Glycosyl-modified fusion protein, nucleic acid molecule, expression vector, host cell, and use

Abstract

A glycosyl-modified fusion protein, a nucleic acid molecule, an expression vector, a host cell, and a use. The glycosyl-modified fusion protein comprises a murine Fc variant and a polypeptide antigen; the murine Fc variant is obtained by carrying out 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 non-mammalian glycosylation modification does not comprise sialic acid modification; site numbering is carried out on the basis of an EU numbering system. The murine Fc variant improves the binding and DC activation of the murine Fc fragment and a DC cell, and comprises 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 the Chinese patent application filed with the China Patent Office on June 30, 2023, with application number 202310798756.4 and application name “Fc variants, fusion proteins and their applications”, and the Chinese patent application filed with the China Patent Office on June 30, 2023, with application number 202310807755.1 and application name “A chimeric fusion protein, nucleic acid molecule, expression vector, host cell and application”, all of which are incorporated by reference into this application. Technical Field

[0002] The present application relates to a glycosyl-modified fusion protein, a nucleic acid molecule, an expression vector, a host cell and applications, and relates to the field of biopharmaceutical technology. Background Art

[0003] FcRs are cell surface proteins that specifically bind to the functional Fc fragment at the carboxyl terminus of immunoglobulin (Ig) heavy chains and are crucial in the antibody-dependent immune response. Different cell types express different types of FcRs, which bind to different structural types of Ig, thereby inducing different types of subsequent immune responses. Immunoglobulin G (IgG) is a powerful antibody in the human body and is the primary antibacterial, antiviral, and antitoxin antibody in serum. The FcRs that bind to IgG are also called FcγRs. Based on the affinity between FcγR and Fc fragment, FcγR can be divided into three subfamilies: FcγRI, FcγRII and FcγRIII (the affinity ranking with IgG is: FcγRI>FcγRIII>FcγRII). Among them, FcγRII is further divided into three subclasses: FcγRIIa (CD32a), FcγRIIb (CD32b) and FcγRIIc (CD32c). FcγRIII can be further divided into two subclasses: FcγRIIIa (CD16a) and FcγRIIIb (CD16b).

[0004] Human FcγRIIa (hFcγRIIA) is an activating Fc receptor. When FcγRIIa binds to the Fc segment of an antibody, it can transmit activation signals and mediate the function of Fc segment effectors, including cell lysis, phagocytosis, degranulation, and cytokine production. At the same time, FcγRIIa can regulate dendritic cell maturation and antigen presentation, mediate T cell memory effects, and participate in stimulating protective CD8 + T cell response promotes long-term protection against viral infection, thereby achieving the effect of clearing target cells and maintaining lasting immune protection.

[0005] To gain a deeper understanding of the binding between the Fc fragment and FcγRIIa, many researchers have modified the structure of the Fc fragment. One researcher found that the Fc fragment mutation (G236A) selectively activates FcγRIIa (the two share 96% sequence identity) while inhibiting FcγRIIb, thereby enhancing ADCP mediated by monocyte-derived macrophages. Adding G236A to the S239D / I332E mutation of the Fc fragment can increase the binding of FcγRIIa by 70 times and the binding ratio of FcγRIIa / FcγRIIb (activation / inhibition ratio) by 13 times, thereby enhancing the phagocytosis of antibody-coated target cells by macrophages (Richards JO, KS, Lazar GA, et al. Molecular cancer therapeutics, 2008, 7(8): 2517-2527., Optimization of antibody binding to FcγRIIa enhances macrophage phagocytosis of tumor cells[J]. Molecular cancer therapeutics, 2008. 7(8): p. 2517-2527). Other researchers have found that Fc mutations induce CD8 + and CD4 + Activation of T cell responses by T cells (Bournazos, S. et al. Fc-optimized antibodies elicit CD8 immunity to viral respiratory infection, Nature, Vol 588 17 December 2020). Therefore, modification of the Fc fragment may affect the effector function of the Fc fragment, thereby changing the biological activity of the antibody.

[0006] The glycosylation pathway in insects differs from that in higher eukaryotes. Recombinant N-glycoproteins typically express simple, non-sialylated high- and low-mannose glycans, rather than the complex sialylated glycans produced at the same glycosylation positions in mammals. This may be due to the high N-acetylglucosaminidase activity, low glycosyltransferase activity, and a limited supply of sugar-nucleosides in these cells. The effector functions elicited by the fragment crystallizable (Fc) region of immunoglobulin G (IgG) antibodies are altered by the presence of a terminal sialic acid (Sia) residue at asparagine-297 (Asn-297). Studies have shown that hypersialylation impairs binding to FcγRIIIa on natural killer (NK) cells, resulting in reduced ADCC efficacy. Sugar chains without terminal sialic acid residues expressed by SF9 cells can also bind to the mannose receptor (CD206), which may increase the uptake and processing ability of dendritic cells for glycoproteins, thereby enhancing the immune response (Scallon BJ, Tam SH, McCarthy SG, et al. Higher levels of sialylated Fc glycans in immunoglobulin G molecules can adversely impact functionality[J]. Molecular immunology, 2007, 44(7): 1524-1534.).

[0007] In addition to a large number of human immunoglobulins, there have also been successful examples of non-humanized antibodies plus antigens as therapeutic vaccines, such as CA-125+ mouse IgG1, Oregovomab (M. Brewer, R. Angioli, G. Scambia, et al., Front-line chemo-immunotherapy with carboplatin-paclitaxel using oregovomab indirect immunization in..., Gynecologic Oncology, https: / / doi.org / 10.1016 / j.ygyno.2019.12.024). This example shows that mouse IgG can increase the immune response to the antigen it carries. Some researchers have directly linked the Fc fragment of mouse IgG1 to various antigens, such as HBV-mFc, and have produced good results (Allan Ma et al. A dendritic cell receptor-targeted chimeric immunotherapeutic protein (C-HBV) for the treatment of chronic hepatitis B, HUMAN VACCINES & IMMUNOTHERAPEUTICS 2020, VOL. 16, NO. 4, 756–778).

[0008] There are many reports in the literature on the modification of the Fc fragment of human immunoglobulins, but there are relatively few reports on the modification of the Fc fragment of mouse immunoglobulins. We believe that it is necessary to study the engineering modification of the Fc fragment of mouse immunoglobulins to see whether engineering modification can increase the presentation effect.

[0009] Summary of the Invention

[0010] The present application provides a glycosylated fusion protein comprising a murine Fc variant and a polypeptide antigen, wherein the murine Fc variant is obtained by modifying a murine IgG1 Fc fragment, which helps to enhance the binding of the murine Fc fragment to DC cells and DC activation, including the proliferation and activation of specific T cells.

[0011] The present application also provides a nucleic acid molecule encoding the above-mentioned fusion protein, as well as an expression vector and a host cell comprising the nucleic acid molecule.

[0012] The present application also provides a use of the above fusion protein in treating one or more of tumors, viral diseases, autoimmune diseases and inflammatory diseases.

[0013] The first aspect of the present application provides a glycosyl-modified fusion protein comprising a murine Fc variant and a polypeptide antigen, wherein the fusion protein improves binding to DC cells and DC activation, including proliferation and activation of specific T cells;

[0014] The murine Fc variant is obtained by subjecting the murine Fc fragment to amino acid mutations and non-mammalian glycosylation modifications;

[0015] 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;

[0016] The non-mammalian glycosylation modification does not include sialic acid modification;

[0017] Amino acid position numbering is according to the EU numbering system.

[0018] In the scheme provided in the present application, the murine Fc variant is based on the wild-type murine Fc fragment shown in SEQ ID NO: 1, and is obtained after amino acid mutation and non-mammalian glycosylation modification.

[0019] In addition, 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 the present application; for example, when based on the Fc segment of mouse IgG2, the Fc variant obtained after amino acid mutation and non-mammalian glycosylation modification has a similar effect of improving binding to DC cells and DC activation.

[0020] In one embodiment, the amino acid sequence of the murine Fc variant is shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[0021] The amino acid sequence shown in SEQ ID NO: 2 includes 232 amino acid residues. The mutation sites are numbered according to the EU numbering system. The alanine at positions 223, 228, and 230 are respectively at the underlined positions in the following sequence:

[0022] The alanine at positions 223, 228, 230, 330, and 332 in SEQ ID NO: 3 are located at the positions indicated by the underlines in the following sequence, respectively:

[0023] Non-mammalian glycosylation is distinguished from mammalian glycosylation in terms of whether it contains sialic acid modification and the content of mannose. In the technical solution provided in this application, glycosylation is mainly N-glycosylation, and the glycosyl group is derived from at least one of mannose, N-acetylglucosamine and fucose; the sugar chain structure formed by the glycosyl connection is selected from one or more of high mannose type, oligomannose type and fucose type, and the high mannose type is composed of GlcNAc and mannose and contains 5 to 9 mannose; oligomannose refers to less than 5 mannose; fucose type refers to containing fucose. In the solution provided in this application, the above-mentioned glycosylation sites are all located on the 297th amino acid of the Fc variant. Non-mammalian glycosylation helps to improve the binding of the fusion protein to DC cells and DC activation.

[0024] Furthermore, the non-mammal is an insect. Furthermore, the non-mammal is Spodoptera frugiperda. Furthermore, the fusion protein is expressed in Sf9 insect cells.

[0025] In the solution provided in this application, the fusion protein also includes a polypeptide antigen, which specifically refers to a polypeptide substance that can induce an immune response in the human body. The fusion expression of the antigen polypeptide and the Fc variant helps to enhance the presentation of the antigen.

[0026] It is understandable that fusion proteins comprising different types of polypeptide antigens can produce different immune responses after binding to DC cells. In a specific embodiment, the polypeptide antigen is a tumor antigen, which refers to different antigenic components present on tumor cells than normal tissue cells, and can induce the body to produce an anti-tumor immune response through tumor antigens. Furthermore, tumor antigens include one of tumor-specific antigens, tumor-associated antigens, and tumor mutation antigens produced by mutation of tumor-associated antigens. Furthermore, the tumor antigen is prostatic acid phosphatase (PAP), which is a glycoprotein in the prostate exocrine secretion that can hydrolyze phosphate esters. Selecting it as an antigen polypeptide can effectively produce an immune response to enhance the human body's immune effect against prostate cancer. Specifically, the amino acid sequence of prostatic acid phosphatase (PAP) is as shown in SEQ ID NO: 5, positions 1-354.

[0027] In another specific embodiment, the polypeptide antigen is a viral antigen. A viral antigen refers to a substance on the surface of a virus that can induce an immune response in the body. The viral antigen varies depending on the type of virus.

[0028] In one embodiment, the viral antigen includes an HBV surface antigen, which includes HBV S1, HBV S2, and HBV core proteins. Specifically, the amino acid sequence of the HBV surface antigen is as shown in SEQ ID NO: 4, positions 36-394.

[0029] In the solutions provided herein, the fusion protein primarily comprises a polypeptide antigen capable of inducing an immune response and a murine Fc variant that binds to DCs. The murine Fc variant can be linked to the C-terminus of the polypeptide antigen, or the polypeptide antigen and the murine Fc variant can be linked via a linker peptide. In one embodiment, the amino acid sequence of the linker peptide is at least one of GGGS, VRPQGGGS, and SRGGGS.

[0030] In the solution provided herein, the fusion protein may further include a protein tag to facilitate expression, detection, and purification of the target gene. Furthermore, 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.

[0031] In the solution provided in this application, the fusion protein further comprises a hydrophilic peptide, which can be linked to the C-terminus of the murine Fc variant to increase the hydrophilicity of the fusion protein. In a specific embodiment, the amino acid sequence of the hydrophilic peptide is QSLSRSTRGS.

[0032] The fusion protein described above is a glycosylated fusion protein comprising a polypeptide antigen capable of inducing a human immune response and a mouse Fc variant. The fusion protein can be obtained by expressing it in insect cells using a baculovirus expression system.

[0033] In a second aspect, the present application provides a nucleic acid molecule encoding any of the above-mentioned fusion proteins.

[0034] The third aspect of the present application provides a recombinant expression vector comprising the above-mentioned nucleic acid molecule.

[0035] The fourth aspect of the present application provides a host cell comprising the above-mentioned recombinant expression vector.

[0036] In a fifth aspect, the present application provides a pharmaceutical composition comprising any of the above-mentioned fusion proteins and a pharmaceutically acceptable carrier.

[0037] In the present application, pharmaceutically acceptable carriers are non-toxic to cells or individuals at the dosage and concentration used. Typically, physiologically acceptable carriers are pH buffered solutions. The pharmaceutical compositions provided herein are further injections, and the carriers may also include diluents such as water, ethanol, or polyethylene glycol, as well as isotonic additives such as sodium chloride, glucose, or glycerol. Furthermore, conventional cosolvents, buffers, and the like may also be added.

[0038] In a sixth aspect, the present application provides the use of any of the above-mentioned fusion proteins in the preparation of drugs for treating one or more of tumors, viral diseases, autoimmune diseases and inflammatory diseases.

[0039] In the solution provided in the present application, different immune responses of the body can be generated according to the different polypeptide antigens contained in the fusion protein, so as to be suitable for the treatment of different diseases.

[0040] In a specific embodiment, the tumor to be treated includes one or more of gastric cancer, pancreatic cancer, prostate cancer, colorectal cancer, cell carcinoma, liver cancer, lung cancer, breast cancer, ovarian cancer, cervical cancer, oral cancer, esophageal cancer, lymphoma, nasopharyngeal cancer, bladder cancer, squamous cell carcinoma, human bone and soft tissue cancer, renal cell carcinoma, glioblastoma, and leukemia. Furthermore, the tumor to be treated is prostate cancer.

[0041] The therapeutic effect on cancer is manifested as inhibiting the growth of tumor cells in the subject and / or limiting their metastatic spread.

[0042] In one embodiment, the viral disease to be treated refers to a disease caused by a virus. Further, the viral disease to be treated includes a disease caused by HBV.

[0043] Autoimmune and inflammatory diseases refer to diseases caused by autoimmune disorders, such as hyperthyroidism, chronic thyroiditis, rheumatoid arthritis, etc.

[0044] The seventh aspect of the present application provides a method for treating viral diseases, wherein the above-mentioned fusion protein is administered to a subject.

[0045] In an eighth aspect, the present application provides a method for treating tumors, wherein the above-mentioned fusion protein is administered to a subject.

[0046] In a ninth aspect, the present application provides any of the above-mentioned murine Fc variants.

[0047] The fusion protein provided in this application is formed by the fusion of a mouse Fc variant and a polypeptide antigen. The mouse Fc variant helps to improve binding to DC cells and DC activation, stimulating the human body to produce an immune response. The fusion protein formed by fusing it with a polypeptide antigen can be used as a vaccine. Based on the different polypeptide antigens, it can activate different immune responses in the human body, thereby playing a role in treating related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1a shows the SDS-PAGE identification results of Seq2-Sf9, where M1 is a protein molecular weight marker and BSA is a protein standard;

[0049] Figure 1b shows the SDS-PAGE identification results of Seq3-Sf9, where M is a protein molecular weight marker and BSA is a protein standard;

[0050] Figure 2a shows the BLI assay results of Seq2-Sf9 and FcγRIIA protein;

[0051] Figure 2b shows the results of BIL assay of Seq3-Sf9 and FcγRIIA protein;

[0052] FIG3 shows the binding ability of Seq2-293 and Seq3-293 to DC2.4 at different concentrations;

[0053] Figure 4a shows the results of a 7.5% reducing SDS-PAGE / Pageblue staining assay of Seq4-Sf9; Line 1 is the sample eluted from the Ni column; Line 2 is the sample dialyzed into storage solution;

[0054] Figure 4b shows the Western blotting results of Seq4-Sf9; the secondary antibody is Anti-mouse IgG (H+L); Line 1 is the sample eluted from the Ni column; Line 2 is the sample dialyzed into the storage solution;

[0055] Figure 5 shows the results of the binding potency test of Seq4-Sf9 to the mouse dendritic cell line DC2.4, with Mouse IgG1 as the control protein;

[0056] Figure 6a shows the expression detection results of CD54 on the cell surface 48 hours after DC were loaded with Seq4-Sf9 fusion protein. Donor#2, Donor#4, and Donor#5 are 3 PBMC volunteers;

[0057] Figure 6b shows the expression detection results of CD83 on the cell surface 48 hours after DC were loaded with Seq4-Sf9 fusion protein. Donor#2, Donor#4, and Donor#5 are 3 PBMC volunteers;

[0058] Figure 6c shows the expression detection results of CD86 on the cell surface 48 hours after DC were loaded with Seq4-Sf9 fusion protein. Donor#2, Donor#4, and Donor#5 are 3 PBMC volunteers;

[0059] Figure 7 shows the results of CD4 + T cell proliferation assays on Seq4-Sf9-loaded DCs. Donor #2, Donor #4, and Donor #5 are three PBMC volunteers;

[0060] FIG8 is the SDS-PAGE gel identification result of the fusion protein Seq5-293, wherein M1 represents the protein molecular weight marker, R represents the reduced protein, and NR represents the non-reduced protein;

[0061] FIG9 is an SDS-PAGE gel identification result of the fusion protein Seq5-Sf9;

[0062] Figure 10 is the SDS-PAGE gel identification results of the fusion protein Seq6-Sf9;

[0063] Figure 11a shows the expression detection results of CD54 on the cell surface after DCs were loaded with Seq6-Sf9 and Seq7-293 for 48 hours;

[0064] Figure 11b shows the expression detection results of CD83 on the cell surface after DCs were loaded with Seq6-Sf9 and Seq7-293 for 48 hours;

[0065] Figure 12a shows the expression detection results of CD54 on the cell surface after DCs were loaded with Seq5-Sf9 for 48 hours;

[0066] Figure 12b shows the expression detection results of CD83 on the cell surface after DCs were loaded with Seq5-Sf9 for 48 hours;

[0067] Figure 13a shows the expression detection results of CD54 on the cell surface after Seq5-Sf9 and Seq5-293 were loaded on DCs;

[0068] Figure 13b shows the expression detection results of CD83 on the cell surface after DCs were loaded with Seq5-Sf9 and Seq5-293;

[0069] Figure 14a shows the expression detection results of CD54 on the cell surface after DCs were loaded with Seq5-Sf9 and Provenge;

[0070] Figure 14b shows the expression detection results of CD83 on the cell surface after DCs were loaded with Seq5-Sf9 and Provenge;

[0071] Figure 15a shows the proliferation detection results of Seq5-Sf9 and Provenge-loaded DCs on CD4+ T cells from volunteer 1;

[0072] Figure 15b shows the proliferation detection results of CD4+ T cells from volunteer 2 by Seq5-Sf9 and Provenge-loaded DCs;

[0073] Figure 16 shows the results of ELISPOT detection of IFNγ secretion by CD8+ T cells activated by Seq5-Sf9 loaded DCs;

[0074] FIG17 is the results of Western Blot detection of PAP protein expression in human prostate cancer cell lines PC3 and LNCap;

[0075] Figure 18a shows the results of the cytotoxic effect test on PC3 tumor cells by CTL induced by Seq5-Sf9 and Provenge-loaded DCs;

[0076] Figure 18b shows the results of the cytotoxic effect test on LNCap tumor cells by CTL induced by Seq5-Sf9 and Provenge-loaded DCs;

[0077] Figure 19a shows the anti-Seq5-Sf9 serum antibody titer test results of SD rats immunized with Seq5-Sf9 subcutaneously (doses of 0 / 2 / 10 / 50 μg); sample collection time points are Day 0 (before the first injection), Day 14 (before the second injection), Day 28 (before the third injection) and Day 35 (one week after the third injection), n=6;

[0078] Figure 19b shows the serum antibody titer test results of SD rats immunized with Seq5-Sf9 subcutaneously (doses of 0 / 2 / 10 / 50 μg) on ​​Day 14;

[0079] Figure 19c shows the serum antibody titer test results of SD rats immunized with Seq5-Sf9 subcutaneously (doses of 0 / 2 / 10 / 50 μg) on ​​Day 28;

[0080] Figure 19d shows the serum antibody titer test results of SD rats immunized with Seq5-Sf9 subcutaneously (doses of 0 / 2 / 10 / 50 ug) on ​​Day 35;

[0081] Figure 20a shows the ELISPOT assay results of IFNγ secretion by SD rat splenocytes after immunization with Seq5-Sf9 on Day 35;

[0082] Figure 20b is an ELISPOT dot plot of IFNγ secretion by SD rat splenocytes after immunization with Seq5-Sf9 on Day 35;

[0083] Figure 21 is a HE pathological staining image of the prostate tissue of SD rats after immunization with Seq5-Sf9 on Day 35 (20× magnification);

[0084] Figure 22a shows the expression detection results of CD54 on the cell surface after 48 hours of DC loading by Seq6-Sf9 compared with Seq5-Sf9;

[0085] Figure 22b shows the expression detection results of CD83 on the cell surface after Seq6-Sf9 compared with Seq5-Sf9 loading DC for 48 hours. DETAILED DESCRIPTION

[0086] To make the purpose, technical solutions, and advantages of this application more clear, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the embodiments of this application. Obviously, the described embodiments are part of the embodiments of this application, not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0087] Example 1 Fc variant affinity detection

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

[0089] To enhance the stability of the mouse IgG Fc fragment, three mutations were made at positions 223, 228, and 230 of the wild-type mouse Fc fragment set forth 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 to DCs, five mutations were made at positions 223, 228, 230, 330, and 332 of the Fc fragment, based on the wild-type mouse Fc fragment. The resulting amino acid sequence is shown in SEQ ID NO: 3.

[0090] The 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. The DNA sequence corresponding to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3 was synthesized and then used For insect cell expression, the recombinant plasmid was subcloned into the pFastBacHTa-gp64 vector digested with Sal I (New England Biolabs, R3138S) and Hind III (New England Biolabs, R0104S). The recombinant plasmid was transformed into DH10Bac chemically competent Escherichia coli cells and 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 overnight incubation, white colonies were picked, and recombinant bacmid DNA was isolated according to standard protocols. Positive bacmids were transiently transfected into 2 ml of Sf9 insect cells using a transfection reagent. The cells were incubated in ESF 921 medium for a period of time. The cells and supernatant were harvested, and the proteins were purified from the supernatant using Protein A. They were designated Seq2-Sf9 and Seq3-Sf9, respectively.

[0091] Seq2-Sf9 and Seq3-Sf9 were identified using Coomassie Brilliant Blue-stained SDS-PAGE gels, using BSA as a control protein. The identification results are shown in Figures 1a and 1b, respectively. The arrows indicate the positions of Seq2-Sf9 and Seq3-Sf9, confirming that the purified proteins were approximately 26 kDa in molecular weight. Biolayer interferometry (BLI) was used to perform multi-concentration affinity testing on the purified Seq2-Sf9 and Seq3-Sf9 proteins, setting a concentration gradient of 5000 nM, 2500 nM, 1250 nM, 625 nM, and 312.5 nM to determine the affinity of Seq2-Sf9 and Seq3-Sf9 proteins for FcγRIIA protein. The test results are shown in Figures 2a and 2b. The curves shown from bottom to top indicate that the concentrations of Seq2-Sf9 and Seq3-Sf9 proteins decrease in sequence. After calculation, the affinities of Seq2-Sf9 and Seq3-Sf9 to FcγRIIA protein are 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 Acquisition of Fc variants expressed in 293 cells and detection of their binding to DCs

[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 with a secretion signal peptide, with EcoRI (New England Biolabs, R0101V) and HindIII (New England Biolabs, R0104S) restriction sites, and electroporated into Escherichia coli trans5α. After ampicillin selection, single clones were sequenced to obtain the correct recombinant plasmids. The host bacteria containing the recombinant plasmids were then expanded and an endotoxin removal kit was used to obtain sterile and endotoxin-free recombinant plasmids. The sterile and 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 culture supernatant was collected and separated and purified using Protein A resin to obtain proteins Seq2-293 and Seq3-293.

[0094] Seq2-293 and Seq3-293 enhance binding to FcRs, which are primarily expressed on the surface of various monocytic cells, including dendritic cells (DCs). Because the Fc variants described above are derived from mouse Fc regions, the mouse dendritic cell line DC2.4 was selected for this study. It expresses relatively stable FcRs and is capable of binding to mouse Fc. Under natural conditions, Fc binds to its receptor, FcR, with weak affinity. Seq2-293 and Seq3-293 were incubated with DC2.4 cells at 4°C for 1 hour. Biotin-labeled anti-mouse IgG1 and streptavidin-HRP were added, respectively, for reaction. The reaction was terminated with TMB substrate for color development. Visible light OD values ​​of 450-570 were measured, indicating Fc / FcR binding on the DC2.4 cell surface.

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

[0096] Example 2 Preparation and Biological Activity Evaluation of Seq4-Sf9

[0097] 2.1 Seq4-Sf9 protein preparation

[0098] The triple mutant mouse Fc with the amino acid sequence shown in SEQ ID NO: 2 was fused with hepatitis B virus antigen (including HBV S1 / S2 / Core) through a connecting peptide, and a hydrophilic peptide was connected to the C-terminus to obtain a fusion protein with the amino acid sequence shown in SEQ ID NO: 4, in which positions 36-394 were hepatitis B virus antigens and positions 403-634 were the triple mutant mouse Fc with the amino acid sequence shown in SEQ ID NO: 2.

[0099] The synthesized 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 insect cell expression and transformed into Escherichia coli DH10Bac for transposition to produce 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, the cell pellet was collected, and ultrasonic lysed. The fusion protein was purified from the infected Sf9 cells using a Ni affinity column and named Seq4-Sf9. The fusion protein was biochemically assayed using Coomassie Brilliant Blue stained SDS-PAGE gel and WB. The results are shown in Figures 4a-4b. The monomeric apparent molecular weight of the fusion protein is approximately 75KD.

[0100] 2.2 Glycosylation identification of Seq4-Sf9

[0101] The fusion protein Seq4-Sf9, expressed in an Sf9 insect cell system, was digested using trypsin or chymotrypsin. The peptide samples were then analyzed by liquid chromatography-mass spectrometry (LC-MS / MS), and the raw data were analyzed using software. By comparing the undeglycosylated peptide maps with the deglycosylated peptide maps (treated with glycosidase), N-glycosylated peptides were identified. Further N-glycosylation of asparagine at position 484 (position 297 of the Fc fragment) was confirmed by primary molecular weight analysis and secondary mass spectrometry. The results showed that the fusion protein expressed in the Sf9 insect cell system was primarily glycosylated with high mannose, accompanied by small amounts of oligomannose and fucose. The specific modification analysis results are shown in Table 1-2.

[0102] Table 1 Glycosylation modification analysis

[0103] Table 2 Analysis of sugar proportion

[0104] Note: Man represents mannose, and the number following it represents the number of mannose; \ represents non-glycosylation modification; Deamidation refers to deamidation modification. In deglycosylated samples, the N at the N-glycosylation site will undergo deamidation to become D (asparagine).

[0105] 2.3 Detection of binding potency of Seq4-Sf9 to DC2.4 FcR

[0106] Fc variants can enhance binding to Fc receptors (FcRs), which are primarily expressed on the surface of various monocytic cells, including dendritic cells (DCs). DC2.4 is a mouse dendritic cell line that stably expresses FcRs and serves as a test for the binding potency of different Fc and Fc variants. To verify the binding potency of the fusion protein to DC2.4 cells, Seq4-Sf9 was incubated with DC2.4 cells at 4°C for 1 hour. Biotin-labeled anti-mouse IgG1 and streptavidin-HRP were added, respectively, for reaction. The reaction was terminated with TMB substrate for color development. Absorbance values ​​of OD450-570 indicate cell surface binding of the fusion protein. Native mouse IgG1 was also used as a control. As shown in Figure 5, Seq4-Sf9 exhibited stronger binding to DC2.4 than the control mouse IgG1, suggesting that the fusion protein has the potential for potent antigen presentation.

[0107] 2.4 Evaluation of the biological activity of Seq4-Sf9

[0108] 2.4.1 Evaluation of the immune activation effect of the fusion protein Seq4-Sf9 using DCs induced by CD14+ monocytes from healthy human peripheral blood PBMCs

[0109] To evaluate the immune-stimulating effects of the Seq4-Sf9 fusion protein, CD14+ monocytes were sorted from PBMCs of three healthy individuals 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 possess potent phagocytic function, and the murine Fc domain contained in the fusion protein enhances their phagocytic uptake, promoting DC maturation and differentiation. Flow cytometry was used to assess changes in surface markers in DCs loaded with the Seq4-Sf9 fusion protein. These included CD54 (intercellular adhesion molecule-1), an adhesion molecule that facilitates the adhesion and interaction of mature DCs with other immune cells, such as T cells; CD83, which is involved in antigen presentation and T cell activation; and CD86, a costimulatory molecule. The test results are shown in Figures 6a-6c. In three different volunteers, DC cells loaded with the Seq4-Sf9 fusion protein highly expressed CD54, CD83, and CD86 maturation markers on their cell surfaces, indicating that the fusion protein has a potent DC activation effect, activating the antigen presentation ability of vaccine-loaded DCs and initiating an immune response as a starting point.

[0110] 2.4.2 Key in vitro evaluation of the effect of Seq4-Sf9-loaded DCs on T cell immune responses

[0111] The primary function of mature DCs (DCs) in CD4+ T cell proliferation is antigen presentation. Antigen peptide / MHC-I or MHC-II complexes on their surface are recognized by T cell surface receptors (TCRs). T cell proliferation is a key step in DC-T activation. Therefore, Seq4-Sf9-loaded DCs were co-cultured with CD4+ T cells labeled with CFSE fluorescent dye. Following antigen presentation, T cells rapidly proliferated and differentiated into Th cells (helper T cells). CD4+ T cell proliferation was thus used to assess immune status. As shown in Figure 7, Seq4-Sf9-loaded DCs significantly promoted T cell proliferation compared to a negative control of CD4+ T cells from the same source (Seq4-Sf9 0 μg / ml), and this effect was positively correlated with the concentration of the active agent.

[0112] Example 3 Preparation and Biological Activity Evaluation of Seq5-293, Seq5-Sf9, and Seq6-Sf9

[0113] 3.1 Preparation of Seq5-293

[0114] The triple-mutant mouse Fc with the amino acid sequence shown in SEQ ID NO:2 was fused and expressed with the prostate cancer-specific marker PAP via a connecting peptide to obtain a fusion protein with the amino acid sequence shown in SEQ ID NO:5, wherein positions 1-354 represent the prostate cancer-specific marker PAP, and positions 361-592 represent the triple-mutant mouse Fc with the amino acid sequence shown in SEQ ID NO:2.

[0115] A gene encoding a fusion protein of the amino acid sequence shown in SEQ ID NO: 5 was synthesized, and the gene sequence encoding the fusion protein was ligated into the expression vector pcDNA3.4 vector with restriction enzyme sites EcoRI (New England Biolabs, R0101V) and HindIII (New England Biolabs, R0104S) to prepare a recombinant expression plasmid, and the recombinant expression plasmid was transiently transfected into eukaryotic cells HD293F, and the cells were cultured, and the cell culture fluid was collected and centrifuged and filtered. The filtered culture supernatant was loaded onto a Protein A affinity chromatography column and purified to obtain a fusion protein, named Seq5-293. Biochemical assays were performed using Coomassie Brilliant Blue stained SDS-PAGE gels, and the assay results are shown in FIG8 .

[0116] 3.2 Preparation of Seq5-Sf9

[0117] The triple mutant mouse Fc of the amino acid sequence shown in SEQ ID NO: 2 was fused with the prostate cancer specific marker PAP through a connecting peptide and named SEQ ID NO: 5. The corresponding coding sequence was connected to the pFastBacHTa-gp64 vector digested with Sal I (New England Biolabs, R3138S) and Hind III (New England Biolabs, R0104S) for insect cell expression and transformed into Escherichia coli DH10Bac for transposition to produce 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 harvested by centrifugation and the protein obtained by Protein A purification was named Seq5-Sf9. Biochemical assays were performed using Coomassie Brilliant Blue stained SDS-PAGE gels, and the assay results are shown in Figure 9.

[0118] 3.3 Preparation of Seq6-Sf9

[0119] The five mutant mouse Fcs with the amino acid sequence shown in SEQ ID NO:3 were fused and expressed with the prostate cancer-specific marker PAP through a connecting peptide to obtain a fusion protein with the amino acid sequence shown in SEQ ID NO:6, wherein positions 1-354 are PAP, a specific marker for prostate cancer, and positions 361-592 are the five mutant mouse Fcs with the amino acid sequence shown in SEQ ID NO:3.

[0120] The same method as 3.2 was used to express the above fusion protein in Sf9 insect cells. The expressed fusion protein was named Seq6-Sf9. Biochemical analysis was performed using Coomassie Brilliant Blue-stained SDS-PAGE gel. The results are shown in FIG10 .

[0121] 3.4 Detection of the effect of Seq6-Sf9 on DC activation

[0122] The human wild-type Fc fragment and the prostate cancer-specific marker PAP were expressed in mammalian 293 cells using the same method as in 3.1 to obtain the Seq7-293 fusion protein, whose amino acid sequence is shown in SEQ ID NO:7. The Seq7-293 fusion protein was used as a control to verify the effects of the Fc variants and insect glycoforms on DC activation.

[0123] CD14+ monocytes were sorted from PBMCs of two healthy individuals and induced to differentiate into immature DCs in a culture system containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Immature DCs, which possess robust phagocytic capabilities, were loaded with Seq6-Sf9 and Seq7-293 fusion proteins, respectively. DCs ingested the fusion proteins and presented the antigen, thereby activating them and expressing activation markers (including CD54 and CD83). Flow cytometry was used to assess changes in surface markers on vaccine-loaded DCs. The results are shown in Figures 11a-11b. At the same concentration, Seq6-Sf9 significantly activated DCs compared to Seq7-293, with high cell surface expression of CD54 and CD83. These results demonstrate that the murine Fc variant and non-mammalian glycoforms in the fusion proteins significantly enhance DC phagocytosis and activation, and contribute to the expression of activation phenotypes such as CD54 and CD83.

[0124] 3.5 Detection of the effect of Seq5-Sf9 on DC activation

[0125] 3.5.1 Evaluation of the immune activation effect of the fusion protein Seq5-Sf9 using DCs induced by CD14+ monocytes in human peripheral blood PBMCs

[0126] To evaluate the immune-stimulating effects of Seq5-Sf9, CD14+ monocytes were isolated from PBMCs of three healthy individuals 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 possess potent phagocytic function, and the murine Fc domain contained in the fusion protein enhances their uptake, promoting DC maturation and differentiation. Flow cytometry was used to assess changes in surface markers in vaccine-loaded DCs, including CD54 (ICAM-1), an adhesion molecule that facilitates the adhesion and interaction of mature DCs with other immune cells, such as T cells, and CD83, which is involved in antigen presentation and T cell activation. The test results are shown in Figures 12a-12b. In three different volunteers, Seq5-Sf9-loaded DCs highly expressed CD54 and CD83 on their cell surface, indicating that the fusion protein has a potent DC activation effect, activating the antigen presentation ability of Seq5-Sf9-loaded DCs, serving as the starting link to initiate an immune response.

[0127] In addition, the immune activation effects of Seq5-Sf9, Seq5-293, and the positive control drug Provenge were compared. The comparison results are shown in Figures 13a-14b. It can be seen that Fc variants and non-mammalian glycosylation enhance the DC activation effect, and Seq5-Sf9 has stronger DC activation function.

[0128] 3.5.2 Key in vitro evaluation of the effect of Seq5-Sf9-loaded DCs on T cell immune responses

[0129] The most important function of mature DCs for CD4+T cell proliferation is antigen presentation. The antigen peptide / MHC-I complex or antigen peptide / MHC-II complex on its surface is recognized by the T cell surface receptor (TCR). An important part of DC-T activation is the proliferation of T cells. Therefore, Seq5-Sf9-loaded DCs and CFSE fluorescent dye-labeled CD4+T cells are co-cultured. After antigen presentation, T cells can rapidly proliferate and differentiate into Th cells (helper T cells). The immune status is thus assessed based on the proliferation of CD4+T cells. As shown in Figures 15a-15b, DC cells from different healthy people can significantly promote the proliferation of T cells after being loaded with Seq5-Sf9, and the effect is positively correlated with its concentration; Provenge under the same experimental conditions also has the same effect.

[0130] 3.5.3 In vitro evaluation of Seq5-Sf9-loaded DC antigen presentation to activated cytotoxic T cells

[0131] The antigen peptide / MHC class I molecule complex presented on the DC membrane surface can directly recognize and bind to the TCR on the surface of CD8+ T cells, thereby activating CD8+ T cells to exert their biological effects, one of the main ways of which is the secretion of interferon gamma (IFNγ). Seq5-Sf9-loaded DCs were co-cultured with CD8+ T cells from the same volunteer, and Seq5-Sf9-loaded DCs were added again for secondary stimulation to activate a large number of CD8+ T cells. IFNγ secretion at the single cell level was detected by ELISPOT. The test results are shown in Figure 16. It can be seen that the Seq5-Sf9-loaded group produced significantly more IFNγ than the volunteer's own CD8+ T cells (negative control), that is, Seq5-Sf9 can effectively promote CD8+ T cell activation.

[0132] 3.5.4 Human prostate cancer cell lines expressing PAP antigen were used as target cells to evaluate the killing effect of CTL activated by Seq5-Sf9

[0133] Prostate cancer treatment has different therapeutic effects 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 higher levels of PAP protein. These cells were used as target cells and co-cultured with effector cells (CTLs activated by DCs loaded with Seq5-Sf9); the absolute count of dead cells was calculated using CFSE and Counting Beads. As shown in Figures 18a-18b, CTLs activated by DCs loaded with Seq5-Sf9 can directly kill target cells, and the killing effect is proportional to the concentration of Seq5-Sf9. In addition, the number of dead cells in LNCap cells, which express higher levels of PAP, is much higher than that in PC3, confirming its targeted killing effect and that the effect is higher than that of Provenge.

[0134] 3.6 Immunogenicity Assessment of Seq5-Sf9

[0135] To evaluate the immune response of Seq5-Sf9 in vivo, 6-8 week-old Sprague Dawley (SD) male rats were immunized with Seq5-Sf9. Sprague Dawley rats were subcutaneously injected with Seq5-Sf9 (Placebo, 2 μg, 10 μg, and 50 μg) every two weeks for a total of three doses. Serum was collected from the animals on Day 14, Day 28, and Day 35 (one week after the third immunization) to assess Seq5-Sf9-specific antibody titers. As shown in Figures 19a-19d, compared to the Placebo group, a low dose of 2 μg was sufficient to induce vaccine-specific serum antibodies, with antibody titers reaching peak levels after the third and one week after the third dose.

[0136] In addition, spleens from rats on Day 35 were re-stimulated with Seq5-Sf9 in vitro to activate and induce effector T cells. ELISPOT assays were performed to measure IFNγ secretion from individual splenocytes. As shown in Figures 20a-20b, splenic immune cells rapidly differentiated into cytotoxic T lymphocytes and secreted IFNγ following secondary stimulation with Seq5-Sf9.

[0137] At the same time, the prostate tissue was observed. The results are shown in FIG21 . Inflammatory cell infiltration was also observed in the prostate tissue of rats in the Seq5-Sf9 group.

[0138] In summary, Seq5-Sf9 can establish a complete in vivo immune response in SD rats and effectively activate T cells, that is, Seq5-Sf9 can complete in vivo immune activation.

[0139] 3.7 Evaluation of the immune activation effect of the fusion protein Seq6-Sf9 using DCs induced by CD14+ monocytes in human peripheral blood PBMCs

[0140] The Fc region of Seq6-Sf9 differs from that of Seq5-Sf9. To evaluate its immune activation effect, CD14+ monocytes sorted from PBMCs of two healthy individuals were induced to differentiate and loaded with Seq6-Sf9. Flow cytometry was used to assess changes in surface markers of antigen-loaded DCs. As shown in Figures 22a-22b, Seq6-Sf9 increased the expression of CD54 and CD83 on the surface of DCs, promoting DC activation, and the expression of these markers was higher than that of Seq5-Sf9.

[0141] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A glycosyl-modified fusion protein, characterized in that: The fusion protein comprises a mouse Fc variant and a polypeptide antigen, wherein the fusion protein improves binding to DC cells and DC activation, including proliferation and activation of specific T cells; The mouse Fc variant is obtained by subjecting the mouse Fc fragment to amino acid mutation and non-mammalian glycosylation modification; 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 non-mammalian glycosylation modification does not include sialic acid modification; The amino acid position numbering is performed according to the EU numbering system.

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

3.

3. The fusion protein according to claim 1 or 2, characterized in that In the glycosylation modification, the sugar group is derived from at least one of mannose, N-acetylglucosamine and fucose.

4. The fusion protein according to claim 3, characterized in that In the glycosylation modification, the sugar chain structure formed by the sugar linkage is selected from at least one of a high mannose type, an oligomannose type, and a fucose type.

5. The fusion protein according to any one of claims 1 to 4, characterized in that The polypeptide antigen is a tumor antigen, including a tumor-specific antigen, a tumor-associated antigen, and a tumor mutation antigen generated by a mutation of a tumor-associated antigen.

6. The fusion protein according to claim 5, characterized in that The tumor antigen is PAP.

7. The fusion protein according to any one of claims 1 to 4, characterized in that The polypeptide antigen is a viral antigen.

8. The fusion protein according to any one of claims 1 to 7, characterized in that The fusion protein further comprises a protein tag.

9. The fusion protein according to any one of claims 1 to 8, characterized in that: The fusion protein further comprises a hydrophilic peptide, which is linked to the C-terminus of the murine Fc variant.

10. A nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 9.

11. A recombinant expression vector, characterized in that: Comprising the nucleic acid molecule of claim 10.

12. A host cell, characterized in that Comprising the recombinant expression vector according to claim 11.

13. A pharmaceutical composition, characterized in that The invention comprises the fusion protein according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

14. Use of the fusion protein according to any one of claims 1 to 9 in the preparation of a medicament for treating one or more of tumors, viral diseases, autoimmune diseases and inflammatory diseases.

15. A method for treating a viral disease, wherein: The fusion protein of claim 7 is administered to a subject.

16. A method for treating a tumor, wherein: The fusion protein according to claim 5 or 6 is administered to a subject.

17. The murine Fc variant according to any one of claims 1 to 9.

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