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

Abstract

The invention provides a glycosyl-modified fusion protein, a nucleic acid molecule, an expression vector, a host cell and application. In the first aspect, the invention provides a glycosyl-modified fusion protein which comprises a mouse Fc variant and a polypeptide antigen, the mouse Fc variant is obtained by performing amino acid mutation and non-mammalian glycosylation modification on a mouse Fc fragment; the mouse Fc variant comprises at least one of alanine at the 223 site, alanine at the 228 site, alanine at the 230 site, leucine at the 330 site and glutamic acid at the 332 site; the non-mammalian glycosylation modification does not comprise sialic acid modification; the site numbering is carried out according to an EU numbering system; the mouse Fc variant improves combination of a mouse Fc fragment and DC cells and DC activation, including proliferation and activation of specific T cells.

Description

[0001] This application claims the priority of 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 priority of 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] FcR is a type of cell surface protein that can specifically bind to the functional region Fc fragment at the carboxyl terminus of the immunoglobulin (Ig) heavy chain, and is crucial in the process of antibody-dependent immune response. Different types of cells can express different types of FcR, and different types of FcR also bind to different structural types of Ig, thereby inducing different types of subsequent immune responses. Immunoglobulin G (IgG) has a very powerful effect in the human body. It is the main antibody in the serum against bacteria, viruses, and toxins. The FcR that binds to IgG is also called FcγR. According to the affinity between FcγR and Fc fragment, FcγR can be divided into three subfamilies: FcγRI, FcγRII and FcγRIII (with IgG affinity ranking: 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 both clearing target cells and maintaining lasting immune protection.

[0005] In order to gain a deeper understanding of the binding between Fc fragment and FcγRIIa, many researchers have modified the structure of Fc fragment. Some researchers have found that the Fc fragment mutation (G236A) selectively activates FcγRIIa (the two have 96% sequence identity) on the basis of 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, increase the binding ratio of FcγRIIa / FcγRIIb by 13 times, and enhance 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 of insects is different from that of higher eukaryotes. The sugar chains of recombinant N-glycoproteins generally expressed are simple, non-sialylated high / low mannose types, rather than the sialylated complex sugar chains produced by mammals at the same glycosylation positions. This may be due to the high N-acetylglucosaminidase activity, low glycosyltransferase activity and fewer sugar-nucleoside sources of their cells. The effector function triggered by the fragment crystallizable (Fc) region of immunoglobulin G (IgG) antibodies changes due to the presence of terminal sialic acid (Sia) residues at asparagine-297 (Asn-297). Studies have found that high sialylation can hinder binding to FcγRIIIa on natural killer (NK) cells, resulting in reduced ADCC efficacy. The 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 connected 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 on the modification of the Fc fragment of human immunoglobulins in the literature, but there are relatively few reports on the modification of the Fc fragment of mouse immunoglobulins. In order to determine whether engineering modification can increase the presentation effect, we believe that it is necessary to study the engineering modification of the Fc fragment of mouse immunoglobulins. Summary of the invention

[0009] The present application provides a glycosylated fusion protein, comprising a mouse Fc variant and a polypeptide antigen, wherein the mouse Fc variant is obtained by modifying a mouse IgG1 Fc fragment, which helps to improve the binding of the mouse Fc fragment to DC cells and DC activation, including the proliferation and activation of specific T cells.

[0010] The present application also provides a nucleic acid molecule encoding the above fusion protein, as well as an expression vector and a host cell comprising the nucleic acid molecule.

[0011] The present application also provides an application of the above fusion protein in treating one or more of tumors, viral diseases, autoimmune diseases and inflammatory diseases.

[0012] 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;

[0013] The mouse Fc variant is obtained by subjecting the mouse Fc fragment to amino acid mutation and non-mammalian glycosylation modification;

[0014] 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;

[0015] The non-mammalian glycosylation modification does not include sialic acid modification;

[0016] The amino acid position numbering is performed according to the EU numbering system.

[0017] 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.

[0018] 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 the Fc segment of mouse IgG2 is used as the basis, the Fc variant obtained after amino acid mutation and non-mammalian glycosylation modification has a similar effect of improving the binding to DC cells and DC activation.

[0019] In a specific embodiment, the amino acid sequence of the murine Fc variant is shown in SEQ ID NO:2 or SEQ ID NO:3.

[0020] 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 positions indicated by the underlines in the following sequence:

[0021] VDKKIVP A DCGC A PCIC AVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTK GRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGL.

[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:

[0023] VDKKIVP A DCGC A PCIC A VPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFP L P E EKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGL.

[0024] Non-mammalian glycosylation is distinguished from mammalian glycosylation in whether it contains sialic acid modification and the content of mannose. In the technical scheme provided in the present application, glycosylation is mainly N-glycosylation, and the glycosyl 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 scheme provided in the present 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 fusion protein to DC cells and DC activation.

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

[0026] In the solution provided in the present 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.

[0027] It is understandable that fusion proteins including 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 antigen components present on normal tissue cells on tumor cells, and can induce the body to produce an anti-tumor immune response through tumor antigens. Further, 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 that can hydrolyze phosphate esters in prostatic exocrine secretions. Selecting it as an antigen polypeptide can effectively produce an immune response to enhance the human body's immune effect on prostate cancer. Specifically, the amino acid sequence of prostatic acid phosphatase (PAP) is as shown in SEQ ID NO: 5, positions 1-354 in the sequence.

[0028] 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.

[0029] In a specific embodiment, the viral antigen includes HBV surface antigen, and the HBV surface antigen includes HBV S1, HBV S2 and HBV core protein. Specifically, the amino acid sequence of the HBV surface antigen is shown in positions 36-394 of the sequence shown in SEQ ID NO:4.

[0030] In the solution provided in the present application, the above fusion protein mainly includes a polypeptide antigen capable of inducing an immune response in the body and a mouse Fc variant that binds to DC cells. The mouse Fc variant can be connected to the C-terminus of the polypeptide antigen, or the polypeptide antigen and the mouse Fc variant can be connected by a connecting peptide. In a specific embodiment, the amino acid sequence of the connecting peptide is at least one of GGGS, VRPQGGGS, and SRGGGS.

[0031] In the solution provided in the present application, the above fusion protein may also 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 connected to the N-terminus of the polypeptide antigen to improve the purification efficiency of the fusion protein.

[0032] In the solution provided in the present application, the fusion protein further comprises a hydrophilic peptide, which can be connected to the C-terminus of the murine Fc variant to improve the hydrophilicity of the fusion protein. In a specific embodiment, the amino acid sequence of the hydrophilic peptide is QSLSRSTRGS.

[0033] 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 in insect cells via a baculovirus expression system.

[0034] The second aspect of the present application provides a nucleic acid molecule encoding any of the above-mentioned fusion proteins.

[0035] The third aspect of the present application provides a recombinant expression vector comprising the above-mentioned nucleic acid molecule.

[0036] The fourth aspect of the present application provides a host cell, comprising the above-mentioned recombinant expression vector.

[0037] The fifth aspect of the present application provides a pharmaceutical composition, comprising any of the above-mentioned fusion proteins and a pharmaceutically acceptable carrier.

[0038] In the present application, the pharmaceutically acceptable carrier is nontoxic to cells or individuals in the dosage and concentration used. Usually, the physiologically acceptable carrier is a pH buffer solution. The pharmaceutical composition provided by the present application is further an injection, and the above-mentioned carrier may also include diluents such as water, ethanol, polyethylene glycol, and isotonic additives such as sodium chloride, glucose or glycerol. In addition, conventional cosolvents, buffers, etc. may also be added.

[0039] The sixth aspect of 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.

[0040] In the scheme 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.

[0041] In a specific embodiment, the tumors treated include 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, leukemia. Further, the tumor treated is prostate cancer.

[0042] The therapeutic effect of cancer is manifested as inhibiting the growth of tumor cells in the subject and / or limiting their metastatic spread.

[0043] In a specific 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.

[0044] Autoimmune and inflammatory diseases refer to diseases caused by autoimmune disorders, such as hyperthyroidism, chronic thyroiditis, rheumatoid arthritis, etc.

[0045] 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.

[0046] The eighth aspect of the present application provides a method for treating tumors, which comprises administering the above-mentioned fusion protein to a subject.

[0047] The ninth aspect of the present application provides any of the above-mentioned murine Fc variants.

[0048] The fusion protein provided in the present application is formed by the fusion of a mouse Fc variant and a polypeptide antigen. The mouse Fc variant helps to improve the binding with DC cells and DC activation, stimulates the human body to produce an immune response, and the fusion protein formed by fusing it with the polypeptide antigen can be used as a vaccine. Based on the difference in polypeptide antigens, different immune responses of the human body can be activated, thereby playing a role in treating related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1a The SDS-PAGE identification results of Seq2-Sf9, where M1 is a protein molecular weight marker and BSA is a protein standard;

[0050] Figure 1b The SDS-PAGE identification results of Seq3-Sf9, where M is a protein molecular weight marker and BSA is a protein standard;

[0051] Figure 2a The results of BLI determination of Seq2-Sf9 and FcγRIIA protein;

[0052] Figure 2b The results of the BIL assay of Seq3-Sf9 and FcγRIIA protein;

[0053] Figure 3 The results of the binding ability test of Seq2-293 and Seq3-293 with DC2.4 at different concentrations;

[0054] Figure 4a The results of Seq4-Sf9 in 7.5% reducing SDS-PAGE / Pageblue staining test; Line 1 is the sample eluted from the Ni column; Line 2 is the sample dialyzed into the storage solution;

[0055] Figure 4b The results of the WB experiment 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;

[0056] Figure 5 The results are for the binding potency test of Seq4-Sf9 to the mouse dendritic cell line DC2.4, and Mouse IgG1 is the control protein;

[0057] Figure 6a This is the result of the detection of CD54 expression on the cell surface 48 hours after DCs were loaded with Seq4-Sf9 fusion protein. Donor#2, Donor#4, and Donor#5 are 3 PBMC volunteers;

[0058] Figure 6b The results of the cell surface CD83 expression test after DCs were loaded with Seq4-Sf9 fusion protein for 48 hours. Donor#2, Donor#4, and Donor#5 are 3 PBMC volunteers;

[0059] Figure 6c This is the result of the detection of CD86 expression on the cell surface 48 hours after DCs were loaded with Seq4-Sf9 fusion protein. Donor#2, Donor#4, and Donor#5 are 3 PBMC volunteers;

[0060] Figure 7 This is the result of the proliferation test of CD4+T cells by Seq4-Sf9-loaded DCs. Donor#2, Donor#4, and Donor#5 are three PBMC volunteers;

[0061] Figure 8 The SDS-PAGE gel identification result of the fusion protein Seq5-293, where M1 represents the protein molecular weight marker, R represents the reduced protein, and NR represents the non-reduced protein;

[0062] Fig. 9 This is the SDS-PAGE gel identification result of the fusion protein Seq5-Sf9;

[0063] Fig.10 This is the SDS-PAGE gel identification result of the fusion protein Seq6-Sf9;

[0064] Fig.11a The results of CD54 expression on the cell surface after DCs were loaded with Seq6-Sf9 and Seq7-293 for 48 hours;

[0065] Fig.11bThe results of the expression of CD83 on the cell surface after DCs were loaded with Seq6-Sf9 and Seq7-293 for 48 hours;

[0066] Fig.12a The results of the detection of CD54 expression on the cell surface 48 hours after DCs were loaded with Seq5-Sf9;

[0067] Figure 12b The results of the expression of CD83 on the cell surface after DCs were loaded with Seq5-Sf9 for 48 hours;

[0068] Fig.13a The results of the cell surface CD54 expression test after DCs were loaded with Seq5-Sf9 and Seq5-293;

[0069] Fig.13b The results of the expression of CD83 on the cell surface after DCs were loaded with Seq5-Sf9 and Seq5-293;

[0070] Fig.14a The results of the cell surface CD54 expression test after DCs were loaded with Seq5-Sf9 and Provenge;

[0071] Fig.14b The results of the cell surface CD83 expression test after DCs were loaded with Seq5-Sf9 and Provenge;

[0072] Fig.15a The results of the proliferation test of CD4+T cells from volunteer 1 by DC loaded with Seq5-Sf9 and Provenge;

[0073] Fig.15b The results of the proliferation test of CD4+T cells from volunteer 2 by DC loaded with Seq5-Sf9 and Provenge;

[0074] Fig.16 The results of ELISPOT detection of IFNγ secretion by CD8+T activated by Seq5-Sf9 loaded DCs;

[0075] Fig.17 The results of Western Blot detection of PAP protein expression in human prostate cancer cell lines PC3 and LNCap;

[0076] Fig.18a The results show the killing effect of CTL induced by Seq5-Sf9 and Provenge-loaded DC on PC3 tumor cells;

[0077] Fig.18bThe results show the killing effect of CTL induced by Seq5-Sf9 and Provenge-loaded DC on LNCap tumor cells;

[0078] Fig.19a The results of anti-Seq5-Sf9 serum antibody titer detection in SD rats immunized with Seq5-Sf9 subcutaneously (doses of 0 / 2 / 10 / 50ug); the sample collection time points were 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;

[0079] Fig.19b The results of serum antibody titer test of SD rats immunized with Seq5-Sf9 subcutaneously (doses of 0 / 2 / 10 / 50ug) on ​​Day 14;

[0080] Fig.19c The results of serum antibody titer test of SD rats immunized with Seq5-Sf9 subcutaneously (doses of 0 / 2 / 10 / 50ug) on ​​Day 28;

[0081] Fig.19d The results of serum antibody titer test of SD rats immunized with Seq5-Sf9 subcutaneously (doses of 0 / 2 / 10 / 50ug) on ​​Day 35;

[0082] Fig.20a The results of ELISPOT assay of IFNγ secretion by spleen cells of SD rats after immunization with Seq5-Sf9 on Day 35;

[0083] Fig.20b This is the ELISPOT detection spot diagram of IFNγ secretion by spleen cells of SD rats after immunization with Seq5-Sf9 on Day 35;

[0084] Fig.21 HE pathological staining of prostate tissue of SD rats after immunization with Seq5-Sf9 on Day 35 (20× magnification);

[0085] Fig.22a The expression detection results of CD54 on the cell surface after 48h of DC loading with Seq5-Sf9 were compared for Seq6-Sf9;

[0086] Figure 22b The expression detection results of cell surface CD83 were compared between Seq6-Sf9 and Seq5-Sf9 after DC was loaded for 48 hours. DETAILED DESCRIPTION

[0087] In order to make the purpose, technical solutions and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below in combination with the embodiments of this application. Obviously, the described embodiments are part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0088] Example 1 Fc variant affinity detection

[0089] 1.1 Acquisition and affinity testing of Fc variants expressed in Sf9 insect cells

[0090] To enhance the stability of the mouse IgG Fc fragment, based on the wild-type mouse Fc fragment shown in SEQ ID NO: 1, three sites at positions 223, 228, and 230 of the Fc fragment were mutated, and the amino acid sequence after mutation is shown in SEQ ID NO: 2. To enhance the binding and activation of the mouse IgG1 Fc fragment to DC cells, five sites at positions 223, 228, 230, 330, and 332 of the Fc fragment were mutated based on the wild-type mouse Fc fragment, and the amino acid sequence after mutation is shown in SEQ ID NO: 3.

[0091] 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 shown in SEQ ID NO:2 or SEQ ID NO:3 was synthesized and then used The baculovirus system was subcloned into the pFastBacHTa-gp64 vector digested with Sal I (New England Biolabs, R3138S) and Hind III (New England Biolabs, R0104S) for insect cell expression. The recombinant plasmid was transformed into DH10Bac chemically competent E. coli 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 picked and recombinant bacmid DNA was isolated according to standard protocols. Positive bacmids were transiently transfected into 2 ml insect cells Sf9 with transfection reagent, and the cells were incubated in ESF 921 medium for a period of time. The cells and supernatant were collected, and the proteins were purified from the supernatant by Protein A and named Seq2-Sf9 and Seq3-Sf9, respectively.

[0092] Seq2-Sf9 and Seq3-Sf9 were identified using Coomassie Brilliant Blue stained SDS-PAGE gel, with BSA as the control protein. The identification results are as follows: Figure 1a-1b As shown, the arrows indicate Seq2-Sf9 and Seq3-Sf9, and it can be determined that the purified protein has a molecular weight of about 26kDa. The purified Seq2-Sf9 and Seq3-Sf9 proteins were subjected to multi-concentration affinity detection using biolayer interferometry (BLI), with a concentration gradient of 5000nM, 2500nM, 1250nM, 625nM, and 312.5nM to determine the affinity of Seq2-Sf9 and Seq3-Sf9 proteins to FcγRIIA protein. The test results are shown in Figure 1. Figure 2a-2b As shown, the curves shown from bottom to top indicate that the concentrations of Seq2-Sf9 and Seq3-Sf9 proteins decrease successively. 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.

[0093] 1.2 Acquisition of Fc variants expressed by 293 cells and detection of their binding ability to DC cells

[0094] The synthesized DNA sequences corresponding to SEQ ID NO:2 and SEQ ID NO:3 were cloned into a eukaryotic expression vector pcDNA3.4 with a secretion signal peptide, with restriction sites EcoRI (New England Biolabs, R0101V) and HindIII (New England Biolabs, R0104S), and electroporated into Escherichia coli trans5α. After ampicillin selection, the correct recombinant plasmid was obtained by sequencing the single clone; then, the host bacteria containing the recombinant plasmid were expanded and cultured, and an endotoxin removal kit was used to obtain a sterile endotoxin-free recombinant plasmid; the sterile endotoxin-free recombinant plasmid was mixed with a 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 Protein A resin was used to separate and purify to obtain proteins Seq2-293 and Seq3-293.

[0095] Seq2-293 and Seq3-293 can enhance the binding with FcR, which is mainly expressed on the surface of various mononuclear cells, including dendritic cells (DC). Since the above Fc variants are derived from the mouse Fc segment, the mouse dendritic cell line DC2.4 was selected as the research object, which expresses relatively stable FcR and can bind to the mouse Fc. Under natural conditions, Fc has a weak affinity with its receptor FcR. The same concentration of Seq2-293 and Seq3-293 were incubated with DC2.4 cells at 4°C for 1 hour, and 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 color development. Finally, the visible light OD450-570 was detected, indicating the Fc / FcR bound on the surface of DC2.4 cells.

[0096] Test results such as Figure 3 As shown, it can be seen that the affinity of Seq3-293 to DC2.4 is significantly higher than that of Seq2-293 at the same concentration, and is proportional to the working concentration; that is, the five-mutated Fc variant can enhance the binding ability to DC and has considerable antigen presentation potential.

[0097] Example 2 Preparation and biological activity evaluation of Seq4-Sf9

[0098] 2.1 Seq4-Sf9 protein preparation

[0099] The triple mutant mouse Fc of the amino acid sequence shown in SEQ ID NO:2 was fused and expressed with hepatitis B virus antigen (including HBV S1 / S2 / Core) through a connecting peptide, and a hydrophilic peptide was connected at the C-terminus to obtain a fusion protein of 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 triple mutant mouse Fc of the amino acid sequence shown in SEQ ID NO:2.

[0100] The synthesized gene encoding the amino acid sequence shown in SEQ ID NO:4 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, the cell pellets were collected, and ultrasonic lysed. The fusion protein was purified from the infected Sf9 cells using a Ni affinity column and named Seq4-Sf9. The biochemical assay was performed using Coomassie Brilliant Blue stained SDS-PAGE gel and WB, and the test results are shown in the figure. Figure 4a-4b As shown, the apparent molecular weight of the monomer of the fusion protein is about 75 KD.

[0101] 2.2 Glycosylation identification of Seq4-Sf9

[0102] The fusion protein Seq4-Sf9 obtained by the Sf9 insect cell expression system was digested by trypsin or chymotrypsin, and then the peptide samples after enzymatic digestion were analyzed by liquid mass spectrometry, and the original data of liquid mass spectrometry were analyzed by software. The N-glycosylated peptides were found by comparing the undeglycosylated peptide map and the deglycosylated peptide map (glycosidase treatment), and the asparagine at position 484 (position 297 of the Fc fragment) was further confirmed by primary molecular weight and secondary mass spectrometry. The results showed that the glycosylation modification of the fusion protein expressed by the Sf9 insect system was mainly high mannose type, accompanied by a small amount of oligomannose type and fucose type. The specific modification analysis results are shown in Table 1-2.

[0103] Table 1 Glycosylation modification analysis

[0104]

[0105] Table 2 Analysis of sugar ratio

[0106]

[0107] Note: Man represents mannose, and the number connected to it represents the number of mannose; \ represents non-glycosylation modification; Deamidation refers to deamidation modification. In the deglycosylated sample, the N-glycosylation site N will undergo deamidation to become D (asparagine).

[0108] 2.3 Detection of binding potency of Seq4-Sf9 to DC2.4 FcR

[0109] Fc variants can enhance binding to Fc receptors (FcR), which are mainly expressed on the surface of various mononuclear cells, including dendritic cells (DC). DC2.4 is a mouse dendritic cell line that stably expresses FcR and can be used as a test object for the binding efficacy of different Fc and Fc variants. To verify the binding efficacy of the fusion protein to DC2.4 cells, Seq4-Sf9 was incubated with DC2.4 cells at 4°C for 1 hour, and biotin-labeled anti-mouse IgG1 (anti-Mouse IgG1 Biotin) and streptavidin-HRP (Streptavidin-HRP) were added for reaction, and the reaction was terminated after color development with TMB substrate; the absorbance value of OD450-570 indicates that the fusion protein binds to the cell surface. At the same time, natural mouse IgG1 was used as a control. Figure 5 As shown, compared with the control Mouse IgG1, Seq4-Sf9 has a stronger binding efficiency with DC2.4, suggesting that the fusion protein has a strong antigen presentation potential.

[0110] 2.4 Evaluation of biological activity of Seq4-Sf9

[0111] 2.4.1 Evaluation of the immune activation effect of the fusion protein Seq4-Sf9 using DCs induced by CD14+ monocytes in healthy human peripheral blood PBMCs

[0112] To evaluate the immune activation effect of Seq4-Sf9 fusion protein, CD14+ monocytes from PBMCs of three healthy individuals were sorted 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 have strong phagocytic function, and the mouse Fc domain contained in the fusion protein will enhance its uptake function and promote DC maturation and differentiation. Flow cytometry was used to detect changes in surface markers of DCs loaded with Seq4-Sf9 fusion protein, including adhesion molecule CD54 (ICAM-1: Intercellular adhesion molecule-1), which helps mature DCs adhere to and interact with other immune cells (such as T cells), and CD83 and co-stimulatory molecule CD86, which are involved in antigen presentation and T cell activation. The test results are as follows: Figure 6a-6cAs shown, in three different volunteers, DC cells loaded with Seq4-Sf9 fusion protein highly expressed CD54, CD83 and CD86 maturation markers on their surface, indicating that the fusion protein has a potent DC activation effect, activating the antigen presentation ability of DC loaded with the vaccine, and initiating an immune response as a starting point.

[0113] 2.4.2 Key points for evaluating the effect of Seq4-Sf9-loaded DC on T cell immune response in vitro

[0114] The main 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). The important link of DC-T activation is the proliferation of T cells. Therefore, DCs loaded with Seq4-Sf9 and CD4+T cells labeled with CFSE fluorescent dye were co-cultured. After antigen presentation, T cells can rapidly proliferate and differentiate into Th cells (helper T cells). Thus, the proliferation of CD4+T cells can be used to evaluate the immune status. The results are shown in Figure 7 As shown, Seq4-Sf9-loaded DCs can significantly promote the proliferation of T cells compared with the negative control of CD4+ T cells from the same source (Seq4-Sf9 0 μg / ml), and the effect is positively correlated with its concentration.

[0115] Example 3 Preparation and biological activity evaluation of Seq5-293, Seq5-Sf9, and Seq6-Sf9

[0116] 3.1 Preparation of Seq5-293

[0117] The triple mutant mouse Fc of the amino acid sequence shown in SEQ ID NO:2 was fused and expressed with the specific marker PAP for prostate cancer through a connecting peptide to obtain a fusion protein of the amino acid sequence shown in SEQ ID NO:5, wherein positions 1-354 are the specific marker PAP for prostate cancer, and positions 361-592 are the triple mutant mouse Fc of the amino acid sequence shown in SEQ ID NO:2.

[0118] 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 connected to the expression vector pcDNA3.4 vector, and the restriction sites were 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, which was named Seq5-293. Biochemical assays were performed using Coomassie Brilliant Blue stained SDS-PAGE gels, and the assay results were shown in FIG. Figure 8 shown.

[0119] 3.2 Preparation of Seq5-Sf9

[0120] 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. The biochemical assay was performed using Coomassie Brilliant Blue stained SDS-PAGE gel. The assay results are shown in the following table. Fig. 9 shown.

[0121] 3.3 Preparation of Seq6-Sf9

[0122] The five mutant mouse Fcs with the amino acid sequence shown in SEQ ID NO:3 were fused and expressed with the specific marker PAP for prostate cancer 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 the specific marker PAP for prostate cancer, and positions 361-592 are the five mutant mouse Fcs with the amino acid sequence shown in SEQ ID NO:3.

[0123] 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. Coomassie brilliant blue stained SDS-PAGE gel was used for biochemical assay. The assay results were as follows: Fig.10 shown.

[0124] 3.4 Detection of the effect of Seq6-Sf9 on DC activation

[0125] The human wild-type Fc segment and the prostate cancer-specific marker PAP were expressed in mammalian 293 cells, and the Seq7-293 fusion protein was obtained in the same manner as in 3.1. The amino acid sequence of the Seq7-293 fusion protein was 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.

[0126] CD14+ monocytes from PBMC of two healthy individuals were sorted 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 have strong phagocytic function. They are loaded with Seq6-Sf9 and Seq7-293 fusion proteins, respectively. DCs engulf the fusion proteins and present antigens, thereby activating DCs and expressing activation markers (including CD54 and CD83, etc.). Flow cytometry was used to detect changes in surface markers of vaccine-loaded DCs. The test results are shown in Figure 2. Figure 11a-11b As shown in the figure, at the same concentration, Seq6-Sf9 can significantly activate DCs compared to Seq7-293, and CD54 and CD83 are highly expressed on the cell surface. The above results prove that the mouse Fc variant and non-mammalian glycoforms in the fusion protein can significantly enhance DC phagocytosis and activation, and express activation phenotypes such as CD54 and CD83.

[0127] 3.5 Detection of the effect of Seq5-Sf9 on DC activation

[0128] 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

[0129] To evaluate the immune activation effect of Seq5-Sf9, CD14+ monocytes from PBMCs of three healthy individuals were sorted 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 have a strong phagocytic function, and the mouse Fc domain contained in the fusion protein will enhance its uptake function and promote 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 helps mature DCs adhere to and interact with other immune cells (such as T cells), and CD83, which is involved in antigen presentation and T cell activation. The test results are as follows: Figure 12a-12b As shown, 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, and initiating an immune response as a starting point.

[0130] In addition, the immune activation effects of Seq5-Sf9, Seq5-293 and the positive control drug Provenge were compared. Figures 13a-14b As shown, it can be seen that Fc variants and non-mammalian glycosylation enhance the DC activation effect, and Seq5-Sf9 has stronger DC activation function.

[0131] 3.5.2 Key points for evaluating the effect of Seq5-Sf9-loaded DC on T cell immune response in vitro

[0132] The main function of CD4+T cell proliferation and mature DC 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). The important link of DC-T activation is the proliferation of T cells. Therefore, Seq5-Sf9-loaded DC 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). Therefore, the proliferation of CD4+T cells can be used to evaluate the immune status. Figure 15a-15b As shown, DC cells from different healthy people loaded with Seq5-Sf9 can significantly promote the proliferation of T cells, and the effect is positively correlated with its concentration; Provenge has the same effect under the same experimental conditions.

[0133] 3.5.3 In vitro evaluation of Seq5-Sf9 loaded DC antigen presentation activated cytotoxic T cells

[0134] The antigen peptide / MHC class I molecule complex presented on the surface of DC membrane 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 is to secrete interferon γ (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. ELISPOT was used to detect the secretion of IFNγ at the single cell level. The test results are shown in the figure. Fig.16 As shown, it can be seen that the IFNγ in the Seq5-Sf9 loading group was significantly more than the volunteers' own CD8+T cells (negative control), that is, Seq5-Sf9 can effectively promote the activation of CD8+T cells.

[0135] 3.5.4 Human prostate cancer cell lines expressing PAP antigen as target cells to evaluate the killing effect of CTL activated by Seq5-Sf9

[0136] The treatment of prostate cancer has different therapeutic effects depending on whether it is androgen sensitive or not. 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 Fig.17 As shown, LNCap expressed more PAP protein than LNCap. They were used as target cells and co-cultured with effector cells (CTL activated by DC loaded with Seq5-Sf9); the absolute count of dead cells was calculated by CFSE and Counting Beads. Figure 18a-18b As shown, CTL activated by DC loaded with Seq5-Sf9 can directly kill target cells, and the effect is proportional to the concentration of Seq5-Sf9; in addition, the number of dead cells in PAP cells expressing higher levels of LNCap is much higher than that in PC3, which verifies its targeted killing effect and the effect is higher than that of Provenge. 3.6 Evaluation of the immunogenicity of Seq5-Sf9

[0137] To evaluate the immune response effect of Seq5-Sf9 in vivo, 6-8 week old Sprague Dawley (SD) male rats were immunized with Seq5-Sf9. Seq5-Sf9 (placebo / rat, 2ug / rat, 10ug / rat, 50ug / rat) was injected subcutaneously every two weeks for a total of 3 injections. Serum was collected from the animals immunized with Seq5-Sf9 (Day 14, Day 28, Day 35 (one week after the third injection)) and the titer of Seq5-Sf9 specific antibodies was detected. Figures 19a-19d As shown, compared with the Placebo group, a low dose of 2ug was enough to produce vaccine-specific serum antibodies, and the antibody titer reached the highest level at the third injection and one week after the third injection.

[0138] In addition, the spleen of rats on Day 35 was taken and re-stimulated with Seq5-Sf9 in vitro to activate and induce effector T cell activation, and ELISPOT was used to detect the secretion of IFNγ by single spleen cells. Figure 20a-20b As shown, spleen immune cells can rapidly differentiate into cytotoxic T lymphocytes and secrete IFNγ under the secondary stimulation of Seq5-Sf9.

[0139] At the same time, the prostate tissue was observed, and the results were as follows: Fig.21 As shown, inflammatory cell infiltration was also observed in the prostate tissues of rats in the Seq5-Sf9 group.

[0140] 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.

[0141] 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

[0142] The Fc segment of Seq6-Sf9 is different from that of Seq5-Sf9. To evaluate its immune activation effect, CD14+ monocytes sorted from PBMCs of two healthy subjects were induced to differentiate and loaded with Seq6-Sf9. Flow cytometry was used to detect changes in surface markers of antigen-loaded DCs, such as Figure 22a-22b As shown, Seq6-Sf9 can increase the expression of CD54 and CD83 on the surface of DC cells, promote DC activation, and the expression level of markers is higher than that of Seq5-Sf9.

[0143] 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 it. 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 replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate 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. The murine Fc variant according to any one of claims 1 to 9.

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