules; and the fucose type contains fucose. In the solution provided in this application, the aforementioned glycosylation sites are all located on the 297th amino acid of the Fc variant.

[0025] Furthermore, the non-mammal is an insect. Further, the non-mammal is the fall armyworm. Even further, the fusion protein is expressed in Sf9 insect cells.

[0026] In this application, hepatitis B virus antigen refers to a protein on the surface of the hepatitis B virus (HBV), which is one of the important markers of HBV infection. These surface antigens—large protein (S1 / S2 / S), medium protein (S2 / S), and small protein (S)—are believed to participate in the binding of the virus to cell receptors for uptake, while the core protein (HBV Core) forms a capsid that encapsulates a portion of the double-stranded DNA genome. In this application, the HBV antigen can be HBV S1 and / or S2 and / or CORE (core antigen). Further, in some specific embodiments of this application, the HBV antigen is HBV S1 / S2 / CORE. HBV S1 / S2 / CORE, also known as "HBV S1 / S2 / core protein chimeric antigen," is a combination of the dominant B-cell epitopes in the pre-S1 and pre-S2 proteins of the HBV surface antigen and the HBV core antigen (HBcAg).

[0027] In this application, the aforementioned fusion protein mainly comprises a hepatitis B virus antigen capable of inducing an immune response in the body and a murine Fc variant that binds to dendritic cells (DCs). The murine Fc variant can be linked to the C-terminus of the hepatitis B virus antigen, or the hepatitis B virus antigen and the murine Fc variant can be linked by a linker peptide. In one embodiment, the amino acid sequence of the linker peptide is at least one of GGGS (SEQ ID NO:13), SRGGGS (SEQ ID NO:14), and VRPQGGGS (SEQ ID NO:15).

[0028] In the scheme provided in this application, the fusion protein may further include a protein tag to facilitate the expression, detection, and purification of the target gene. Further, the protein tag may be a His tag, which is attached to the N-terminus of the polypeptide antigen to improve the purification efficiency of the fusion protein.

[0029] The fusion protein described above is a glycosylated fusion protein that includes a hepatitis B virus antigen and a murine Fc variant that can elicit an immune response in humans. The fusion protein can be obtained by expression in insect cells using a baculovirus expression system.

[0030] A second aspect of this application provides a nucleic acid molecule encoding any of the fusion proteins described above.

[0031] A third aspect of this application provides a recombinant expression vector comprising the aforementioned nucleic acid molecule.

[0032] A fourth aspect of this application provides a host cell comprising the aforementioned recombinant expression vector.

[0033] The fifth aspect of this application provides a pharmaceutical composition comprising any of the fusion proteins described above and a pharmaceutically acceptable carrier.

[0034] In this application, the pharmaceutically acceptable carrier is non-toxic to cells or individuals at the dosage and concentration used. A physiologically acceptable carrier is typically a pH buffer solution. The pharmaceutical composition provided in this application is further formulated as an injectable preparation, and the aforementioned carrier may also include diluents such as water, ethanol, and polyethylene glycol, as well as isotonic additives such as sodium chloride, glucose, or glycerol. Furthermore, conventional solubilizers and buffers may be added.

[0035] The sixth aspect of this application provides the use of any of the above-described fusion proteins in the preparation of medicaments for treating hepatitis B virus.

[0036] The seventh aspect of this application provides the use of any of the above-described fusion proteins in the preparation of medicaments for treating any hepatitis B virus expressing HBV S1 and / or S2 and / or core protein antigens.

[0037] In this application, the drug for treating hepatitis B virus can be an HBV antiviral agent. In some instances, the HBV antiviral agent can be a pharmaceutically acceptable agent comprising one or more compounds selected from tenofovir, tenofovir disoproxil fumarate, tenofovir alafenamide, entecavir, tebivelin, adefovir dipivoxil, and lamivudine. In other instances, the HBV antiviral agent is an HBV antiviral RNAi agent that inhibits HBV replication. In still other instances, the HBV antiviral agent is a core capsid assembly inhibitor. In yet another instance, the HBV antiviral agent is a TLR agonist.

[0038] In other embodiments of the method, the method further includes administering an immunomodulatory agent to the host. In some instances, the immunomodulatory agent is interferon, such as pegylated interferon alpha2-a. In other instances, the immunomodulatory agent is an immune checkpoint inhibitor, such as a PD1 / PDL1 inhibitor.

[0039] In other embodiments of this method, the host is a human subject. In still other embodiments, the viral infection is HBV infection.

[0040] The fusion protein provided in this application is formed by fusing a murine Fc variant with hepatitis B virus antigen. This fusion protein can bind to dendritic cells (DCs), activate DCs, and promote the proliferation and activation of specific T cells, thereby achieving the effect of treating hepatitis B virus infection in the host. Attached Figure Description

[0041] Figure 1a shows the SDS-PAGE identification results of Seq2-Sf9, where M1 is the protein molecular weight marker and BSA is the protein standard;

[0042] Figure 1b shows the SDS-PAGE identification results of Seq3-Sf9, where M is the protein molecular weight marker and BSA is the protein standard;

[0043] Figure 2a shows the BLI assay results of Seq2-Sf9 and FcγRIIA proteins;

[0044] Figure 2b shows the BLI assay results of Seq3-Sf9 and FcγRIIA proteins;

[0045] Figure 3 shows the results of the binding force determination of Seq2-293 and Seq3-293 with DC2.4 at different concentrations;

[0046] Figure 4a shows the results of the Seq4-Sf9 staining assay using SDS-PAGE / Pageblue.

[0047] Figure 4b shows the results of Seq4-Sf9 protein blot analysis; the primary antibody was His tag polyclonal antibody (Rabbit); the secondary antibody was HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L);

[0048] Figure 4c shows the results of SDS-PAGE / Pageblue staining assays for Seq5-Sf9 and Seq6-Sf9.

[0049] Figure 4d shows the results of Western blot analysis for Seq5-Sf9 and Seq6-Sf9 proteins; the primary antibody was His tag Polvclonal antibody; the secondary antibody was HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L).

[0050] Figure 4e shows the N-glycosylation modification result at position 484 (position 297 of Fc fragment) of Seq4-Sf9;

[0051] Figure 4f shows the percentage results of fluorescence intensity detected by flow cytometry for Seq5-Sf9 and Seq6-Sf9.

[0052] Figure 5a shows the results of SDS-PAGE / Pageblue staining of Seq7-Sf9; lane 1 is the sample eluted from the Ni column; lane 2 is the sample dialyzed into the storage solution;

[0053] Figure 5b shows the WB results of Seq7-Sf9; the antibody is Goat anti-mouse IgG (H+L), lane 1 is the sample eluted from the Ni column, and lane 2 is the sample dialyzed into the storage solution.

[0054] Figure 5c shows the binding efficacy of Seq7-Sf9 with the mouse dendritic cell line DC2.4, with Mouse IgG1 as the control protein;

[0055] Figure 6a shows the expression of CD54 on the cell surface after Seq7-Sf9 fusion protein was loaded with DC for 48 h. Among them, Donor#2, Donor#4 and Donor#5 were 3 PBMC volunteers.

[0056] Figure 6b shows the expression of CD83 on the cell surface after Seq7-Sf9 fusion protein was loaded with DC for 48 h. Among them, Donor#2, Donor#4, and Donor#5 were 3 PBMC volunteers.

[0057] Figure 6c shows the expression results of CD86 on the cell surface after Seq7-Sf9 fusion protein was loaded with DC for 48 h. Among them, Donor#2, Donor#4, and Donor#5 were 3 PBMC volunteers.

[0058] Figure 6d shows the proliferation detection results of CD4+ T cells by Seq7-Sf9 loaded DCs, where Donor#2, Donor#4, and Donor#5 are 3 PBMC volunteers;

[0059] Figure 7 shows the serum titer of rabbits immunized with Seq7-Sf9;

[0060] Figure 8 shows the SDS-PAGE gel identification results of the fusion protein Seq8-293, where M1 represents the protein molecular weight marker, R is the reduced protein, and NR is the non-reduced protein.

[0061] Figure 9 shows the SDS-PAGE gel identification results of the fusion protein Seq8-Sf9;

[0062] Figure 10 shows the SDS-PAGE gel identification results of the fusion protein Seq9-Sf9;

[0063] Figure 11a shows the expression of CD54 on the cell surface after Seq9-Sf9 and Seq10-293 loaded with DC for 48 h;

[0064] Figure 11b shows the expression results of CD83 on the cell surface after Seq9-Sf9 and Seq10-293 loaded with DC for 48 h;

[0065] Figure 12a shows the expression of CD54 on the cell surface after Seq8-Sf9 loaded DC for 48 h;

[0066] Figure 12b shows the expression detection results of CD83 on the cell surface after Seq8-Sf9 loaded DC for 48 h;

[0067] Figure 13a shows the expression detection results of CD54 on the cell surface after Seq8-Sf9 and Seq8-293 were loaded with DC;

[0068] Figure 13b shows the expression detection results of CD83 on the cell surface after Seq8-Sf9 and Seq8-293 were loaded with DC;

[0069] Figure 14a shows the expression detection results of CD54 on the cell surface after Seq8-Sf9 and Provenge loaded DC;

[0070] Figure 14b shows the expression detection results of CD83 on the cell surface after Seq8-Sf9 and Provenge loaded DC;

[0071] Figure 15a shows the proliferation detection results of Seq8-Sf9 and Provenge loaded DCs on CD4+ T cells derived from volunteer 1;

[0072] Figure 15b shows the proliferation detection results of CD4+ T cells derived from volunteer 2 using Seq8-Sf9 and Provenge loaded DCs;

[0073] Figure 16 shows the results of ELISPOT detection of IFNγ secretion by DC-activated CD8+ T cells loaded with Seq8-Sf9;

[0074] Figure 17 shows the results of Western Blot analysis of PAP protein expression in human prostate cancer cell lines PC3 and LNCap.

[0075] Figure 18a shows the results of detecting the killing effect of Seq8-Sf9 and Provenge loaded DC-induced CTLs on PC3 tumor cells;

[0076] Figure 18b shows the results of detecting the killing effect of Seq8-Sf9 and Provenge loaded DC-induced CTLs on LNCap tumor cells;

[0077] Figure 19a shows the serum antibody titer of anti-Seq6-Sf9 in SD rats immunized with Seq8-Sf9 subcutaneously (dose of 0 / 2 / 10 / 50ug); the sample collection time points were 0 days (before the first injection), 14 days (before the second injection), 28 days (before the third injection) and 35 days (one week after the third injection), n=6.

[0078] Figure 19b shows the serum antibody titer of SD rats immunized with Seq8-Sf9 subcutaneously (dose of 0 / 2 / 10 / 50ug) 14 days later.

[0079] Figure 19c shows the serum antibody titer of SD rats immunized with Seq8-Sf9 subcutaneously (dose of 0 / 2 / 10 / 50ug) 28 days later.

[0080] Figure 19d shows the serum antibody titer of SD rats immunized with Seq8-Sf9 subcutaneously (dose of 0 / 2 / 10 / 50ug) 35 days later.

[0081] Figure 20a shows the ELISPOT detection results of IFNγ secretion by spleen cells of SD rats after Day 35 immunization with Seq8-Sf9;

[0082] Figure 20b shows the ELISPOT speckle pattern of IFNγ secreted by spleen cells of SD rats after Day 35 immunization with Seq8-Sf9.

[0083] Figure 21 shows the HE staining of prostate tissue from SD rats after Day 35 immunization with Seq8-Sf9 (20× magnification).

[0084] Figure 22a shows the expression of CD54 on the cell surface after Seq9-Sf9 was compared with that of Seq8-Sf9 loaded with DC for 48 h;

[0085] Figure 22b shows the expression detection results of CD83 on the cell surface after Seq9-Sf9 was loaded with DC for 48 h, compared with Seq8-Sf9. Detailed Implementation

[0086] To enable those skilled in the art to better understand the solutions of this application, a further detailed description of this application is provided below. The specific embodiments listed below are merely descriptions of the principles and features of this application; the examples are only for explaining this application and are not intended to limit its scope. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this application.

[0087] Example 1: Fc Variant Affinity Detection

[0088] 1.1 Acquisition and affinity detection of Fc variants expressed in Sf9 insect cells

[0089] To enhance the stability of the mouse IgG Fc fragment, mutations were made at positions 223, 228, and 230 of the wild-type mouse Fc fragment shown in SEQ ID NO:1. The resulting amino acid sequence is shown in SEQ ID NO:2. To enhance the binding and activation of the mouse IgG1 Fc fragment with DC cells, mutations were made at positions 223, 228, 230, 330, and 332 of the wild-type mouse Fc fragment. The resulting amino acid sequence is shown in SEQ ID NO:3.

[0090] The signal sequence (amino acid sequence shown in SEQ ID NO:11) of the AcMNPV gp64 protein was cloned into the pFastBacHTa (Thermo Fisher Scientific; Cat#10584-027) vector digested with RsrII (New England Biolabs, R051S) to obtain pFastBacHTa-gp64. Similarly, the signal sequence (amino acid sequence shown in SEQ ID NO:12) of the AcMNPV gp67 protein was cloned into the pFastBacHTa (Thermo Fisher Scientific; Cat#10584-027) vector digested with RsrII (New England Biolabs, R051S) to obtain pFastBacHTa-gp67. The DNA sequence corresponding to the amino acids shown in SEQ ID NO:2 or SEQ ID NO:3 was synthesized, and the fragment was subcloned into the pFastBacHTa-gp64 vector digested with Sal I (New England Biolabs, R3138S) and Hind III (New England Biolabs, R0104S) for expression in insect cells. The recombinant plasmid was transformed into DH10Bac competent cells and then cultured on fresh LB agar plates containing 50 μg / ml kanamycin, 7 μg / ml gentamicin, 10 μg / ml tetracycline, 100 μg / ml Bluo-gal, and 40 μg / ml IPTG. After incubation overnight, white colonies were selected, and recombinant rod-granule DNA was isolated according to standard protocol. Positive rod particles were transiently transfected into 2 ml of insect Sf9 cells using a transfection reagent. The transfected cells were incubated in ESF 921 medium for a period of time, and the cells and supernatant were collected. The proteins were purified from the supernatant using Protein A and named Seq2-Sf9 and Seq3-Sf9, respectively.

[0091] Seq2-Sf9 and Seq3-Sf9 were identified using Coomassie blue-stained SDS-PAGE gels, with BSA as a control protein. The identification results are shown in Figures 1a-1b, with the arrows indicating the positions of Seq2-Sf9 and Seq3-Sf9, confirming that the purified protein was obtained with a molecular weight of approximately 26 kDa. Biolayer Interferometry (BLI) was used to detect the affinity of purified Seq2-Sf9 and Seq3-Sf9 proteins at multiple concentrations. Concentration gradients of 5000 nM, 2500 nM, 1250 nM, 625 nM, and 312.5 nM were set to determine the affinity of Seq2-Sf9 and Seq3-Sf9 proteins for FcγRIIA protein. The detection results are shown in Figures 2a-2b. The curves from bottom to top indicate that the concentrations of Seq2-Sf9 and Seq3-Sf9 proteins increase sequentially. The calculated affinity (KD value) between Seq2-Sf9 and Seq3-Sf9 and FcγRIIA protein were 7.893E-07 and 4.496E-07, respectively. The smaller the value, the higher the affinity, indicating that the five mutations in the mouse Fc domain can enhance the affinity for FcγRIIA.

[0092] 1.2 Obtaining the Fc variant expressed in 293 cells and detecting its binding affinity to DC cells

[0093] The synthesized DNA sequences corresponding to SEQ ID NO:2 and SEQ ID NO:3 were cloned into the eukaryotic expression vector pcDNA3.4 containing a secretion signal peptide, with EcoRI (New England Biolabs, R0101V) and HindIII (New England Biolabs, R0104S) restriction sites. The clones were electroporated into *E. coli* trans5α, screened with ampicillin, and sequenced to obtain the correct recombinant plasmids. The host bacteria containing the recombinant plasmids were then expanded, and sterile, endotoxin-free recombinant plasmids were obtained using an endotoxin-free kit. The sterile, endotoxin-free recombinant plasmids were mixed with Polyplus suspension cell transfection reagent and transfected into HEK293F cells. After 5 days of expansion culture in serum-free medium, the supernatant was collected and purified using Protein A resin to obtain proteins Seq2-293 and Seq3-293.

[0094] Seq2-293 and Seq3-293 enhanced the binding to FcR, which is mainly expressed on the surface of various monocytes, including dendritic cells (DCs). Since the aforementioned Fc variants are derived from the mouse Fc segment, the mouse dendritic cell line DC2.4 was chosen as the research subject, as it expresses relatively stable FcR and can bind to mouse-derived Fc. Under natural conditions, Fc has a weak affinity for its receptor FcR. DC2.4 cells were incubated with the same concentrations of Seq2-293 and Seq3-293 at 4°C for 1 h. Biotin-labeled anti-mouse IgG1 (anti-Mouse IgG1 Biotin) and streptavidin-HRP (streptavidin-HRP) were then added, respectively, for reaction. The reaction was terminated after TMB substrate color development. Finally, visible light OD450-570 was measured to indicate the Fc / FcR binding on the surface of DC2.4 cells.

[0095] The test results are shown in Figure 3. It can be seen that the affinity of Seq3-293 for DC2.4 at the same concentration is significantly higher than that of Seq2-293, and is directly proportional to the working concentration; that is, the Fc variant with the five mutations can enhance the binding ability with DC and has considerable antigen presentation potential.

[0096] Example 2: Preparation of HBV-mFc protein and evaluation of DC binding effect

[0097] 2.1 Preparation of Seq4-Sf9, Seq5-Sf9, and Seq6-Sf9 proteins

[0098] The five-mutant mouse Fc of the amino acid sequence shown in SEQ ID NO:3 was fused with hepatitis B virus antigen (including HBV S1 / S2 / Core) via a linker peptide and a hydrophilic peptide (SEQ ID NO:16) was attached to the C-terminus to obtain a fusion protein with the amino acid sequence shown in SEQ ID NO:4, where hepatitis B virus antigen is located at positions 36-394 and the five-mutant mouse Fc of the amino acid sequence shown in SEQ ID NO:2 is located at positions 403-634.

[0099] The triple mutant mouse Fc of the amino acid sequence shown in SEQ ID NO:2 was fused with hepatitis B virus antigen (including HBV S1 / S2) via a linker peptide to obtain a fusion protein with the amino acid sequence shown in SEQ ID NO:5, where positions 36-209 are hepatitis B virus antigen and positions 218-449 are triple mutant mouse Fc of the amino acid sequence shown in SEQ ID NO:2.

[0100] The five-mutant mouse Fc of the amino acid sequence shown in SEQ ID NO:3 was fused with hepatitis B virus antigen (including HBV S1 / S2) via a linker peptide to obtain a fusion protein with the amino acid sequence shown in SEQ ID NO:6, where positions 36-209 are hepatitis B virus antigen and positions 218-449 are the five-mutant mouse Fc of the amino acid sequence shown in SEQ ID NO:2.

[0101] The gene encoding the amino acid sequence shown in SEQ ID NO:4 was ligated into the pFastBacHTa-gp64 vector digested with Sal I (New England Biolabs, R3138S) and Hind III (New England Biolabs, R0104S) for expression in insect cells. Similarly, the genes encoding the amino acid sequences shown in SEQ ID NO:5 and SEQ ID NO:6 were ligated into the pFastBacHTa-gp67 vector digested with Sal I (New England Biolabs, R3138S) and Hind III (New England Biolabs, R0104S) for expression in insect cells. These vectors were then transformed into *E. coli* DH10Bac for transposition to produce recombinant baculovirus. The recombinant baculovirus was isolated and transfected into Sf9 insect cells. Cells were incubated in ESF921 medium at 27°C for 72 hours. The cell pellet was collected and lysed by sonication. The fusion proteins were purified from infected Sf9 cells using affinity chromatography and named Seq4-Sf9, Seq5-Sf9, and Seq6-Sf9, respectively. SDS-PAGE gels were stained with Coomassie blue and Western blotting was performed. The results are shown in Figures 4a-4d. The apparent monomeric molecular weights of the Seq4-Sf9, Seq5-Sf9, and Seq6-Sf9 fusion proteins were 77 kDa, 57 kDa, and 57 kDa, respectively.

[0102] 2.2 Identification of Glycosylation of Seq4-Sf9

[0103] The fusion protein Seq4-Sf9 obtained from the Sf9 insect cell expression system was sequentially processed using Lys-C protease and Trypsin. The peptide samples after enzymatic digestion were then analyzed using liquid chromatography-mass spectrometry (LC-MS). The raw LC-MS data were analyzed using software, confirming that the N-glycosylation modification ratio of asparagine at position 484 (position 297 of the Fc fragment) was 68.93%. The glycoform distribution ratio is shown in Figure 4e, indicating that the glycosylation modification of the fusion protein expressed by the Sf9 insect system is predominantly high-mannose, with small amounts of oligomannose and fucose (glycoforms according to Oxford nomenclature).

[0104] 2.3 Detection of the protein binding efficacy of the fusion protein HBV-mFc

[0105] To assess the binding level of the fusion protein HBV-mFc to dendritic cells, imDCs were induced using the classic adherent method. Under serum-free conditions, monocytes exhibit some adhesion; after removing non-adherent cells (mainly T and B cells), differentiation into immature dendritic cells (imDCs) was induced in a culture system containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). The imDCs were incubated with Seq5-Sf9 and Seq6-Sf9 fusion proteins (50 μg / mL each) on ice (2-8℃) for 1 h. After gently washing away unbound portions, the cells were incubated with the secondary antibody PE Rat anti-Mouse IgG1 for a period of time. The fluorescence percentage of Seq5-Sf9 and Seq6-Sf9 fusion proteins bound to the DCs was then detected by flow cytometry. The Seq5-Sf9 mouse Fc fragment is a triple mutation, while the Seq6-Sf9 mouse Fc fragment is a pentamutation. The fluorescence percentage represents the proportion of fusion proteins bound to DC cells. As shown in Figure 4f, the percentage of fluorescence intensity detected by flow cytometry indicates that the fluorescence of the imDC stealth control is low, suggesting that the nonspecific background of this cell is low. The binding of the pentamutation (Seq6-Sf9) to imDC is significantly higher than that of the triple mutation (Seq5-Sf9).

[0106] 2.4 Evaluation of the in vitro activation effect of HBV-mFc protein

[0107] 2.4.1 Preparation of Seq7-Sf9 fusion protein

[0108] Triple-mutant mouse Fc cells with the amino acid sequence shown in SEQ ID NO:2 were fused with hepatitis B virus antigens (including HBV S1 / S2 / Core) via a linker peptide, and a hydrophilic peptide was attached to the C-terminus to obtain a fusion protein with the amino acid sequence shown in SEQ ID NO:7. Positions 36-394 represent the hepatitis B virus antigen, and positions 403-634 represent the triple-mutant mouse Fc cells with the amino acid sequence shown in SEQ ID NO:2. The protein expression method is shown in 2.1, using the pFastBacHTa-gp64 vector, and the protein was named Seq7-Sf9. Biochemical assays were performed using Coomassie Brilliant Blue staining on SDS-PAGE gels and Western blotting. The results are shown in Figures 5a-5b. The apparent molecular weight of the monomer of the fusion protein was approximately 77 kDa.

[0109] 2.4.2 Assessing the binding affinity of Seq7-Sf9 to DC cells

[0110] Different concentrations of Seq7-Sf9 were incubated with DC2.4 cells at 4°C for 1 h (Seq7-Sf9 concentrations were 0 μg / mL, 5 μg / mL, 25 μg / mL, and 50 μg / mL). Biotin-labeled anti-mouse IgG1 (anti-Mouse IgG1 Biotin) and streptavidin-HRP (streptavidin-HRP) were added for reaction, and the reaction was terminated after TMB substrate treatment for color development. The OD450-570 absorbance value represents the cell surface binding of the fusion protein. Natural mouse IgG1 was used as a control. As shown in Figure 5c, compared with the control Mouse IgG1, Seq7-Sf9 showed stronger binding efficacy to DC2.4, suggesting that the fusion protein has the potential for potent antigen presentation.

[0111] 2.4.3 Evaluation of the immune activation effect of the fusion protein Seq7-Sf9 using dendritic cells induced by CD14+ monocytes in peripheral blood PBMCs from healthy individuals.

[0112] To assess the immune-activating effect of the Seq7-Sf9 fusion protein, CD14+ monocytes from three healthy human PBMCs were selected and induced to differentiate into immature dendritic cells (DCs) in a culture system containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Immature DCs exhibited strong phagocytic function, and the murine Fc domain contained in the fusion protein enhanced their uptake function, promoting DC maturation and differentiation. Flow cytometry was used to detect changes in surface markers of Seq7-Sf9 fusion protein-loaded DCs, including the adhesion molecule CD54 (ICAM-1: Intercellular adhesion molecule-1), which facilitates adhesion and interaction between mature DCs and other immune cells (such as T cells), and CD83 and the co-stimulatory molecule CD86, which are involved in antigen presentation and T cell activation. The detection results are shown in Figures 6a-6c. In three different volunteers, the DCs loaded with the Seq7-Sf9 fusion protein highly expressed the maturation markers CD54, CD83, and CD86 on their surface, indicating that the fusion protein has a strong DC activation effect, which activates the antigen presentation ability of the vaccine-loaded DCs and initiates the immune response as the starting point.

[0113] 2.4.2 Key to in vitro assessment of the effect of Seq7-Sf9-loaded DCs on T cell immune responses

[0114] The primary function of proliferating and maturing dendritic cells (DCs) of CD4+ T cells is antigen presentation. The antigen peptide / MHC-I or antigen peptide / MHC-II complexes on their surface are recognized by T cell surface receptors (TCRs). A crucial step in DC-T cell activation is T cell proliferation. Therefore, co-culturing Seq7-Sf9-loaded DCs with CFSE-labeled CD4+ T cells, after antigen presentation, allowed T cells to rapidly proliferate and differentiate into Th cells (helper T cells). The proliferation of CD4+ T cells was then used to assess immune status. As shown in Figure 6d, Seq7-Sf9-loaded DCs significantly promoted T cell proliferation compared to a negative control of CD4+ T cells from the same source (Seq7-Sf9 0 μg / ml), and this promotion was positively correlated with the concentration of Seq7-Sf9.

[0115] 2.5 Determination of rabbit serum titer

[0116] Antibody levels in rabbit serum were assessed using ELISA. Two healthy male New Zealand rabbits aged 3-4 months were selected as experimental animals. 15 μg of the test substance Seq7-Sf9 protein was dissolved in 0.5-1 mL of PBS solution and emulsified thoroughly with complete / incomplete Freund's adjuvant (4 mg / mL) at a 1:1 volume ratio. The mixture was then injected subcutaneously at four points on the back of the rabbit, resulting in a total of six immunizations (D1, D15, D29, D43, D64, D92), with each immunization spaced 2-4 weeks apart. The immunization dose was 15 μg / rabbit / immunization. (The first immunization used complete Freund's adjuvant, while the second, third, fourth, fifth, and sixth immunizations used incomplete Freund's adjuvant).

[0117] After incubating Seq7-Sf9 (200 ng / well) protein-coated plates overnight, the plates were blocked with PBS + 5% (w / v) BSA for 2 hours. The plates were then incubated for one hour with serially diluted serum samples (starting at 1:300 for each serum sample, with 12 serial dilutions in 2-fold increments). Binding of Seq7-Sf9 to IgG was detected using goat anti-rabbit IgG antibody. The TMB (3,3',5,5'-tetramethylbenzidine) two-component peroxidase substrate kit (KPL) was used for colorimetric development, and the reaction was terminated by adding stop solution. The absorbance at 450 nm was immediately recorded using a SpectraMax Plus spectrophotometer (Molecular Devices) (Figure 7). The results showed that antibody levels increased after persistent Seq7-Sf9 infection in rabbits.

[0118] Example 3: Preparation and Bioactivity Evaluation of Seq8-293, Seq8-Sf9, and Seq9-Sf9

[0119] 3.1 Preparation of Seq8-293

[0120] The triple-mutant mouse Fc of the amino acid sequence shown in SEQ ID NO:2 was fused with a linker peptide to PAP, a specific marker of prostate cancer, to obtain a fusion protein with the amino acid sequence shown in SEQ ID NO:8, wherein positions 1-354 are PAP, a specific marker of prostate cancer, and positions 361-592 are the triple-mutant mouse Fc of the amino acid sequence shown in SEQ ID NO:2.

[0121] A gene encoding the amino acid sequence shown in SEQ ID NO:8 was synthesized. The gene sequence encoding this fusion protein was ligated into the expression vector pcDNA3.4. A recombinant expression plasmid was prepared by digesting EcoRI (New England Biolabs, R0101V) and HindIII (New England Biolabs, R0104S) restriction enzymes. The recombinant expression plasmid was transiently transfected into eukaryotic HD293F cells, and the cells were cultured. The cell culture medium was collected, centrifuged, and filtered. The filtered culture supernatant was loaded onto a Protein A affinity chromatography column to purify the fusion protein, named Seq8-293. Biochemical assays were performed using Coomassie blue-stained SDS-PAGE gels, and the results are shown in Figure 8.

[0122] 3.2 Preparation of Seq8-Sf9

[0123] The triple-mutant mouse Fc protein with the amino acid sequence shown in SEQ ID NO:2 was fused with a linker peptide to PAP, a specific marker of prostate cancer, to obtain a fusion protein with the amino acid sequence shown in SEQ ID NO:8. The corresponding coding sequence was then ligated into the pFastBacHTa-gp67 vector digested with Sal I (New England Biolabs, R3138S) and Hind III (New England Biolabs, R0104S) for expression in insect cells. This vector was transformed into E. coli DH10Bac for transposition to generate recombinant baculovirus. The recombinant baculovirus was isolated and transfected into Sf9 insect cells. The cells were incubated in ESF921 medium at 27°C for 72 hours to express the target protein. The supernatant was collected by centrifugation, and the protein was purified by Protein A and named Seq8-Sf9. Biochemical assays were performed using Coomassie blue-stained SDS-PAGE gels. The results are shown in Figure 9.

[0124] 3.3 Preparation of Seq9-Sf9

[0125] The five-mutant mouse Fc of the amino acid sequence shown in SEQ ID NO:3 was fused with a linker peptide to PAP, a specific marker of prostate cancer, to obtain a fusion protein with the amino acid sequence shown in SEQ ID NO:9, wherein positions 1-354 are PAP, a specific marker of prostate cancer, and positions 361-592 are the five-mutant mouse Fc of the amino acid sequence shown in SEQ ID NO:3.

[0126] Using the same method as in 3.2, the above fusion protein was expressed in Sf9 insect cells. The expressed fusion protein was named Seq9-Sf9. Biochemical assays were performed using Coomassie Brilliant Blue staining of SDS-PAGE gels. The results are shown in Figure 10.

[0127] 3.4 Detection of the activation effect of Seq9-Sf9 on DC

[0128] The human wild-type Fc fragment and the prostate cancer-specific marker PAP were expressed in mammalian 293 cells. The Seq10-293 fusion protein was obtained using the same method as in 3.1. Its amino acid sequence is shown in SEQ ID NO:10. The Seq10-293 fusion protein was used as a control to verify the effect of Fc variants and insect glycoforms on DC activation.

[0129] CD14+ monocytes from two healthy human PBMCs were selected and induced to differentiate into immature dendritic cells (DCs) in a culture system containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Immature DCs exhibited strong phagocytic function and were loaded with Seq9-Sf9 and Seq10-293 fusion proteins, respectively. DCs phagocytosed the fusion proteins and presented antigens, thereby activating the DCs and expressing activation markers (including CD54 and CD83). Flow cytometry was used to detect changes in surface markers on vaccine-loaded DCs, and the results are shown in Figures 11a-11b. At the same concentration, compared to Seq10-293, Seq9-Sf9 significantly activated DCs, and the cell surface highly expressed CD54 and CD83. These results demonstrate that the murine Fc variant and non-mammalian glycoform in the fusion protein can significantly enhance DC phagocytosis and activation, and express activation phenotypes such as CD54 and CD83.

[0130] 3.5 Detection of DC activation effect by Seq8-Sf9

[0131] 3.5.1 Using CD14+ monocytes induced by human peripheral blood PBMCs to assess the immune activation effect of the fusion protein Seq8-Sf9

[0132] To assess the immune activation effect of Seq8-Sf9, CD14+ monocytes from three healthy human PBMCs were selected and induced to differentiate into immature dendritic cells (DCs) in a culture system containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Immature DCs exhibited strong phagocytic function, and the murine Fc domain contained in the fusion protein enhanced their uptake function, promoting DC maturation and differentiation. Flow cytometry was used to detect changes in surface markers of vaccine-loaded DCs, including the adhesion molecule CD54 (ICAM-1: Intercellular adhesion molecule-1), which facilitates adhesion and interaction between mature DCs and other immune cells (such as T cells), and CD83, which is involved in antigen presentation and T cell activation. The detection results are shown in Figures 12a-12b. In three different volunteers, the surface of DCs loaded with Seq8-Sf9 highly expressed CD54 and CD83, indicating that the fusion protein has a strong DC activation effect, which activates the antigen presentation ability of DCs loaded with Seq8-Sf9, and initiates the immune response as the starting point.

[0133] Furthermore, the immune activation effects of Seq8-Sf9, Seq8-293, and the positive control drug Provenge (sipuleucel-T) were compared, and the results are shown in Figures 13a-13b and 14a-14b. It can be seen that the Fc variant and non-mammalian glycosylation enhanced the DC activation efficacy, and Seq8-Sf9 had a stronger DC activation function.

[0134] 3.5.2 Key to in vitro assessment of the effect of Seq8-Sf9-loaded DCs on T cell immune responses

[0135] The primary function of proliferating and maturing dendritic cells (DCs) of CD4+ T cells is antigen presentation. The antigen peptide / MHC-I or antigen peptide / MHC-II complexes on their surface are recognized by T cell surface receptors (TCRs). A crucial step in DC-T cell activation is T cell proliferation. Therefore, co-culturing Seq8-Sf9-loaded DCs with CFSE-labeled CD4+ T cells, after antigen presentation, allows T cells to rapidly proliferate and differentiate into Th cells (helper T cells). This CD4+ T cell proliferation can then be used to assess immune status. As shown in Figures 15a-15b, loading DCs from different healthy individuals with Seq8-Sf9 significantly promotes T cell proliferation, with the effect positively correlated with the concentration of Seq8-Sf9. Provenge under the same experimental conditions also exhibits the same effect.

[0136] 3.5.3 In vitro assessment of Seq8-Sf9-loaded DC antigen-presenting activated cytotoxic T cells

[0137] The antigen peptide / MHC class I molecule complex presented on the surface of dendritic cells (DCs) can directly recognize and bind to the TCRs on the surface of CD8+ T cells, thereby activating CD8+ T cells to exert their biological effects, one of the main mechanisms being the secretion of interferon-γ (IFNγ). Seq8-Sf9-loaded DCs were co-cultured with CD8+ T cells from the same volunteer, and then Seq8-Sf9-loaded DCs were added again for secondary stimulation, activating a large number of CD8+ T cells. The level of IFNγ secreted by individual cells was detected by ELISPOT. The results are shown in Figure 16. It can be seen that the Seq8-Sf9-loaded group had significantly more IFNγ than the volunteer's own CD8+ T cells (negative control), indicating that Seq8-Sf9 can effectively promote CD8+ T cell activation.

[0138] 3.5.4 Using human prostate cancer cell lines expressing PAP antigen as target cells to evaluate the killing effect of Seq8-Sf9 activated CTLs.

[0139] Treatment of prostate cancer varies depending on androgen sensitivity. PC3 is a common androgen-independent human prostate cancer cell line, while LNCap is androgen-dependent. Both cell lines express a certain amount of PAP, and as shown in Figure 17, LNCap expresses relatively more PAP protein. These cells were used as target cells and co-cultured with effector cells (CTLs activated by DCs loaded with Seq8-Sf9). The absolute count of dead cells was determined using CFSE and counting beads. As shown in Figures 18a-18b, the DC-activated CTLs loaded with Seq8-Sf9 could directly kill target cells, and the effect was directly proportional to the concentration of Seq8-Sf9. Furthermore, the number of dead cells in LNCap, which expresses a higher level of PAP, was significantly higher than that in PC3, verifying its targeted killing effect, which is superior to that of Provenge.

[0140] 3.6 Immunogenicity assessment of Seq8-Sf9

[0141] To assess the immune response to Seq8-Sf9 in vivo, male Sprague Dawley (SD) rats aged 6-8 weeks were immunized with Seq8-Sf9. SD rats were subcutaneously injected with Seq8-Sf9 (Placebo / rat, 2ug / rat, 10ug / rat, 50ug / rat) every two weeks for a total of three injections. Serum samples were collected from the immunized animals at Day 14, Day 28, and Day 35 (one week after the third injection) to detect Seq8-Sf9-specific antibody titers. As shown in Figures 19a-19d, compared to the Placebo group, a low dose of 2ug was sufficient to produce vaccine-specific serum antibodies, with antibody titers reaching their highest levels one week after the third and fourth injections.

[0142] In addition, the spleen of rats on day 35 was harvested and restimulated in vitro with Seq8-Sf9 to activate and induce effector T cell activation. ELISPOT analysis was performed to detect IFNγ secretion by individual spleen cells. As shown in Figures 20a-20b, spleen immune cells, under the secondary stimulation of Seq8-Sf9, can rapidly differentiate into cytotoxic T lymphocytes and secrete IFNγ.

[0143] Meanwhile, the prostate tissue was observed, and the results are shown in Figure 21. Inflammatory cell infiltration was also observed in the prostate tissue of rats in the Seq8-Sf9 group.

[0144] In summary, Seq8-Sf9 can establish a complete in vivo immune response in SD rats and effectively activate T cells, meaning that Seq8-Sf9 can complete in vivo immune activation.

[0145] 3.7 Assessment of the immune activation effect of Seq9-Sf9 fusion protein using dendritic cells induced by CD14+ monocytes from human peripheral blood PBMCs

[0146] The Fc segment of Seq9-Sf9 differs from that of Seq8-Sf9. To assess its immune activation effect, CD14+ monocytes sorted from PBMCs of two healthy individuals were induced to differentiate and loaded with Seq9-Sf9. Flow cytometry was used to detect changes in antigen-loaded DC surface markers, as shown in Figures 22a-22b. Seq9-Sf9 increased the expression of CD54 and CD83 on the surface of DCs, promoting DC activation, and the expression levels of these markers were higher than those of Seq8-Sf9.

[0147] Finally, it should be noted that other embodiments of this application will readily conceive of by those skilled in the art upon consideration of the specification and practice of the application disclosed herein. This application is intended to cover any variations, uses, or adaptations of this application that follow the general principles of this application and include common knowledge or customary techniques in the art not disclosed herein, and is not limited to what has been described above, and various modifications and changes may be made without departing from its scope. The scope of this application is limited only by the appended claims.

Claims

1. A glycosylated fusion protein, wherein, Including a murine Fc variant and hepatitis B virus antigen, the fusion protein binds to dendritic cells (DCs), activates DCs, and promotes the proliferation and activation of specific T cells; The mouse-derived Fc variant is obtained by amino acid mutation and glycosylation modification of a mouse-derived Fc fragment; The mouse-derived Fc variant includes at least one of alanine at position 223, alanine at position 228, alanine at position 230, leucine at position 330, and glutamic acid at position 332. In the glycosylation modification, the glycosyl group is derived from at least one of mannose, N-acetylglucosamine, and fucose; In the glycosylation modification, the sugar chain structure formed by the glycosyl linkage is selected from at least one of high-mannose type, oligomannose type, and fucose type; The glycosylation modification does not include sialic acid modification; The amino acid sites were numbered according to the EU numbering system.

2. The fusion protein according to claim 1, wherein, The amino acid sequence of the mouse Fc variant is shown in SEQ ID NO:2 or SEQ ID NO:

3.

3. The fusion protein according to claim 1 or 2, wherein, The hepatitis B virus antigen is HBV S1 and / or S2 and / or CORE.

4. The fusion protein according to any one of claims 1-3, wherein, The fusion protein also includes a protein tag.

5. The fusion protein according to any one of claims 1-4, wherein, The fusion protein also contains linker peptides.

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

7. A recombinant expression vector, wherein, Includes the nucleic acid molecule as described in claim 6.

8. A host cell, wherein, Includes the recombinant expression vector as described in claim 7.

9. A pharmaceutical composition, wherein, Includes the fusion protein as described in any one of claims 1-5 and a pharmaceutically acceptable carrier.

10. The use of the fusion protein according to any one of claims 1-5 in the preparation of a medicament for treating hepatitis B virus and hepatitis B-related liver cancer.

11. The use of the fusion protein according to any one of claims 1-5 in the preparation of a medicament for treating any hepatitis B virus expressing HBV S1 and / or S2 and / or core protein antigens and hepatitis B-related hepatocellular carcinoma.

12. A method for treating hepatitis B virus infection, wherein, The fusion protein according to any one of claims 1 to 5 is administered to a subject.

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