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

Abstract

The present application provides glycosyl-modified fusion proteins, nucleic acid molecules, expression vectors, host cells, and uses thereof. In a first aspect, the present application provides a glycosyl-modified fusion protein comprising a mouse-derived Fc variant and a polypeptide antigen, wherein the mouse-derived Fc variant is obtained by amino acid mutation of the mouse-derived Fc fragment and non-mammalian glycosylation modification, the mouse-derived 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, and the non-mammalian glycosylation modification does not comprise a sialic acid modification, the positions are numbered according to the EU numbering system, and the mouse-derived Fc variant improves binding of the mouse-derived Fc fragment to DC cells and DC activation, including proliferation and activation of specific T cells.

Description

[Technical Field]

[0001] The present application relates to glycosyl-modified fusion proteins, nucleic acid molecules, expression vectors, host cells and uses in the field of biopharmaceutical technology. [Background technology]

[0002] FcRs are cell surface proteins that specifically bind to the carboxyl-terminal functional domain, the Fc fragment, of immunoglobulin (Ig) heavy chains and are crucial in antibody-dependent immune responses. Different cell types can express different types of FcRs, which bind to different structural forms of Ig and subsequently induce different types of immune responses. Immunoglobulin G (IgG) plays a very powerful role in the human body and is the major antibacterial, antiviral, and antitoxin antibody in serum. FcRs that bind to IgG are also called FcγRs. Based on the strength of affinity between FcγR and Fc fragments, FcγR is classified into three subfamilies: FcγRI, FcγRII, and FcγRIII (ranking of affinity to IgG: FcγRI > FcγRIII > FcγRII). FcγRII is further classified into three subtypes: FcγRIIa (CD32a), FcγRIIb (CD32b), and FcγRIIc (CD32c). FcγRIII is further classified into two subtypes: FcγRIIIa (CD16a) and FcγRIIIb (CD16b).

[0003] Human FcγRIIa (hFcγRIIA) is an activating Fc receptor. Upon binding to antibody Fc fragments, FcγRIIa presents activation signals and mediates Fc fragment effector function, including cell lysis, internalization, degranulation, and cytokine production. At the same time, FcγRIIa regulates dendritic cell maturation and antigen presentation, mediates T cell memory effectors, and is involved in stimulating protective CD8+ T cell responses, thereby promoting long-term protection against viral infections and maintaining long-term immune defense while eliminating target cells.

[0004] To gain a deeper understanding of the binding between the Fc fragment and FcγRIIa, many researchers have improved the structure of the Fc fragment. One researcher has shown that a mutation (G236A) in the Fc fragment selectively activates FcγRIIa (they share 96% sequence identity) while suppressing FcγRIIb, leading to the ADCP mediated by monocyte-derived macrophages. (antibody-dependent cellular phagocytosis) The addition of G236A to the S239D / I332E mutation in the Fc fragment increases FcγRIIa binding by 70-fold, increases the FcγRIIa / FcγRIIb binding ratio (activation / inhibition ratio) by 13-fold, and can enhance phagocytosis of antibody-coated target cells by macrophages (Richards JO, KS, Lazar GA, et al., O optimization of antibody binding to FcγRIIa enhances macrophage phagocytosis of tumor cells. M Cholecular cancer therapeutics, 2008. 7(8): p. 2517-2527). Also, Fc mutations have been shown to inhibit CD8 + and CD4 + Some researchers have found that Fc-optimized antibodies induce the activation of T cell responses (Bournazos, S. et al. Fc-optimized antibodies elicit CD8 immunity to viral respiratory infection, Nature, Vol 588 17 December 2020). Therefore, improving the Fc fragment may affect the effector function of the Fc fragment and change the biological activity of the antibody.

[0005] Insect glycosylation strategies differ from those of higher eukaryotes, and the glycans of commonly expressed recombinant N-glycoproteins are unsialylated, simple high / low mannose glycans, rather than the sialylated complex glycans produced by mammals at the same glycosylation sites. This is likely due to the fact that these cells have high N-acetylglucosaminidase activity, low glycosyltransferase activity, and a reduced source of sugar-nucleosides. The effector functions elicited by the fragment crystallizable (Fc) domain 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 inhibits binding to FcγRIIIa on natural killer (NK) cells and inhibits ADCC. (antibody-dependent cell-mediated cytotoxicity) The glycans without terminal sialic acid residues expressed by SF9 cells can also bind to the mannose receptor (CD206), which may enhance the ability of dendritic cells to take up and process glycoproteins, thereby enhancing immune responses (Scallon BJ, Tam SH, McCarthy SG, et al. Higher levels of sialylated Fc glycans in immunoglobulin G molecules can adversely impact functional Y.M. Olecular immunology, 2007, 44(7): 1524-1534.).

[0006] In addition to large amounts of human immunoglobulins, there have been successful examples of the use of non-humanized antibodies and antigens as therapeutic vaccines, such as IgG1 from CA-125+ mice, oregovomab (M. Brewer, R. Angioli, G. Scambia, et al., Front-Line chemo-immunotherapy with carboplatin-paclitaxel using oregovomab indirect immunization in advanced ovarian cancer: A randomized phase II study,Gynecologic Oncology, https: / / doi.org / 10.1016 / j.ygyno.2019.12.024). This example shows that mouse IgG can enhance the immune response to antigens carried by the mouse. One researcher used the Fc fragment of mouse IgG1 to bind to HBV. (Hepatitis B virus) It has been directly conjugated to various antigens, including HBV-mFc, and has produced favorable 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).

[0007] Although there are many references to the modification of human immunoglobulin Fc fragments, there are relatively few references to the modification of murine immunoglobulin Fc fragments. We believe that further research is needed into the modification of murine immunoglobulin Fc fragments to determine whether such modifications can enhance presentation. Summary of the Invention [Problem to be solved by the invention]

[0008] The present application provides a glycosylated fusion protein comprising a mouse-derived Fc variant and a polypeptide antigen, wherein the mouse-derived Fc variant is obtained by improving a mouse-derived IgG1 Fc fragment, and contributes to improving binding of the mouse-derived Fc fragment to DC cells and DC activation, including proliferation and activation of specific T cells.

[0009] The present application further provides a nucleic acid molecule encoding the above-mentioned fusion protein, as well as an expression vector and a host cell containing the nucleic acid molecule.

[0010] The present application further provides a use of the above fusion protein in the treatment of one or more of tumors, viral diseases, autoimmune diseases, and inflammatory diseases. [Means for solving the problem]

[0011] In a first aspect, the present application provides a glycosyl-modified fusion protein comprising a murine-derived Fc variant and a polypeptide antigen, The binding of antigens to DC cells and the activation of DC cells include the proliferation and activation of specific T cells. The mouse-derived Fc variant was obtained by performing amino acid mutations and non-mammalian glycosylation modifications on the mouse-derived Fc fragment, The murine-derived 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 modifications do not include sialic acid modifications; Amino acid positions are numbered according to the EU numbering system.

[0012] In the solution provided by the present application, the mouse-derived Fc variant was obtained by performing amino acid mutation and non-mammalian glycosylation modification on the wild-type mouse-derived Fc fragment shown in SEQ ID NO: 1.

[0013] Furthermore, the wild-type mouse-derived Fc fragment shown in SEQ ID NO: 1 is an Fc fragment of mouse-derived IgG1, and any Fc fragment of mouse-derived IgG other than the Fc fragment of IgG1 is applicable to the present application. For example, an Fc variant obtained by subjecting the Fc fragment of mouse-derived IgG2 to amino acid mutation and non-mammalian glycosylation modification has a similar effect of improving binding to DC cells and DC activation.

[0014] In one specific embodiment, the amino acid sequence of the murine-derived Fc variant is shown in SEQ ID NO:2 or SEQ ID NO:3.

[0015] The amino acid sequence shown in SEQ ID NO: 2 contains 232 amino acid residues, and the mutation positions are listed according to the EU numbering system, with positions 223, Alanine at positions 228 and 230 are, in order, the underlined positions in the sequence below. VDKKIVP A DCGC A PCIC A VPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTK GRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGL.

[0016] Positions 223 and 228 of SEQ ID NO: 3 , and 230th place Alanine, Leucine at position 330, glutamic acid at position 332 are, in order, the underlined positions in the sequence below. VDKKIVP A DCGC A PCIC A VPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFP L P E EKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGL.

[0017] Non-mammalian glycosylation is distinguished from mammalian glycosylation by the presence or absence of sialic acid modification and the mannose content. In the technical solution provided by the present application, glycosylation is mainly N-glycosylation, and glycosyl is derived from at least one of mannose, N-acetylglucosamine, and fucose. The sugar chain structure formed by glycosyl binding is one or more selected from high-mannose type, oligomannose type, and fucose type. The high-mannose type consists of GlcNAc and mannose and contains 5 to 9 mannoses, oligomannose refers to less than 5 mannoses, and fucose type refers to a type containing fucose. In the solution provided by the present application, all of the above glycosylation sites are located at amino acid position 297 of the Fc variant. Non-mammalian glycosylation contributes to the binding of the fusion protein to DC cells and improved DC activation.

[0018] Furthermore, the non-mammal is an insect. Furthermore, the non-mammal is the armyworm. Furthermore, the fusion protein is obtained by expression in Sf9 insect cells.

[0019] In the solution provided by the present application, the fusion protein further comprises a polypeptide antigen, which specifically refers to a polypeptide-based substance capable of eliciting an immune response in vivo. The fusion expression of the antigen polypeptide and the Fc variant contributes to enhanced antigen presentation.

[0020] It should be understood that fusion proteins containing different types of polypeptide antigens can generate different immune responses when bound to DC cells. In one specific embodiment, the polypeptide antigen is a tumor antigen. Tumor antigens refer to antigenic components present on tumor cells that differ from those on normal tissue cells, and can induce anti-tumor immune responses in the body. Furthermore, tumor antigens include tumor-specific antigens, tumor-associated antigens, and tumor-mutated antigens generated by tumor-associated antigen mutations. Furthermore, the tumor antigen is prostatic acid phosphatase (PAP), a glycoprotein found in exocrine prostate secretions that can hydrolyze phosphate esters. Selecting PAP as an antigen polypeptide can effectively generate an immune response and enhance the immune effect against prostate cancer in the body. Specifically, the amino acid sequence of prostatic acid phosphatase (PAP) is shown as positions 1 to 354 of the sequence shown in SEQ ID NO: 5.

[0021] In another specific embodiment, the polypeptide antigen is a viral antigen, which refers to a substance on the surface of a virus that can induce an immune response in the body, and the viral antigen varies depending on the type of virus.

[0022] In one specific embodiment, the viral antigen comprises an HBV surface antigen, which comprises HBV S1, HBV S2, and HBV core protein. Specifically, the amino acid sequence of the HBV surface antigen is set forth as positions 36 to 394 of the sequence set forth in SEQ ID NO:4.

[0023] In the solution provided by the present application, the fusion protein mainly comprises a polypeptide antigen capable of inducing an immune response in vivo and a mouse-derived Fc variant that binds to DC cells, and the mouse-derived Fc variant may be linked to the C-terminus of the polypeptide antigen, or the polypeptide antigen and the mouse-derived Fc variant may be linked via a binding peptide. In a specific embodiment, the amino acid sequence of the binding peptide is at least one of GGGS, VRPQGGGS, and SRGGGS.

[0024] In the solution provided by the present application, the fusion protein further comprises 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.

[0025] In the solution provided by the present application, the fusion protein further comprises a hydrophilic peptide, which can be attached to the C-terminus of the mouse-derived Fc variant to increase the hydrophilicity of the fusion protein. In one specific embodiment, the amino acid sequence of the hydrophilic peptide is QSLSRSTRGS.

[0026] The above-mentioned fusion protein is a glycosylated fusion protein that contains a polypeptide antigen capable of eliciting an immune response in vivo and a mouse-derived Fc variant, and the above-mentioned fusion protein can be obtained after being expressed in insect cells using a baculovirus expression system.

[0027] In a second aspect, the present application provides a nucleic acid molecule encoding any of the fusion proteins described above.

[0028] In a third aspect, the present application provides a recombinant expression vector comprising the above-described nucleic acid molecule.

[0029] In a fourth aspect, the present application provides a host cell comprising the recombinant expression vector described above.

[0030] In a fifth aspect, the present application provides a pharmaceutical composition comprising any of the fusion proteins described above and a pharmaceutically acceptable carrier.

[0031] In the present application, a pharmaceutically acceptable carrier is non-toxic to cells or individuals at the dosage or concentration used. Typically, a physiologically acceptable carrier is a pH buffer solution. The pharmaceutical composition provided by the present application can also be an injection, and the above carriers include diluents such as water, ethanol, polyethylene glycol, and the like, and diluents such as sodium chloride, glucose, or glycerin. additives Conventional cosolvents, buffers, etc. may also be added.

[0032] In a sixth aspect, the present application provides the use of any of the fusion proteins described above in the preparation of a medicament for the treatment of one or more of tumors, viral diseases, autoimmune and inflammatory diseases.

[0033] In the solution provided by the present application, the polypeptide antigen contained in the fusion protein can generate different immune responses in the body, so as to be applicable to the treatment of different diseases.

[0034] In one particular embodiment, the tumor to be treated comprises one or more of gastric cancer, pancreatic cancer, prostate cancer, colorectal cancer, carcinoma, liver cancer, lung cancer, breast cancer, ovarian cancer, cervical cancer, oral cancer, esophageal cancer, lymphoma, nasopharyngeal cancer, bladder cancer, squamous cell carcinoma, soft tissue and bone tumor, renal cell carcinoma, glioblastoma, and leukemia. Further, the tumor to be treated is prostate cancer.

[0035] The therapeutic effect on cancer is manifested as the inhibition of growth and / or limitation of metastatic spread of tumor cells in the subject.

[0036] In one particular 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.

[0037] Autoimmune and inflammatory diseases refer to diseases formed by autoimmune disorders, such as hyperthyroidism, chronic thyroiditis, and rheumatoid arthritis.

[0038] In a seventh aspect, the present application provides a method for treating a viral disease, comprising administering to a subject the fusion protein described above.

[0039] In an eighth aspect, the present application provides a method for treating tumors, comprising administering the above-described fusion protein to a subject.

[0040] In a ninth aspect, the present application provides any of the above-mentioned murine-derived Fc variants. [Effects of the Invention]

[0041] The fusion proteins provided herein are obtained by fusing a mouse-derived Fc variant with a polypeptide antigen. The mouse-derived Fc variant contributes to binding to DC cells, improving DC activation and stimulating the human immune response. The fusion proteins formed by fusing the mouse-derived Fc variant with the polypeptide antigen can be used as vaccines, activating different immune responses in the human body depending on the polypeptide antigen, thereby achieving therapeutic effects against related diseases. [Brief explanation of the drawings]

[0042] [Figure 1a] This is the result of SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) identification of Seq2-Sf9. M1 is a protein molecular weight marker, and BSA (bovine serum albumin) is a protein standard. [Figure 1b] SDS-PAGE identification results of Seq3-Sf9, where M is a protein molecular weight marker and BSA is a protein standard. [Figure 2a] These are the results of BLI (Biolayer Interferometry) measurements of Seq2-Sf9 and FcγRIIA proteins. [Figure 2b] This shows the results of BLI measurements of Seq3-Sf9 and FcγRIIA protein. [Figure 3] This shows the results of measuring the binding affinity of Seq2-293 and Seq3-293 to DC2.4 at different concentrations. [Figure 4a] Seq4-Sf9 staining test results using 7.5% reduced SDS-PAGE / Pageblue. Lane 1 is the sample eluted from the Ni column, and lane 2 is the sample dialyzed against the storage solution. [Figure 4b] This is the result of a WB experiment for Seq4-Sf9. The secondary antibody was anti-mouse IgG (H+L). Lane 1 is the sample eluted from the Ni column, and lane 2 is the sample dialyzed against the storage solution. [Figure 5] This shows the binding efficacy of Seq4-Sf9 to the mouse dendritic cell line DC2.4, with mouse IgG1 as the control protein. [Figure 6a] This shows the results of detecting cell surface CD54 expression after loading DCs with Seq4-Sf9 fusion protein for 48 hours. Donors #2, #4, and #5 are each from three volunteers with peripheral blood mononuclear cells (PBMCs). [Figure 6b] This shows the results of detecting cell surface CD83 expression after loading DCs with Seq4-Sf9 fusion protein for 48 hours. Donors #2, #4, and #5 are each three volunteers with PBMCs. [Figure 6c] This shows the results of detecting cell surface CD86 expression after loading DCs with Seq4-Sf9 fusion protein for 48 hours. Donors #2, #4, and #5 are each three volunteers with PBMCs. [Figure 7] This shows the results of detecting proliferation of CD4+ T cells when DCs were loaded with Seq4-Sf9. Donors #2, #4, and #5 are each three PBMC volunteers. [Figure 8] SDS-PAGE gel identification of the fusion protein Seq5-293, where M1 indicates a protein molecular weight marker, R indicates the reduced protein, and NR indicates the non-reduced protein. [Figure 9] 1 shows the results of SDS-PAGE gel identification of the fusion protein Seq5-Sf9. [Figure 10] 1 shows the results of SDS-PAGE gel identification of the fusion protein Seq6-Sf9. [Figure 11a] This shows the results of detecting cell surface CD54 expression after loading Seq6-Sf9 and Seq7-293 into DCs for 48 hours. [Figure 11b] This shows the results of detecting cell surface CD83 expression after loading Seq6-Sf9 and Seq7-293 into DCs for 48 hours. [Figure 12a] This shows the results of detecting cell surface CD54 expression after loading Seq5-Sf9 onto DCs for 48 hours. [Figure 12b] This shows the results of detecting cell surface CD83 expression after loading Seq5-Sf9 into DCs for 48 hours. [Figure 13a] This shows the results of detecting cell surface CD54 expression after loading Seq5-Sf9 and Seq5-293 into DCs. [Figure 13b] This shows the results of detecting cell surface CD83 expression after loading Seq5-Sf9 and Seq5-293 into DCs. [Figure 14a] This shows the results of detecting cell surface CD54 expression after loading DCs with Seq5-Sf9 and Provenge. [Figure 14b] This shows the results of detecting cell surface CD83 expression after loading DCs with Seq5-Sf9 and Provenge. [Figure 15a] Seq5-Sf9, proliferation detection results for CD4+ T cells derived from volunteer 1 after loading DCs with Provenge. [Figure 15b] Seq5-Sf9, proliferation detection results for CD4+ T cells from volunteer 2 after loading DCs with Provenge. [Figure 16] ELISPOT detection of IFNγ secretion by CD8+ T cells activated by Seq5-Sf9-loaded DCs. [Figure 17] This shows the results of Western blot detection of PAP protein expression levels in human prostate cancer cell lines PC3 and LNCap. [Figure 18a] This shows the results of detecting the killing effect of CTLs induced when DCs are loaded with Seq5-Sf9 and Provenge on PC3 tumor cells. [Figure 18b] This shows the results of detecting the killing effect of CTLs induced when DCs were loaded with Seq5-Sf9 and Provenge on LNCap tumor cells. [Figure 19a] Serum antibody titer detection results of Seq5-Sf9 immunized SD rats after subcutaneous injection of anti-Seq5-Sf9 (dose 0 / 2 / 10 / 50 μg). Samples were collected on 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. [Figure 19b] This shows the serum antibody titer detection results on the 14th day after subcutaneous injection of Seq5-Sf9 into immunized SD rats (doses of 0 / 2 / 10 / 50 μg). [Figure 19c] This shows the serum antibody titer detection results on the 28th day after subcutaneous injection of Seq5-Sf9 into immunized SD rats (doses of 0 / 2 / 10 / 50 μg). [Figure 19d] This shows the serum antibody titer detection results on day 35 after subcutaneous injection of Seq5-Sf9 into immunized SD rats (doses of 0 / 2 / 10 / 50 μg). [Figure 20a] This shows the results of ELISPOT detection of IFNγ secretion by splenocytes from SD rats immunized with Seq5-Sf9 on day 35. [Figure 20b] ELISPOT detection spot diagram of IFNγ secretion by splenocytes from SD rats immunized with Seq5-Sf9 on day 35. [Figure 21] This is an HE pathological staining image (magnification 20x) of prostate tissue from an SD rat immunized with Seq5-Sf9 on day 35. [Figure 22a] This shows the results of detecting cell surface CD54 expression in Seq6-Sf9 compared with Seq5-Sf9 after loading DCs for 48 hours. [Figure 22b] This shows the results of detecting cell surface CD83 expression in Seq6-Sf9 compared with Seq5-Sf9 after loading DCs for 48 hours. DETAILED DESCRIPTION OF THE INVENTION

[0043] In order to make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions will be described clearly and completely below with reference to the drawings of the embodiments of the present application. Of course, the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. Those skilled in the art can obtain all other embodiments without creative work based on the embodiments of the present application, and all other embodiments will fall within the scope of protection of the present application.

[0044] Example 1 Affinity detection of Fc variants

[0045] 1.1 Obtaining Fc variants expressed in Sf9 insect cells and detecting their affinity

[0046] To increase the stability of the mouse IgG Fc fragment, mutations were introduced into three positions, 223, 228, and 230, of the Fc fragment starting from the wild-type mouse-derived Fc fragment shown in SEQ ID NO: 1, and the mutated amino acid sequence is shown in SEQ ID NO: 2. To enhance the binding of the mouse IgG1 Fc fragment to DC cells and DC activation, mutations were introduced into five positions, 223, 228, 230, 330, and 332, of the Fc fragment starting from the wild-type mouse-derived Fc fragment, and the mutated amino acid sequence is shown in SEQ ID NO: 3.

[0047] The gp64 protein was cloned into pFastBacHTa (Thermo Fisher Scientific, Cat# 10584-027) vector digested with RsrII (New England Biolabs, R051S) to yield 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 the recombinant plasmid was then transformed into chemically competent E. coli cells, DH10Bac, using the Bac-to-Bac® baculovirus system and subcloned into pFastBacHTa-gp64 vector digested with SalI (New England Biolabs, R3138S) and HindIII (New England Biolabs, R0104S) for insect cell expression. The cells were then plated 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 selected, and recombinant bacterial DNA was isolated according to standard protocols. Positive bacilli were transiently transfected with transfection reagent into 2 ml of Sf9 insect cells. The cells were incubated in ESF 921 medium for a period of time, and the cells and supernatant were harvested. Proteins were purified from the supernatant using Protein A and designated Seq2-Sf9 and Seq3-Sf9.

[0048] Coomassie Brilliant Blue-stained SDS-PAGE gels were used to identify Seq2-Sf9 and Seq3-Sf9, with BSA as a control protein. The results are shown in Figures 1a-1b. The arrows indicate Seq2-Sf9 and Seq3-Sf9, confirming the acquisition of purified proteins with a molecular weight of approximately 26 kDa. Biolayer interferometry (BLI) was used to perform affinity detection of the purified Seq2-Sf9 and Seq3-Sf9 proteins at multiple concentrations. A concentration gradient of 5000 nM, 2500 nM, 1250 nM, 625 nM, and 312.5 nM was used to determine the affinity of Seq2-Sf9 and Seq3-Sf9 proteins with FcγRIIA protein. The results are shown in Figures 2a and 2b. The curves from bottom to top represent the sequentially decreasing concentrations of Seq2-Sf9 and Seq3-Sf9 proteins. The calculated affinity of Seq2-Sf9 and Seq3-Sf9 for FcγRIIA protein was 7.893E-07 and 4.496E-07, respectively. Lower values indicate higher affinity. The five-fold mutation in the mouse Fc domain can enhance the affinity of FcγRIIA.

[0049] 1.2 Obtaining Fc variants expressed in 293 cells and detecting their binding to DC cells

[0050] The synthesized DNA sequences corresponding to SEQ ID NO:2 and SEQ ID NO:3 were cloned into the eukaryotic expression vector pcDNA3.4, which contains a secretory signal peptide and is cleaved with EcoRI (New England Biolabs, R0101V) and HindIII (New England Biolabs, R0104S). The correct recombinant plasmids were then electrotransformed into E. coli trans5α, screened with ampicillin, and sequenced. The host cells containing the recombinant plasmids were then expanded and sterile, endotoxin-free recombinant plasmids were obtained using an endotoxin removal kit. The sterile, endotoxin-free recombinant plasmids were then mixed with Polyplus suspension cell transfection reagent and transfected into HEK293F cells. After expanding the cultures in serum-free medium for 5 days, the culture supernatants were collected and isolated and purified using Protein A resin to yield proteins Seq2-293 and Seq3-293.

[0051] Seq2-293 and Seq3-293 can enhance FcR binding, which is primarily expressed on the surface of various mononuclear cells, including dendritic cells (DCs). Because the above Fc variants are derived from mouse Fc fragments, the murine dendritic cell line DC2.4 was used as the study subject. It expresses a relatively stable FcR and can bind to mouse-derived Fc. Under natural conditions, Fc has weak affinity for its receptor, FcR. Equal concentrations of Seq2-293 and Seq3-293 were incubated with DC2.4 cells at 4°C for 1 hour, followed by the addition of biotin-labeled anti-mouse IgG1 (anti-mouse IgG1 biotin) and streptavidin-HRP (streptavidin-HRP), respectively. The reaction was terminated by color development using TMB substrate. Finally, visible light OD450–570, indicating Fc / FcR binding on the DC2.4 cell surface, was detected.

[0052] The detection results are shown in Figure 3. The affinity of Seq3-293 for DC2.4 was significantly higher than that of Seq2-293 at the same concentration, and was directly proportional to the concentration used. This indicates that the five-fold mutated Fc mutant has enhanced binding ability to DCs and considerable antigen-presenting ability.

[0053] Example 2 Preparation of Seq4-Sf9 and biological activity evaluation

[0054] 2.1 Preparation of Seq4-Sf9 proteins

[0055] A triple-mutated mouse Fc having the amino acid sequence shown in SEQ ID NO: 2 was fused with hepatitis B virus antigen (containing HBV S1 / S2 / core) via a binding peptide and expressed, and a hydrophilic peptide was attached to the C-terminus to obtain a fusion protein having the amino acid sequence shown in SEQ ID NO: 4. Positions 36 to 394 represent the hepatitis B virus antigen, and positions 403 to 634 represent the triple-mutated mouse Fc having the amino acid sequence shown in SEQ ID NO: 2.

[0056] The synthetic gene encoding the amino acid sequence shown in SEQ ID NO:4 was expressed in insect cells by ligating it into the pFastBacHTa-gp64 vector digested with SalI (New England Biolabs, R3138S) and HindIII (New England Biolabs, R0104S). The vector was then transformed into Escherichia coli DH10Bac and translocated to generate recombinant baculovirus. The isolated recombinant baculovirus was then used to infect Sf9 insect cells in ESF921 medium. The infected Sf9 insect cells were cultured at 27°C for 72 hours, harvested by centrifugation, and sonicated. The infected Sf9 insect cells were purified using a Ni affinity column to obtain a fusion protein, designated Seq4-Sf9. Biochemical analysis was performed using Coomassie Brilliant Blue-stained SDS-PAGE gels and WB, and the results are shown in Figures 4a-4b. The apparent molecular weight of the fusion protein alone was approximately 75 KD.

[0057] 2.2 Glycosylation Identification of Seq4-Sf9

[0058] The fusion protein Seq4-Sf9 obtained from the Sf9 insect cell expression system was enzymatically cleaved using trypsin or chymotrypsin. The enzymatically digested peptide segment samples were then analyzed using a liquid mass meter, and the raw liquid mass data was detected and analyzed using software. N-glycan-modified peptide segments were identified by comparing the profiles of undeglycosylated and deglycosylated peptides (treated with glycosidase). Furthermore, N-glycosylation of asparagine at position 484 (position 297 in the Fc fragment) was confirmed by primary molecular weight and secondary mass spectrometry. The glycosylation of the fusion protein expressed in the Sf9 insect cell system was primarily high-mannose, with small amounts of oligomannose and fucose. Specific modification analysis results are shown in Tables 1 and 2.

[0059] [Table 1]

[0060] [Table 2]

[0061] Note: Man represents mannose, the number following it represents the number of mannose units, \ represents non-glycosylation modification, and Deamidation refers to deamidation modification; in deglycosylated samples, N-glycosylation position N is deamidated to D (asparagine).

[0062] 2.3 Detection of binding efficiency between Seq4-Sf9 and DC2.4 FcR

[0063] Fc variants can enhance binding to Fc receptors (FcRs), which are primarily expressed on the surface of mononuclear cells, including dendritic cells (DCs). DC2.4 is a murine dendritic cell line that stably expresses FcRs and can be used to detect the binding efficacy of different Fc variants to DC2.4 cells. To verify the binding efficacy of fusion proteins to DC2.4 cells, Seq4-Sf9 and DC2.4 cells were incubated at 4°C for 1 hour, then incubated with biotin-labeled anti-mouse IgG1 (anti-mouse IgG1 biotin) and streptavidin-HRP (streptavidin-HRP), respectively. The reaction was terminated by color development using TMB substrate. The absorbance values at OD450–570 indicate the amount of fusion protein bound to the cell surface. Native mouse IgG1 was also used as a control. As shown in Figure 5, compared with the control mouse IgG1, Seq4-Sf9 had stronger binding efficacy to DC2.4, suggesting that the fusion protein has potent antigen-presenting ability.

[0064] 2.4 Biological activity evaluation of Seq4-Sf9

[0065] 2.4.1 Evaluating the immune stimulatory effect of the fusion protein Seq4-Sf9 using DCs induced by CD14+ monocytes from peripheral blood PBMCs of healthy volunteers

[0066] To evaluate the immune-stimulating effect of the Seq4-Sf9 fusion protein, CD14+ monocytes from three healthy donors' PBMCs were selected 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 endocytic function, and the mouse Fc structural domain contained in the fusion protein enhances this endocytic function and promotes DC maturation and differentiation. Changes in surface markers of DCs loaded with the Seq4-Sf9 fusion protein were detected by flow cytometry. These markers included the adhesion molecule CD54 (intercellular adhesion molecule-1), which contributes to the adhesion and interaction of mature DCs with other immune cells (e.g., T cells), and CD83 and the costimulatory molecule CD86, which are involved in antigen presentation and T cell activation. The detection results are shown in Figures 6a to 6c. In three different volunteers, the cell surface of DCs loaded with Seq4-Sf9 fusion protein highly expressed maturation markers CD54, CD83, and CD86, indicating that the fusion protein has a strong DC activation effect, activating the antigen-presenting ability of vaccine-loaded DCs and initiating an immune response as the first step.

[0067] 2.4.2 Key points for in vitro evaluation of the effect of Seq4-Sf9-loaded DCs on T cell immune responses

[0068] The most important function of mature DCs is antigen presentation and promotion of CD4+ T cell proliferation, and the antigen peptide / MHC (major histocompatibility)The antigen peptide / MHC-II complex or the antigen peptide / MHC-II complex is recognized by the T cell surface receptor (TCR). A key step in DC-T activation is T cell proliferation. Therefore, when Seq4-Sf9-loaded DCs were cocultured with CD4+ T cells labeled with CFSE fluorescent dye, T cells rapidly proliferated and differentiated into Th cells (helper T cells) after antigen presentation. This allowed us to assess the immune status based on CD4+ T cell proliferation. The results are shown in Figure 7. Seq4-Sf9-loaded DCs significantly promoted T cell proliferation compared to the negative control (0 μg / ml Seq4-Sf9) for CD4+ T cells from the same source, and this was positively correlated with the effective concentration.

[0069] Example 3 Preparation of Seq5-293, Seq5-Sf9, and Seq6-Sf9 and evaluation of their biological activities

[0070] 3.1 Preparation of Seq5-293

[0071] The triple mutant mouse Fc having the amino acid sequence shown in SEQ ID NO: 2 was fused with the prostate cancer-specific marker PAP via a binding peptide and expressed to obtain a fusion protein having the amino acid sequence shown in SEQ ID NO: 5. Positions 1 to 354 of the fusion protein represent the prostate cancer-specific marker PAP, and positions 361 to 592 represent the triple mutant mouse Fc having the amino acid sequence shown in SEQ ID NO: 2.

[0072] A gene encoding a fusion protein with 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. A recombinant expression plasmid containing EcoRI (New England Biolabs, R0101V) and HindIII (New England Biolabs, R0104S) enzyme cleavage sites was prepared. The recombinant expression plasmid was transiently transfected into eukaryotic cells HD293F, followed by cell culture. The cell culture medium was collected, centrifuged, and filtered. The filtered culture supernatant was applied to a Protein A affinity chromatography column to purify the fusion protein, designated Seq5-293. Biochemical analysis was performed using a Coomassie Brilliant Blue-stained SDS-PAGE gel, and the results are shown in Figure 8.

[0073] 3.2 Preparation of Seq5-Sf9

[0074] The triple mutant mouse Fc having the amino acid sequence shown in SEQ ID NO: 2 was fused with a specific marker for prostate cancer, PAP, via a binding peptide and expressed. 5 to obtain a fusion protein having the amino acid sequence shown in The corresponding coding sequence was ligated into pFastBacHTa-gp64 vector digested with Sal I (New England Biolabs, R3138S) and Hind III (New England Biolabs, R0104S) and expressed in insect cells. The vector was then transformed into E. coli DH10Bac to generate recombinant baculovirus. The recombinant baculovirus was isolated and transfected into Sf9 insect cells. The cells were incubated in ESF921 medium at 27°C for 72 hours to express the target protein. The supernatant was collected by centrifugation, and the resulting protein, designated Seq5-Sf9, was purified with Protein A. Biochemical analysis was performed using a Coomassie Brilliant Blue-stained SDS-PAGE gel, and the results are shown in Figure 9.

[0075] 3.3 Preparation of Seq6-Sf9

[0076] The five-fold mutant mouse Fc having the amino acid sequence shown in SEQ ID NO: 3 was fused with the prostate cancer-specific marker PAP via a binding peptide and expressed to obtain a fusion protein having the amino acid sequence shown in SEQ ID NO: 6. Positions 1 to 354 represent the prostate cancer-specific marker PAP, and positions 361 to 592 represent the five-fold mutant mouse Fc having the amino acid sequence shown in SEQ ID NO: 3.

[0077] The fusion protein was expressed in Sf9 insect cells using the same method as in 3.2. The resulting fusion protein was designated Seq6-Sf9. Biochemical analysis was performed using a Coomassie Brilliant Blue-stained SDS-PAGE gel. The results are shown in Figure 10.

[0078] 3.4 Detection of DC activation effect by Seq6-Sf9

[0079] A human wild-type Fc fragment and a prostate cancer-specific marker, PAP, were expressed in mammalian 293 cells, and the Seq7-293 fusion protein was obtained using the same method as in 3.1. The amino acid sequence is shown in SEQ ID NO: 7. Using the Seq7-293 fusion protein as a control, the effects of the Fc mutants and insect glycosylated forms on DC activation were examined.

[0080] CD14+ monocytes from PBMCs of two healthy individuals were selected 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 internalizing capacity, and when loaded with Seq6-Sf9 or Seq7-293 fusion proteins, DCs internalized the fusion proteins and presented antigens, activating them and expressing activation markers (including CD54 and CD83). Changes in surface markers of vaccine-loaded DCs were detected by flow cytometry, and the results are shown in Figures 11a and 11b. Compared to Seq7-293 at the same concentration, Seq6-Sf9 significantly activated DCs, demonstrating higher expression of CD54 and CD83 on the cell surface. These results demonstrate that the mouse-derived Fc variants and non-mammalian glycosylated forms of the fusion proteins significantly enhance DC uptake and activation, and can express activation phenotypes such as CD54 and CD83.

[0081] 3.5 Detection of DC activation effect by Seq5-Sf9

[0082] 3.5.1 Evaluation of the immune stimulatory effect of the fusion protein Seq5-Sf9 using DCs induced by CD14+ monocytes from human peripheral blood PBMCs

[0083] To evaluate the immune stimulatory effects of Seq5-Sf9, CD14+ monocytes from PBMCs of three healthy individuals were selected 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 endocytic capacity, and the mouse Fc structural domain contained in the fusion protein enhances DC endocytic function and promotes DC maturation and differentiation. Flow cytometry was used to detect changes in surface markers of vaccine-loaded DCs, including the adhesion molecule CD54 (intercellular adhesion molecule-1), which contributes to the adhesion and interaction of mature DCs with other immune cells (e.g., T cells), and CD83, which is involved in antigen presentation and T cell activation. The detection results are shown in Figures 12a-12b. The high expression of CD54 and CD83 on the surface of Seq5-Sf9-loaded DCs from three different volunteers indicates that the fusion protein has a strong DC activation effect and activates the antigen-presenting ability of Seq5-Sf9-loaded DCs, which is the first step in initiating an immune response.

[0084] The immune activation effects of Seq5-Sf9, Seq5-293, and the positive control drug Provenge were compared, and the results are shown in Figures 13a-14b. The Fc variants and non-mammalian glycosylation enhanced the DC activation effect, with Seq5-Sf9 exhibiting stronger DC activation function.

[0085] 3.5.2 Key points for in vitro evaluation of the effect of Seq5-Sf9-loaded DCs on T cell immune responses

[0086] The most important function of mature DCs is antigen presentation and promotion of CD4+ T cell proliferation. The antigen peptide / MHC-I or MHC-II complexes on the surface of mature DCs are recognized by the T cell surface receptor (TCR). The key step in DC-T activation is T cell proliferation. Therefore, co-culture of Seq5-Sf9-loaded DCs with CFSE fluorescent dye-labeled CD4+ T cells rapidly proliferates and differentiates into Th cells (helper T cells) after antigen presentation, allowing for the assessment of immune status based on CD4+ T cell proliferation. As shown in Figures 15a-15b, after loading Seq5-Sf9 onto DCs derived from different healthy individuals, T cell proliferation was significantly promoted and correlated positively with the Seq5-Sf9 concentration. Provenge also had a similar effect under the same experimental conditions.

[0087] 3.5.3 In vitro evaluation of antigen presentation by Seq5-Sf9-loaded DCs and activated cytotoxic T cells

[0088] The antigen peptide / MHC-I molecule complexes presented on the DC membrane surface can directly recognize and bind to the TCR on CD8+ T cells, thereby activating CD8+ T cells and exerting biological effects. One of the main mechanisms of effect is the secretion of interferon-γ (IFNγ). Seq5-Sf9-loaded DCs were co-cultured with CD8+ T cells from the same volunteer, and then Seq5-Sf9-loaded DCs were added again for secondary stimulation, activating a large number of CD8+ T cells. IFNγ secretion was detected at the individual cell level by ELISPOT. The results are shown in Figure 16. IFNγ levels in the Seq5-Sf9-loaded group were significantly higher than those in the volunteer's own CD8+ T cells (negative control). Therefore, Seq5-Sf9 can effectively promote CD8+ T cell activation.

[0089] 3.5.4 Evaluation of the killing effect of Seq5-Sf9-activated CTLs against human prostate cancer cell lines expressing the PAP antigen

[0090] The therapeutic efficacy of prostate cancer depends on the androgen sensitivity of the prostate cancer cells. 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, with LNCap expressing higher amounts of PAP protein, as shown in Figure 17. These two cell lines were used as target cells and co-cultured with effector cells (CTLs activated by Seq5-Sf9-loaded DCs). The absolute number of dead cells was measured using CFSE and counting beads. As shown in Figures 18a and 18b, CTLs activated by Seq5-Sf9-loaded DCs could directly kill target cells, and the killing effect was directly proportional to the concentration of Seq5-Sf9. Furthermore, the number of dead cells in LNCap cells, which express higher levels of PAP, was significantly higher than in PC3 cells, demonstrating the target killing effect of CTLs activated by Seq5-Sf9-loaded DCs, which was more effective than Provenge.

[0091] 3.6 Immunogenicity assessment of Seq5-Sf9

[0092] To evaluate the immune response effects of Seq5-Sf9 in vitro, 6- to 8-week-old male Sprague Dawley (SD) rats were immunized with Seq5-Sf9. Seq5-Sf9 was administered subcutaneously to the SD rats three times every two weeks (placebo / rat, 2 μg / rat, 10 μg / rat, 50 μg / rat). Serum samples were collected from Seq5-Sf9-immunized animals on days 14, 28, and 35 (1 week after the third injection). Seq5-Sf9-specific antibody titers were detected in the collected animal sera. As shown in Figures 19a-19d, vaccine-specific serum antibodies appeared at a low dose of 2 μg compared to the placebo group, and antibody titers reached their highest levels after the third injection and 1 week after the third injection.

[0093] Furthermore, rat spleens were harvested on day 35 and re-stimulated and activated in vitro with Seq5-Sf9 to induce effector T cell activation, and IFNγ secretion by individual splenocytes was detected by ELISPOT. As shown in Figures 20a-20b, under the influence of secondary stimulation with Seq5-Sf9, splenic immune cells rapidly differentiated into cytotoxic T lymphocytes and were able to secrete IFNγ.

[0094] At the same time, prostate tissues were observed, and the results are shown in Figure 21. Infiltration of inflammatory cells was also observed in the prostate tissues of rats in the Seq5-Sf9 group.

[0095] As described above, Seq5-Sf9 can establish a complete in vivo immune response in SD rats and effectively activate T cells, i.e., Seq5-Sf9 can complete in vivo immune activation.

[0096] 3.7 Evaluation of the immune stimulatory effect of the fusion protein Seq6-Sf9 using DCs induced by CD14+ monocytes from human peripheral blood PBMCs

[0097] The Fc fragment of Seq6-Sf9 differs from that of Seq5-Sf9. To evaluate its immune activation effects, CD14+ monocytes selected from PBMCs derived from two healthy volunteers were induced and differentiated and loaded with Seq6-Sf9. Changes in surface markers of antigen-loaded DCs were detected by flow cytometry. As shown in Figures 22a and 22b, Seq6-Sf9 increased the expression of CD54 and CD83 on DC cell surfaces, promoting DC activation and demonstrating higher marker expression levels than Seq5-Sf9.

[0098] Finally, it should be noted that the above embodiments are for illustrating the technical solutions of the present application, not for limiting the same, and the present application will be described in detail with reference to the above embodiments. However, those skilled in the art may still modify the technical solutions described in the above embodiments or make equivalent substitutions for some or all of the technical features thereof, and it should be understood that such modifications or substitutions will not deviate from the essence of the corresponding technical solutions and the scope of the technical solutions of the embodiments of the present application.

[0099] This application claims priority from a Chinese patent application bearing application number 202310798756.4 and entitled "Fc variants, fusion proteins and uses thereof" filed with the Patent Office of the People's Republic of China on June 30, 2023, and application number 202310807755.1 and entitled "Chimeric fusion proteins, nucleic acid molecules, expression vectors, host cells, and uses thereof," the entire contents of which are incorporated herein by reference.

Claims

1. a glycosyl-modified fusion protein comprising a murine Fc variant and a polypeptide antigen, said fusion protein improving DC cell binding and DC activation, including specific T cell proliferation and activation; The mouse-derived Fc variant is obtained by performing amino acid mutation and non-mammalian glycosylation modification on a mouse-derived Fc fragment; the murine-derived 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 modifications do not include sialic acid modifications; A glycosyl-modified fusion protein, characterized in that the amino acid positions are numbered according to the EU numbering system.

2. The fusion protein described in claim 1, characterized in that the amino acid sequence of the mouse-derived Fc variant is as 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, glycosyl is derived from at least one of mannose, N-acetylglucosamine, and fucose.

4. The fusion protein according to claim 3, wherein in the glycosylation modification, the sugar chain structure formed by the glycosyl bond is selected from at least one of high mannose type, oligomannose type, and fucose type.

5. The fusion protein according to any one of claims 1 to 4, wherein the polypeptide antigen is a tumor antigen, and includes one of a tumor-specific antigen, a tumor-associated antigen, and a tumor-mutated antigen generated by tumor-associated antigen mutation.

6. The fusion protein according to claim 5, wherein the tumor antigen is PAP.

7. The fusion protein according to any one of claims 1 to 4, wherein 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, further comprising a hydrophilic peptide bound to the C-terminus of the mouse-derived 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 comprising the nucleic acid molecule of claim 10.

12. A host cell comprising the recombinant expression vector of claim 11.

13. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

14. Use of the fusion protein of any one of claims 1 to 4 in the preparation of a medicament for the treatment of one or more of the following diseases: tumors, viral diseases, autoimmune diseases and inflammatory diseases.

15. A method for treating a viral disease, comprising administering the fusion protein of claim 7 to a subject.

16. A method for treating tumors, comprising administering the fusion protein of claim 5 or 6 to a subject.

17. A mouse-derived Fc variant according to any one of claims 1 to 9.

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