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

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 a fusion protein modified by a glycosyl, a nucleic acid molecule, an expression vector, a host cell and uses thereof, and relates to the field of biopharmaceutical technology.

Background Art

[0002] FcR is a type of cell surface protein that can specifically bind to the functional domain Fc fragment at the carboxyl terminus of the heavy chain of immunoglobulin (Ig), and is extremely important in the process of antibody-dependent immune response. Different types of cells can express different types of FcR, and different types of FcR bind to different conformational Igs, thereby inducing different types of immune responses later. Immunoglobulin G (IgG) plays a very powerful role in the human body and is the main antibody against bacteria, viruses, and toxins in serum. The FcR that binds to IgG is also called FcγR. From the strength of the affinity between FcγR and the Fc fragment, FcγR is classified into three subfamilies: FcγRI, FcγRII, and FcγRIII (rank of affinity with 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. When FcγRIIa binds to the antibody Fc fragment, it presents an activation signal and can mediate Fc fragment effector factor functions including cell lysis, uptake, degranulation, and cytokine generation. At the same time, FcγRIIa controls the maturation and antigen presentation of dendritic cells, mediates the memory effector of T cells, is involved in the stimulation of defensive CD8+ T cell responses, and promotes long-term defense against viral infections, thereby achieving the effect of maintaining immune defense in the long term while removing target cells.

[0004] To gain a deeper understanding of the binding between the Fc fragment and FcγRIIa, many researchers have refined the structure of the Fc fragment. One researcher found that a mutation in the Fc fragment (G236A) selectively activates FcγRIIa (with 96% sequence identity between the two) based on the suppression of FcγRIIb, thereby mediated by monocyte-derived macrophages in ADCP. (antibody-dependent cellular phagocytosis) We discovered that adding G236A to the S239D / I332E mutation in the Fc fragment increased FcγRIIa binding by 70-fold, increased the FcγRIIa / FcγRIIb binding ratio (activation / inhibition ratio) by 13-fold, and enhanced the phagocytosis of antibody-coated target cells by macrophages (Richards JO, KS, Lazar GA, et al.). al., O optimization of antibody binding to FcγRIIa enhances macrophage phagocytosis of tumor Cells. Olecular Cancer Therapeutics, 2008. 7(8): p. 2517-2527). Also, Fc mutations are CD8 + and CD4 + Some researchers have discovered that it induces activation of the T cell response in T cells (Bournazos, S. et al. Fc-optimized antibodies elicit CD8 immunity to viral respiratory infection, Nature, Vol 588 17 December 2020). Therefore, improvements to the Fc fragment may affect the effector function of the Fc fragment and alter the biological activity of the antibody.

[0005] Insect glycosylation mechanisms differ from those of higher eukaryotes; the glycans of commonly expressed recombinant N-glycoproteins are simple, unsialized high / low mannose types, rather than the sialylated complex glycans produced at the same glycosylation site in mammals. This is thought to be because their cells have high N-acetylglucosaminidase activity, low glycosyltransferase activity, and limited sugar-nucleoside sources. The effector function induced by the fragment crystallizable (Fc) domain of immunoglobulin G (IgG) antibodies is altered by the presence of the terminal sialic acid (Sia) residue of asparagine-297 (Asn-297). Studies have shown that high sialylation inhibits binding to FcγRIIIa on natural killer (NK) cells and ADCC. (antibody-dependent cell-mediated cytotoxicity) It was found that this reduced the effect. Since glycans without terminal sialic acid residues expressed by SF9 cells can also bind to the mannose receptor (CD206), they may enhance the ability of dendritic cells to take up and process glycoproteins, thereby potentially 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 functionalit). y. M Olecular immunology, 2007, 44(7): 1524-1534.).

[0006] In addition to large amounts of human immunoglobulin, there have been successful cases of using non-humanized antibodies and antigens as therapeutic vaccines, for example, IgG1 from CA-125+ mice, and 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,A notable example is Gynecologic Oncology (https: / / doi.org / 10.1016 / j.ygyno.2019.12.024). This example demonstrates that mouse IgG can enhance the immune response to antigens carried by mice. One researcher used the Fc fragment of mouse IgG1 to study HBV (Hepatitis B virus) -Good results were obtained by directly binding to various antigens such as mFc (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] While there are many references describing the improvement of human immunoglobulin Fc fragments, there are relatively few descriptions of the improvement of mouse immunoglobulin Fc fragments. We believe it is necessary to conduct research on the engineering of mouse immunoglobulin Fc fragments to determine whether their presentation effect can be enhanced through engineering. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] This invention provides a glycosyl-modified fusion protein comprising a mouse-derived Fc variant and a polypeptide antigen, wherein the mouse-derived Fc variant was obtained by modifying a mouse-derived IgG1 Fc fragment, and contributes to improved binding between the mouse-derived Fc fragment and DC cells, as well as DC activation, including the proliferation and activation of specific T cells.

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

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

[0011] In a first aspect, the present invention provides a glycosyl-modified fusion protein comprising a mouse-derived Fc mutant and a polypeptide antigen, and the fusion protein The binding of quality to DC cells and the activation of DC cells include the proliferation and activation of specific T cells. Mouse-derived Fc mutants were obtained by performing amino acid mutations and non-mammalian glycosylation modifications on mouse-derived Fc fragments. The mouse-derived Fc mutant contains at least one of the following: alanine at position 223, alanine at position 228, alanine at position 230, leucine at position 330, and glutamic acid at position 332. Non-mammalian glycosylation modifications do not include sialic acid modifications. The amino acid positions were numbered according to the EU numbering system.

[0012] In the solution provided by this application, the mouse-derived Fc mutant is obtained by performing amino acid mutations and non-mammalian glycosylation modifications on the wild-type mouse-derived Fc fragment shown in Sequence ID No. 1.

[0013] Furthermore, the wild-type mouse-derived Fc fragment shown in Sequence ID No. 1 is an Fc fragment of mouse-derived IgG1, and any other mouse-derived IgG Fc fragment, excluding those derived from IgG1, is applicable to this invention. For example, Fc mutants obtained by performing amino acid mutations and non-mammalian glycosylation modifications on a mouse-derived IgG2 Fc fragment have a similar effect of improving binding to DC cells and DC activation.

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

[0015] The amino acid sequence shown in Sequence ID No. 2 contains 232 amino acid residues, and the mutation site is described according to the EU numbering system, at position 223. Alanine ranked 228th and 230th. These correspond to the underlined positions in the following sequence, in order. VDKKIVP A DCGC A PCIC A VPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTK GRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGL.

[0016] Sequence ID 3, ranks 223 and 228 , and 230th place alanine, Leucine is ranked 330th, and glutamic acid is ranked 332nd. These correspond to the underlined positions in the following sequence, in order. 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 the glycan is derived from at least one of mannose, N-acetylglucosamine, and fucose. The sugar chain structure formed by the binding of glycans 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. The oligomannose refers to less than 5 mannoses, and the fucose type refers to the type containing fucose. In the solution provided by the present application, all of the above glycosylation positions are located at the amino acid at position 297 of the Fc variant. Non-mammalian glycosylation contributes to the binding of the fusion protein to DC cells and the improvement of DC activation.

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

[0019] In the solution provided by the present application, the fusion protein further contains a polypeptide antigen, and the polypeptide antigen specifically refers to a polypeptide substance capable of inducing an immune response in a living body. The fusion expression of the antigen polypeptide and the Fc variant contributes to the enhancement of antigen presentation.

[0020] It should be understood that a fusion protein containing different types of polypeptide antigens can generate different immune responses when binding to DC cells. In one specific embodiment, the polypeptide antigen is a tumor antigen, and a tumor antigen refers to an antigen component that exists on tumor cells and is different from normal tissue cells, and the tumor antigen can induce an anti-tumor immune response in the living body. Furthermore, the tumor antigen includes one of tumor-specific antigens, tumor-associated antigens, and tumor mutant antigens generated by tumor-associated antigen mutations. Furthermore, the tumor antigen is prostate acid phosphatase (PAP), and PAP is a glycoprotein that can hydrolyze phosphate esters in prostatic exocrine secretions. When it is selected as an antigen polypeptide, it can effectively generate an immune response and enhance the immune effect of the living body against prostate cancer. Specifically, the amino acid sequence of prostate 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, and a viral antigen refers to a substance that can induce an immune response of the living body on the viral surface, and the viral antigen varies depending on the virus type.

[0022] In one 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 as positions 36 to 394 of the sequence shown in SEQ ID NO: 4.

[0023] In the solution provided by the present application, the above-mentioned fusion protein mainly includes a polypeptide antigen capable of inducing an immune response of the living body and a mouse-derived Fc mutant that binds to DC cells. The mouse-derived Fc mutant may bind to the C-terminus of the polypeptide antigen, or the polypeptide antigen and the mouse-derived Fc mutant may be bound by a linking peptide. In one specific embodiment, the amino acid sequence of the linking peptide is at least one of GGGS, VRPQGGGS, and SRGGGS.

[0024] In the solution provided by this application, the fusion protein further comprises a protein tag to facilitate the expression, detection, and purification of the target gene. Furthermore, the protein tag may be a His tag, which is bound to the N-terminus of the polypeptide antigen to enhance the purification efficiency of the fusion protein.

[0025] In the solution provided by this application, the fusion protein further comprises a hydrophilic peptide, which may be bound to the C-terminus of a mouse-derived Fc mutant to enhance the hydrophilicity of the fusion protein. In one particular embodiment, the amino acid sequence of the hydrophilic peptide is QSLSRSTRGS.

[0026] The fusion protein described above is a glycosylated fusion protein containing a polypeptide antigen capable of eliciting an immune response in living organisms and a mouse-derived Fc variant. This 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 above-described fusion proteins.

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

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

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

[0031] In this application, a pharmaceutically acceptable carrier is non-toxic to cells or organisms at the dose and concentration used. Typically, a physiologically acceptable carrier is a pH buffered solution. The pharmaceutical composition provided herein can be further made into an injectable, and the carrier may contain a diluent such as water, ethanol, polyethylene glycol, and sodium chloride, glucose, or glycerin. additives These may be included. Conventional solvents, buffers, etc., may also be added.

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

[0033] In the solution provided by this application, polypeptide antigens contained in the fusion protein can generate different biological immune responses, making them applicable to the treatment of different diseases.

[0034] In one particular embodiment, the tumors to be treated include one or more of the following: 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, bone and soft tissue tumors, renal cell carcinoma, glioblastoma, and leukemia. Furthermore, the tumor to be treated is prostate cancer.

[0035] The therapeutic effect of cancer treatment manifests itself as the suppression of tumor cell growth and / or the limitation of their metastasis and spread in the subject.

[0036] In one particular embodiment, the viral disease to be treated refers to a disease caused by a virus. Furthermore, the viral disease to be treated includes diseases 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 invention provides a method for treating a viral disease, comprising administering the above-mentioned fusion protein to a subject.

[0039] In an eighth aspect, the present invention provides a method for treating a tumor by administering the above-mentioned fusion protein to a subject.

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

[0041] The fusion protein provided in this application is obtained by fusing a mouse-derived Fc mutant with a polypeptide antigen. The mouse-derived Fc mutant contributes to improved binding to DC cells, enhanced DC activation, and stimulation of the human immune response. The fusion protein formed by fusing with the polypeptide antigen can be used as a vaccine, and by activating different polypeptide antigens, it is possible to activate different immune responses in the human body and achieve therapeutic effects for related diseases. [Brief explanation of the drawing]

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

[0043] To clarify the purpose, technical solutions, and advantages of the embodiments of this application, the technical solutions will be described clearly and completely below with reference to the drawings relating to the embodiments of this application. Naturally, the embodiments described are only a part of the embodiments of this application, not all of them. A person skilled in the art will know that all other embodiments obtained without creative work based on the embodiments of this application are all within the scope of protection of this application.

[0044] Example 1: Affinity detection of Fc variants

[0045] 1.1 Acquisition and affinity detection of Fc mutants expressed in Sf9 insect cells

[0046] To enhance the stability of the mouse IgG Fc fragment, mutations were introduced at three positions (223, 228, and 230) of the Fc fragment derived from wild-type mice, as shown in Sequence ID No. 1. The mutated amino acid sequences are shown in Sequence ID No. 2. To enhance the binding of the mouse IgG1 Fc fragment to DC cells and DC activation, mutations were introduced at five positions (223, 228, 230, 330, and 332) of the Fc fragment derived from wild-type mice. The mutated amino acid sequences are shown in Sequence ID No. 3.

[0047] The gp64 protein was cloned into a pFastBacHTa (Thermo Fisher Scientific, Cat# 10584-027) vector, which was enzymatically cleaved with RsrII (New England Biolabs, R051S), to obtain pFastBacHTa-gp64. DNA sequences corresponding to the amino acids shown in SEQ ID NO: 2 or 3 were synthesized, and the recombinant plasmid was then transformed into chemically competent E. coli cells, DH10Bac, using the Bac-to-Bac® baculovirus system. For insect cell expression, the plasmid was subcloned into pFastBacHTa-gp64 vectors, which were enzymatically cleaved with Sal I (New England Biolabs, R3138S) and Hind III (New England Biolabs, R0104S). Subsequently, the cells were cultured on fresh LB agar plates containing 50 μg / ml kanamycin, 7 μg / ml gentamicin, 10 μg / ml tetracycline, 100 μg / ml Bluo-gal, and 40 μg / ml IPTG. After overnight incubation, white colonies were selected, and recombinant bacillus DNA was isolated according to the standard protocol. Positive bacilluses were transiently transfected into 2 ml of insect cells Sf9 with transfection reagent. The cells were incubated in ESF 921 medium for a period of time, and the cells and supernatant were collected. Protein was purified from the supernatant with Protein A and named Seq2-Sf9 and Seq3-Sf9.

[0048] Seq2-Sf9 and Seq3-Sf9 were identified using Coomassie brilliant blue stained SDS-PAGE gels, with BSA used as a control protein. The identification results are shown in Figures 1a and 1b, respectively. The positions indicated by the arrows are Seq2-Sf9 and Seq3-Sf9, allowing for the determination of how to obtain purified proteins with a molecular weight of approximately 26 kDa. Multi-concentration affinity detection of purified Seq2-Sf9 and Seq3-Sf9 proteins was performed using biolayer interferometry (BLI). Concentration gradients were set to 5000 nM, 2500 nM, 1250 nM, 625 nM, and 312.5 nM to determine the affinity between Seq2-Sf9 and Seq3-Sf9 proteins and FcγRIIA protein. The detection results are shown in Figures 2a and 2b, respectively. The curves shown from bottom to top indicate a sequential decrease in the concentrations of Seq2-Sf9 and Seq3-Sf9 proteins. Calculations show that the affinities between Seq2-Sf9 and Seq3-Sf9 and the FcγRIIA protein are 7.893E-07 and 4.496E-07, respectively. A smaller number indicates higher affinity, suggesting that the quintuple mutation in the mouse Fc structural domain can increase the affinity of FcγRIIA.

[0049] 1.2 Acquisition of Fc mutants expressed in 293 cells and detection of their binding affinity to DC cells

[0050] The DNA sequences corresponding to the synthesized Sequence ID No. 2 and Sequence ID No. 3 were cloned into the eukaryotic cell expression vector pcDNA3.4 containing a secretory signal peptide. The enzymatic cleavage sites were EcoRI (New England Biolabs, R0101V) and HindIII (New England Biolabs, R0104S). E. coli trans5α was electrotransformed, screened with ampicillin, and monoclonal sequencing was performed to obtain the correct recombinant plasmid. Subsequently, the host bacteria containing the recombinant plasmid were cultured in large quantities, and a sterile endotoxin-free recombinant plasmid was obtained using an endotoxin removal kit. The sterile endotoxin-free recombinant plasmid was mixed with polyplus suspension cell transfection reagent, HEK293F cells were transfected, and the culture was expanded in serum-free medium for 5 days. After collecting the supernatant of the medium, it was isolated and purified using Protein A resin to obtain proteins Seq2-293 and Seq3-293.

[0051] Seq2-293 and Seq3-293 can enhance binding to FcR, which is mainly expressed on the surface of mononuclear cells, including dendritic cells (DCs). Since the above Fc mutants are derived from mouse Fc fragments, the mouse dendritic cell line DC2.4 was used as the study target. It expresses relatively stable FcR and can bind to mouse-derived Fc. Under natural conditions, Fc has a weak affinity for its receptor FcR. Seq2-293 and Seq3-293 at the same concentration were incubated with DC2.4 cells at 4°C for 1 hour. Biotin-labeled anti-mouse IgG1 and streptavidin-HRP were added, respectively, and the reaction was terminated when color development occurred due to the action of a TMB substrate. Finally, visible light OD450-570, indicating Fc / FcR binding to the surface of DC2.4 cells, was detected.

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

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

[0054] 2.1 Preparation of Seq4-Sf9 protein

[0055] A mouse Fc molecule with a triple mutation in the amino acid sequence shown in Sequence ID No. 2 was fusion-expressed with hepatitis B virus antigen (including HBV S1 / S2 / core) via a binding peptide. A hydrophilic peptide was then attached to the C-terminus to obtain a fusion protein with the amino acid sequence shown in Sequence ID No. 4. Positions 36-394 are the hepatitis B virus antigen, and positions 403-634 are the mouse Fc molecule with the triple mutation in the amino acid sequence shown in Sequence ID No. 2.

[0056] A synthetic gene encoding the amino acid sequence shown in Sequence ID No. 4 was enzymatically cleaved with Sal I (New England Biolabs, R3138S) and Hind III (New England Biolabs, R0104S) and bound to a pFastBacHTa-gp64 vector for expression in insect cells. Recombinant baculovirus was produced by transforming and translocating E. coli DH10Bac. The recombinant baculovirus was isolated. The isolated recombinant baculovirus was used to infect Sf9 insect cells in ESF921 medium. The infected Sf9 insect cells were cultured at 27°C for 72 hours, collected by centrifugation, and degraded by sonication. The infected Sf9 insect cells were purified using a Ni affinity column to obtain a fusion protein, which was named Seq4-Sf9. Biochemical measurements were performed using Coomassie brilliant blue stained SDS-PAGE gel and WB, and the detection results are shown in Figures 4a-4b. The apparent molecular weight of the fusion protein alone was approximately 75 kD.

[0057] 2.2 Identification of glycosylation of Seq4-Sf9

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

[0059] [Table 1]

[0060] [Table 2]

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

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

[0063] Fc mutants can enhance binding to the Fc receptor (FcR), which is primarily expressed on the surface of mononuclear cells, including dendritic cells (DCs). DC2.4 is a mouse dendritic cell line that stably expresses FcR and can be used to detect the binding effectiveness of different Fc and Fc mutants. To verify the binding effectiveness of the fusion protein to DC2.4 cells, Seq4-Sf9 and DC2.4 cells were incubated at 4°C for 1 hour. Biotin-labeled anti-mouse IgG1 (anti-Mouse IgG1 Biotin) and streptavidin-HRP were added and reacted, respectively. The reaction was terminated when color development occurred due to the action of a TMB substrate. Absorbance values ​​from OD450 to 570 indicate the amount of fusion protein bound to the cell surface. Natural mouse IgG1 was used as a control at the same time. As shown in Figure 5, Seq4-Sf9 showed stronger binding efficacy to DC2.4 compared to control mouse IgG1, suggesting that the fusion protein possesses potent antigen-presenting ability.

[0064] 2.4 Evaluation of the biological activity of Seq4-Sf9

[0065] 2.4.1 Evaluate the immunoactivating effect of the fusion protein Seq4-Sf9 using dendritic cells (DCs) induced by CD14+ monocytes from healthy peripheral blood PBMCs.

[0066] To evaluate the immunoactivating effect of the Seq4-Sf9 fusion protein, three CD14+ monocytes from healthy human PBMCs were selected and differentiated into immature dendritic cells (DCs) in a culture system containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Immature DCs have a strong uptake capacity, and the mouse-derived Fc structural domain contained in the fusion protein enhances this uptake function, promoting DC maturation and differentiation. Changes in surface markers of DCs loaded with the Seq4-Sf9 fusion protein were detected by flow cytometry. These markers include the adhesion molecule CD54 (ICAM-1: Intercellular adhesion molecule-1), which contributes to the adhesion and interaction between mature DCs and other immune cells (e.g., T cells), and CD83 and the co-stimulatory molecule CD86, which are involved in antigen presentation and T cell activation. The detection results are shown in Figures 6a-6c. In three different volunteers, the cell surface of DCs loaded with the Seq4-Sf9 fusion protein showed high expression of CD54, CD83, and CD86 maturation markers, indicating that the fusion protein has a potent DC-activating effect. This activates the antigen-presenting ability of vaccine-loaded DCs, initiating an immune response as the first step.

[0067] 2.4.2 Key points for in vitro evaluation of the effect of T cell immune response by dendritic cells loaded with Seq4-Sf9

[0068] The most important functions of mature DCs are antigen presentation and promotion of CD4+ T cell proliferation, and antigen peptides / MHC on the surface of mature DCs. (major histocompatibility)-I complexes or antigen peptide / MHC-II complexes are recognized by T cell surface receptors (TCRs). A crucial step in DC-T activation is T cell proliferation. Therefore, when DCs loaded with Seq4-Sf9 were co-cultured with CD4+ T cells labeled with CFSE fluorescent dye, the T cells rapidly proliferated and differentiated into Th cells (helper T cells) after antigen presentation, allowing for the assessment of immune status based on CD4+ T cell proliferation. The results are shown in Figure 7, where DCs loaded with Seq4-Sf9 clearly promoted T cell proliferation compared to negative controls of CD4+ T cells from the same source (Seq4-Sf9 0 μg / ml), and this was positively correlated with the effective concentration.

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

[0070] 3.1 Preparation of Seq5-293

[0071] Triple mutant mouse Fc with 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 the fusion protein with the amino acid sequence shown in SEQ ID NO: 5. Positions 1-354 are the prostate cancer-specific marker PAP, and positions 361-592 are the triple mutant mouse Fc with the amino acid sequence shown in SEQ ID NO: 2.

[0072] A gene encoding a fusion protein with the amino acid sequence shown in Sequence ID No. 5 was synthesized, and the gene sequence encoding this fusion protein was ligated to the expression vector pcDNA3.4 vector. Recombinant expression plasmids with enzymatic cleavage sites EcoRI (New England Biolabs, R0101V) and HindIII (New England Biolabs, R0104S) were prepared. These recombinant expression plasmids were transiently transfected into eukaryotic cells HD293F, and cell culture was performed. The cell culture medium was collected, centrifuged, and filtered. The filtered culture supernatant was subjected to Protein A affinity chromatography to purify the fusion protein, which was named Seq5-293. Biochemical measurements were 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 with the amino acid sequence shown in Sequence ID No. 2 was expressed by fusing it with the prostate cancer-specific marker PAP via a binding peptide, and Sequence ID No. 2 was used. Obtain a fusion protein with the amino acid sequence shown in 5, The corresponding coding sequence was enzymatically cleaved with Sal I (New England Biolabs, R3138S) and Hind III (New England Biolabs, R0104S) and bound to a pFastBacHTa-gp64 vector. This vector was expressed in insect cells, and Escherichia coli DH10Bac was transformed by translocation to generate recombinant baculovirus. The recombinant baculovirus was isolated, transfected into Sf9 insect cells, and incubated in ESF921 medium at 27°C for 72 hours to express the target protein. The supernatant was collected by centrifugation, and the protein obtained by Protein A purification was named Seq5-Sf9. Biochemical measurements were 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] A quintuple mutant mouse Fc with 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 with the amino acid sequence shown in SEQ ID NO: 6. Positions 1-354 are the prostate cancer-specific marker PAP, and positions 361-592 are the quintuple mutant mouse Fc with the amino acid sequence shown in SEQ ID NO: 3.

[0077] Using the same method as in 3.2, the above fusion protein was expressed in Sf9 insect cells, and the resulting fusion protein was named Seq6-Sf9. Biochemical measurements were performed using a Coomassie brilliant blue stained SDS-PAGE gel, and the results are shown in Figure 10.

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

[0079] Human wild-type Fc fragments and the prostate cancer-specific marker PAP were expressed in 293 mammalian cells, and the Seq7-293 fusion protein was obtained using the same method as in 3.1. Its amino acid sequence is shown in SEQ ID NO: 7, and its effect on Fc mutants and insect glycosyl-type DC activation was examined using the Seq7-293 fusion protein as a control.

[0080] CD14+ monocytes were selected from two healthy PBMCs and differentiated into immature dendritic cells (DCs) in a culture system containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Immature DCs have strong uptake capacity, and when loaded with Seq6-Sf9 and Seq7-293 fusion proteins, respectively, the DCs take up the fusion proteins and present antigens, thereby activating the DCs and expressing activation markers (including CD54 and CD83). Changes in surface markers of vaccine-loaded DCs were detected by flow cytometry, and the detection results are shown in Figures 11a-11b. Compared with Seq7-293 at the same concentration, Seq6-Sf9 significantly activated DCs, resulting in higher expression of CD54 and CD83 on the cell surface. These results demonstrate that mouse-derived Fc mutants and non-mammalian glycosyl forms in the fusion protein significantly enhance uptake and activation by dendritic cells (DCs), enabling the expression of activated phenotypes such as CD54 and CD83.

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

[0082] 3.5.1 Evaluation of the immunoactivating effect of the fusion protein Seq5-Sf9 using CD14+ monocyte-induced dendritic cells (DCs) from human peripheral blood PBMCs.

[0083] To evaluate the immunoactivating effect of Seq5-Sf9, CD14+ monocytes were selected from three healthy PBMCs and differentiated into immature dendritic cells (DCs) in a culture system containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Immature DCs exhibit strong uptake capacity, and the mouse-derived Fc structural domain contained in the fusion protein enhances DC uptake function, promoting DC maturation and differentiation. Flow cytometry was used to detect changes in vaccine-loaded DC surface markers, including the adhesion molecule CD54 (ICAM-1: Intercellular adhesion molecule-1), which contributes to the adhesion and interaction between mature DCs and other immune cells (such as T cells), and CD83, which is involved in antigen presentation and T cell activation. The detection results are shown in Figures 12a-12b. Among the three different volunteers, the high expression of CD54 and CD83 on the surface of Seq5-Sf9-loaded DC cells indicates that the fusion protein has a potent DC-activating effect, activating the antigen-presenting ability of Seq5-Sf9-loaded DCs and initiating an immune response as a first step.

[0084] Furthermore, the immunoactivating effects of Seq5-Sf9, Seq5-293, and the positive control drug Provenge were compared, and the results are shown in Figures 13a-14b. It was observed that Fc mutants and non-mammalian glycosylation enhanced the DC activation effect, and Seq5-Sf9 had a more potent DC activation function.

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

[0086] The most important functions of mature DCs are antigen presentation and promotion of CD4+ T cell proliferation. Antigen peptide / MHC-I or antigen peptide / MHC-II complexes on the surface of mature DCs are recognized by T cell surface receptors (TCRs). A crucial step in DC-T activation is T cell proliferation. Therefore, when DCs loaded with Seq5-Sf9 and CD4+ T cells labeled with CFSE fluorescent dye are co-cultured, T cells can rapidly proliferate and differentiate into Th cells (helper T cells) after antigen presentation, thereby allowing for assessment of immunological status based on CD4+ T cell proliferation. As shown in Figures 15a-15b, after loading Seq5-Sf9 into DC cells from different healthy individuals, T cell proliferation was clearly promoted and showed a positive correlation with Seq5-Sf9 concentration, and Provenge under the same experimental conditions showed a similar effect.

[0087] 3.5.3 In vitro evaluation of antigen-presenting activated cytotoxic T cells from DCs loaded with Seq5-Sf9

[0088] The antigen peptide / MHC-I molecule complex presented on the DC membrane surface can directly recognize and bind to the TCR on the CD8+ T cell surface, thereby activating CD8+ T cells and exerting biological effects. One of the main ways in which these effects are exerted is by secreting interferon-gamma (IFNγ). DCs loaded with Seq5-Sf9 were co-cultured with CD8+ T cells from the same volunteer, and secondary stimulation was performed by adding Seq5-Sf9-loaded DCs again, activating a large number of CD8+ T cells. IFNγ secreted at the individual cell level was detected using ELISPOT. The detection results are shown in Figure 16, and it was confirmed that the IFNγ in the Seq5-Sf9-loaded group was significantly higher than that of the volunteer's own CD8+ T cells (negative control), meaning that Seq5-Sf9 can effectively promote the activation of CD8+ T cells.

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

[0090] The therapeutic effect of prostate cancer treatment depends on whether prostate cancer cells are sensitive to androgens. PC3 is a common androgen-independent human prostate cancer cell line, while LNCap is androgen-dependent. Both cell lines express a certain amount of PAP, and as shown in Figure 17, LNCap expresses a higher amount of PAP protein. These two cell lines were co-cultured with effector cells (CTLs activated by DCs loaded with Seq5-Sf9) as target cells, and the absolute number of dead cells was statistically determined using CFSE and Counting Beads. As shown in Figures 18a-18b, CTLs activated by DCs loaded with Seq5-Sf9 can directly kill target cells, and the killing effect is directly proportional to the concentration of Seq5-Sf9. Furthermore, the number of dead cells in LNCap cells, which express higher levels of PAP, was far greater than in PC3 cells, verifying the targeted killing effect of CTLs activated by DCs loaded with Seq5-Sf9, and demonstrating a higher effect than Provenge.

[0091] 3.6 Immunogenicity evaluation of Seq5-Sf9

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

[0093] Furthermore, rat spleens were collected on day 35, stimulated again using Seq5-Sf9 in vitro to activate them and induce the activation of effector T cells, and IFNγ secretion by individual splenic cells was detected by ELISPOT. As shown in Figures 20a-20b, splenic immune cells can rapidly differentiate into cytotoxic T lymphocytes and secrete IFNγ under the action of secondary stimulation with Seq5-Sf9.

[0094] Simultaneously, prostate tissue was observed, and the results are shown in Figure 21. Infiltration of inflammatory cells was also observed in the prostate tissue 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; in other words, Seq5-Sf9 can complete in vivo immune activation.

[0096] 3.7 Evaluation of the immunoactivating effect of the fusion protein Seq6-Sf9 using CD14+ monocyte-induced dendritic cells (DCs) from human peripheral blood PBMCs.

[0097] Unlike Seq5-Sf9, the Fc fragment of Seq6-Sf9 was used to evaluate its immunoactivating effect. CD14+ monocytes selected from two healthy human PBMCs were induced and differentiated, and then loaded with Seq6-Sf9. Changes in DC surface markers were detected by flow cytometry. As shown in Figures 22a-22b, Seq6-Sf9 increased the expression of CD54 and CD83 on the surface of DC cells, promoting DC activation, and the marker expression levels were higher than those of Seq5-Sf9.

[0098] Finally, it should be noted that the above embodiments are intended to illustrate, and not limit, the technical solutions of the present application. While the present application will be described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments or make equivalent substitutions to some or all of their technical features, and these modifications or substitutions should be understood not to deviate from the essence of the corresponding technical solutions within the scope of the technical solutions of the embodiments of the present application.

[0099] This application claims priority to the Chinese patent application filed with the Chinese National Patent Office on June 30, 2023, with application number 202310798756.4 and title "Fc variants, fusion proteins and their use", and also claims priority to the Chinese patent application filed with the Chinese National Patent Office on June 30, 2023, with application number 202310807755.1 and title "Chimeric fusion proteins, nucleic acid molecules, expression vectors, host cells and their use", all of which are incorporated into this application by reference.

Claims

1. A glycosyl-modified fusion protein comprising a mouse-derived Fc variant and a polypeptide antigen, wherein the fusion protein binds to DC cells and promotes the activation of the DC cells. The mouse-derived Fc mutant is a mouse-derived Fc fragment having amino acid mutations and non-mammalian glycosylation modifications. The amino acid sequence of the mouse-derived Fc mutant is shown in Sequence ID No.

3. The aforementioned non-mammalian glycosylation modification does not include sialic acid modification. A glycosyl-modified fusion protein characterized in that the sugar chain structure formed by the aforementioned non-mammalian glycosylation modification is at least one selected from high-mannose type, oligomannose type, and fucose type.

2. The fusion protein according to claim 1, characterized in that, in the non-mammalian glycosylation modification, the glycosyl is derived from at least one of mannose, N-acetylglucosamine, and fucose.

3. The fusion protein according to claim 1 or 2, characterized in that the polypeptide antigen is a tumor antigen and comprises one of a tumor-specific antigen, a tumor-associated antigen, or a tumor mutation antigen produced by a tumor-associated antigen mutation.

4. The fusion protein according to claim 3, characterized in that the tumor antigen is PAP.

5. The fusion protein according to claim 1 or 2, characterized in that the polypeptide antigen is a viral antigen.

6. The fusion protein according to claim 1 or 2, further comprising a protein tag.

7. The fusion protein according to claim 1 or 2, further comprising a hydrophilic peptide bound to the C-terminus of the mouse-derived Fc mutant.

8. A nucleic acid molecule encoding the fusion protein according to claim 1 or 2.

9. A recombinant expression vector characterized by comprising the nucleic acid molecule described in claim 8.

10. A host cell characterized by containing the recombinant expression vector described in claim 9.

11. A pharmaceutical composition comprising a fusion protein according to claim 1 or 2 and a pharmaceutically acceptable carrier.

12. Use of the fusion protein according to claim 1 or 2 in the preparation of a therapeutic drug for one or two diseases among tumors and viral diseases.

13. A fusion protein for treating viral diseases, which is the fusion protein described in claim 5.

14. A fusion protein for treating tumors, which is the fusion protein described in claim 3.

15. A mouse-derived Fc mutant, wherein the mouse-derived Fc mutant is a mouse-derived Fc fragment having amino acid mutations and non-mammalian glycosylation modifications. The amino acid sequence of the mouse-derived Fc mutant is shown in Sequence ID No.

3. The aforementioned non-mammalian glycosylation modification does not include sialic acid modification. A mouse-derived Fc mutant characterized in that the sugar chain structure formed by the aforementioned non-mammalian glycosylation modification is selected from at least one of high-mannose type, oligomannose type, and fucose type.

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