An Fc signaling protein and its uses

Abstract

This invention relates to the field of biopharmaceuticals, and in particular to an Fc signaling protein and its uses. The Fc signaling protein is the Fc segment of an engineered IgG antibody or an engineered FcγRIIA. The engineered IgG antibody's Fc segment is obtained by mutating the Fc segment of a wild-type IgG antibody. When the engineered IgG antibody's Fc segment binds to FcγRIIA, the reverse bond interaction is stronger than when the wild-type FcγRIIA binds to the Fc segment of the IgG antibody. Similarly, the engineered FcγRIIA, obtained by mutating wild-type FcγRIIA, also exhibits a stronger reverse bond interaction when binding to the Fc segment of an IgG antibody than when the wild-type FcγRIIA binds to the Fc segment of the IgG antibody.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceuticals, and in particular to an Fc signaling protein and its uses. Background Technology

[0002] Human antibodies are classified into five subtypes: IgA, IgE, IgM, IgG, and IgD. IgG accounts for the largest proportion and plays a major role in the immune response. The vast majority of existing antibody drugs also use IgG monoclonal antibodies as their core framework. IgG is a homodimer, and its structure can be specifically divided into a Fab segment containing the CDR region and an Fc segment containing only the heavy chain constant region. The Fab segment determines the antibody's target antigen, while the Fc segment is highly conserved.

[0003] The Fc region of IgG can bind to Fc receptors on the cell surface, mediating an immune response against antibody-bound target antigens / cells. In general, Fc binding to Fc receptors can produce either activating or inhibitory effects, depending on the specific antibody and Fc receptor subtype (Falk Nimmerjahn et al., 2008). Fcγ receptor IIA (FcγRIIA) is expressed on the surface of macrophages and neutrophils and can bind to the Fc region of IgG1. When IgG binds to cells or pathogens carrying its specific target antigens, macrophages / neutrophils can mediate immune regulation through FcγRIIA on their surface, promoting the killing of target cells and the phagocytosis of target antigens, further facilitating downstream immune responses and achieving the clearance of pathogens or abnormal cells.

[0004] Therefore, the binding of the antibody Fc fragment to the corresponding Fc receptor is an important part of the antibody exerting its immune effect. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide an Fc signaling protein and its uses to solve the problems in the prior art.

[0006] To achieve the above and other related objectives, the present invention provides an Fc signaling protein, wherein the Fc signaling protein is the Fc segment of an engineered IgG antibody, the Fc segment of which is obtained by mutating the Fc segment of a wild-type IgG antibody, and the reverse locking force formed when the Fc segment of the engineered IgG antibody binds to FcγRIIA is stronger than that formed when the wild-type antibody binds to FcγRIIA.

[0007] The Fc segment of the engineered IgG antibody is obtained by mutating one or more of the following sites in the Fc segment of the wild-type IgG antibody to histidine (H): L234, L235, G236, G237, S239, V264, D265, V266, S267, N297, S298, A327, L328, P329.

[0008] The Fc signaling protein is engineered FcγRIIA, which is obtained by mutating wild-type FcγRIIA. When engineered FcγRIIA binds to the Fc segment of IgG antibody, the reverse locking force formed is stronger than that formed when wild-type FcγRIIA binds to the Fc segment of IgG antibody.

[0009] The present invention also provides an isolated polynucleotide encoding the Fc segment of the engineered IgG antibody.

[0010] The present invention also provides a nucleic acid construct containing the isolated polynucleotides described above.

[0011] The present invention also provides a cell containing the aforementioned nucleic acid construct or having an exogenous polynucleotide integrated into its genome or expressing the aforementioned Fc signaling protein.

[0012] The present invention also provides the use of the Fc signaling protein, isolated polynucleotides, nucleic acid constructs, and cells in the preparation of therapeutic drugs or in the preparation of diagnostic drugs.

[0013] The present invention provides a drug comprising the Fc segment of the engineered IgG antibody.

[0014] As described above, the Fc signaling protein and its uses of the present invention have the following beneficial effects: This patent successfully achieved signal enhancement and reverse bond activation of Fc and FcγRIIA using histidine scanning technology. Attached Figure Description

[0015] Figure 1 The figure shown is an incubation result of the Fc and reporter cell lines of the present invention.

[0016] Figure 2 The image shown represents the results of incubation and activation detection of the wild-type Fc reporter cell line FcγRIIA as described in this invention. Detailed Implementation

[0017] This invention provides an Fc signaling protein, wherein the Fc signaling protein is the Fc segment of an engineered IgG antibody, and the Fc segment of the engineered IgG antibody is obtained by mutating the Fc segment of a wild-type IgG antibody. When the Fc segment of the engineered IgG antibody binds to FcγRIIA, the reverse locking force formed is stronger than that formed when the wild-type IgG antibody binds to FcγRIIA.

[0018] In some embodiments of the present invention, the Fc segment of the engineered IgG antibody is the Fc segment of IgG1, IgG2, IgG3 or IgG4, that is, it is obtained by mutating the Fc segment of wild-type IgG1, IgG2, IgG3 or IgG4 antibody respectively.

[0019] In some embodiments of the present invention, the Fc segment of the engineered IgG antibody is the Fc segment of IgG1, and the amino acid sequence from position 223 to position 447 of the Fc segment of the wild-type IgG1 antibody is shown in SEQ ID NO.1.

[0020] In some embodiments of the present invention, the Fc segment of the engineered IgG antibody is obtained by mutating one or more of the following sites in the Fc segment of the wild-type IgG antibody to histidine (H): L234, L235, G236, G237, S239, V264, D265, V266, S267, N297, S298, A327, L328, P329.

[0021] The Fc segment of the engineered IgG antibody is an Fc segment that can bind to wild-type FcγRIIA or engineered FcγRIIA.

[0022] The Fc signaling protein is engineered FcγRIIA, which is obtained by mutating wild-type FcγRIIA. When engineered FcγRIIA binds to the Fc segment of IgG antibody, the reverse locking force formed is stronger than that formed when wild-type FcγRIIA binds to the Fc segment of IgG antibody.

[0023] The amino acid sequence of wild-type FcγRIIA is shown in SEQ ID NO.6. The engineered FcγRIIA is obtained by mutating one or more of the following sites in wild-type FcγRIIA to histidine: W123, W146, P150, V152, K153, F165, L168, Y193. Preferably, the engineered FcγRIIA is an FcγRIIA that can bind to the Fc segment of wild-type IgG antibody or the Fc segment of engineered IgG antibody.

[0024] The present invention also provides an isolated polynucleotide encoding the Fc segment of the engineered IgG antibody.

[0025] In some embodiments of the present invention, the polynucleotide encoding the Fc segment of the engineered IgG antibody comprises a nucleotide sequence as described in any of SEQ ID NO. 3 to 5.

[0026] The present invention also provides a nucleic acid construct containing the isolated polynucleotides described above.

[0027] The term "nucleic acid construct" refers to an artificially constructed nucleic acid segment that can be introduced into target cells or tissues. The nucleic acid construct can be various expression vectors, which include a vector backbone (empty vector) and an expression frame. The term "expression frame" refers to a sequence with the potential to encode a protein.

[0028] There is no specific limitation on the type of expression vector. An expression vector is a nucleic acid molecule that allows the insertion of foreign nucleotides without disrupting its ability to replicate and / or integrate into the host cell. Expression vectors may include nucleic acid sequences that allow them to replicate in the host cell, such as origins of replication. Expression vectors may also include one or more selective marker genes and other genetic factors. An expression vector is a vector containing the necessary regulatory sequences to enable the transcription and translation of one or more inserted genes. Expression vectors are selected from eukaryotic expression vectors or prokaryotic expression vectors.

[0029] The prokaryotic expression vector is selected from Escherichia coli expression vectors, Bacillus subtilis expression vectors, or Streptomyces expression vectors. In a preferred embodiment, the prokaryotic expression vector is selected from Escherichia coli expression vectors.

[0030] The eukaryotic expression vector is selected from yeast expression vectors, insect expression vectors, or mammalian expression vectors. The mammalian expression vector is either a non-viral expression vector or a viral expression vector. The viral expression vector is selected from retroviral expression vectors, lentiviral expression vectors, adenovirus expression vectors, and adeno-associated virus expression vectors.

[0031] The host cells are selected from eukaryotic or prokaryotic host cells. Eukaryotic host cells are selected from fungi such as yeast, insects, birds, plants, *C. elegans* or nematodes, or mammalian host cells. A non-limiting example of insect cells is *Noctua przewalskii* cells. Examples of yeast host cells are *Saccharomyces cerevisiae*, *Kluyveromyces lactis*, or *Yersinia lipolytica*. Examples of mammalian cells are COS cells, juvenile hamster kidney cells, mouse L cells, LNCaP cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, African green monkey cells, CV1 cells, Vero, or Hep-2 cells. Examples of prokaryotic host cells include bacterial cells such as *Escherichia coli*, *Streptomyces*, *Bacillus subtilis*, *Salmonella typhi*, or mycobacteria.

[0032] Those skilled in the art can transfect the expression vector into host cells using methods well-known in the art to obtain cells containing the encoding gene of the antibody of the present invention. For example, the expression vector can be introduced into eukaryotic cells via liposome transfection.

[0033] The present invention also provides a cell containing the aforementioned nucleic acid construct or having an exogenous polynucleotide integrated into its genome or expressing the aforementioned Fc signaling protein.

[0034] The cell can be any cell that can serve as a host cell. In some embodiments, the cell is a 293F cell.

[0035] The present invention also provides the use of the Fc signaling protein, isolated polynucleotides, nucleic acid constructs, and cells in the preparation of therapeutic drugs or in the preparation of diagnostic drugs.

[0036] In some embodiments of the present invention, the therapeutic drug is a tumor treatment drug or a viral infection (e.g., HIV infection) treatment drug, an autoimmune disease treatment drug, an infectious disease treatment drug, a nervous system disease treatment drug, an ophthalmic disease treatment drug, or a blood disease treatment drug.

[0037] The tumors are selected from melanoma, leukemia, synovial sarcoma, multiple myeloma, lung cancer, liver cancer, cervical cancer, pancreatic cancer, ovarian cancer, etc.

[0038] These autoimmune diseases include rheumatoid arthritis, inflammatory bowel disease, and systemic lupus erythematosus. Antibodies typically modulate the immune system by targeting specific immune cells or cytokines, reducing inflammation and tissue damage.

[0039] In the treatment of these infectious diseases, IgG antibodies can be used to neutralize pathogens or modulate immune responses. For example, antibody drugs used to treat hepatitis C.

[0040] The aforementioned neurological diseases include multiple sclerosis.

[0041] The ophthalmic diseases mentioned include wet age-related macular degeneration.

[0042] The present invention provides a drug comprising the Fc segment of the engineered IgG antibody.

[0043] The drug is any one or more of engineered IgG antibodies, Fc fusion proteins, ADCs, and bispecific antibodies.

[0044] The engineered IgG antibody includes the Fc fragment and the Fab fragment, which are linked by disulfide bonds.

[0045] The engineered IgG antibody can be an antibody that specifically binds to any antigen, such as any tumor antigen, any viral antigen, etc., as long as the antibody that specifically binds to the antigen can bind to FcγRIIA.

[0046] The engineered IgG antibody is, for example, an anti-CD19 or anti-CD20 antibody.

[0047] The Fc fusion protein comprises the Fc segment of the engineered IgG antibody and a polypeptide linked thereto. The polypeptide may be a coagulation factor, growth hormone, GLP-1, granulocyte colony-stimulating factor, etc.

[0048] In some implementations, the use of the Fc fragment in the Fc fusion protein can prolong the drug's half-life in vivo, improving its stability and bioavailability. Fc fusion proteins can be used to treat a variety of diseases, including cancer, autoimmune diseases, and infections. Fc fusion proteins can enhance antibody-dependent cell-mediated cytotoxicity (ADCC), ADCP, and / or complement-dependent cytotoxicity (CDC) effects, thereby improving therapeutic efficacy.

[0049] The present invention also provides a treatment method for a disease, the treatment method comprising administering a therapeutically effective amount of the Fc signaling protein or the drug to a subject in need.

[0050] "Subjects" include, but are not limited to, animals, preferably mammals; said mammals are preferably rodents, even-toed ungulates, perissodactyls, lagomorphs, primates, etc. The mammals include, for example, humans, non-human primates (e.g., monkeys), mice, pigs, cattle, goats, rabbits, rats, guinea pigs, hamsters, horses, monkeys, sheep, or other non-human mammals; non-mammals include, for example, non-mammal vertebrates, such as birds (e.g., chickens or ducks) or fish, and non-mammal invertebrates. Subjects can be humans, such as patients with weakened immune systems or cancer.

[0051] The disease described varies depending on the type of drug used; for example, it could be cancer, diabetes, etc.

[0052] In this application, "cancer" refers to any medical condition mediated by the growth, proliferation, or metastasis of tumor or malignant cells, resulting in solid tumors and non-solid tumors such as leukemia. In this invention, "tumor" refers to the solid matter of tumors and / or malignant cells.

[0053] "Treatment" or "therapy" for a condition includes preventing or alleviating the condition, slowing the onset or progression of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, reducing or terminating symptoms associated with the condition, achieving complete or partial reversal of the condition, curing the condition, or a combination of the above. For cancer, "treatment" or "therapy" can refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination of the above. For tumors, "treatment" or "therapy" includes eliminating all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor development, or some combination of the above.

[0054] The cancers mentioned include, for example, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer; leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma and other hematologic malignancies such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocytic B-cell lymphoma, EBV-positive and negative PTLD and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma and HHV8-associated primary exudative lymphoma.

[0055] In this invention, "therapeutic effective amount" or "effective dose" refers to the dose or concentration at which a certain drug is effective in treating an antigen-related disease or state. For example, for the use of the antibody or its antigen-binding fragment disclosed in this invention, a therapeutic effective amount is a dose or concentration at which the antibody or antigen conjugate can eliminate all or part of a tumor, inhibit or slow tumor growth, inhibit the growth or proliferation of cells mediating a cancerous state, inhibit tumor cell metastasis, alleviate any symptoms or markers associated with a tumor or cancerous state, prevent or delay the development of a tumor or cancerous state, or some combination thereof.

[0056] Specifically, when administering the medication to subjects, the dosage varies depending on the patient's age and weight, disease characteristics and severity, and route of administration. The results of animal experiments and various other factors can be referenced, and the total dosage should not exceed a certain range.

[0057] In some embodiments, the methods described herein may further include administration in combination with other compounds or other cancer treatment regimens in the prior art.

[0058] Other cancer treatment options may include, but are not limited to: surgery, radiation therapy, chemotherapy, toxin therapy, immunotherapy, cryotherapy, cancer vaccines (e.g., HPV vaccine, hepatitis B vaccine), and gene therapy, as well as any combination thereof. Immunotherapy includes, but is not limited to, adoptive cell therapy, stem cell and / or dendritic cell derivatization, blood transfusion, irrigation, and / or other therapies, including but not limited to cryotherapy of tumors.

[0059] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0060] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0061] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0062] Brief description of the implementation example: First, the complex structure of the human IgG1 Fc fragment and human FcγRIIA (PDB: 3RY6) was selected as the basis for analysis, and ligands with a distance of less than [missing information] were screened. . residues.

[0063] Then, the Fc mutant with a single-site histidine mutation was cloned and constructed onto the pD649 plasmid.

[0064] Furthermore, the wild-type FcγRIIA was constructed in the JRT T3.5 cell line containing the NFAT-GFP reporter system; the wild-type Fc protein and its mutant protein were obtained by expression in Expi293F cells.

[0065] Furthermore, by coating well plates with Fc protein and then incubating NFAT-GFP reporter cell lines in the well plates, the effect of screening for single-site histidine mutations on the interaction between Fc and FcγRIIA was detected.

[0066] Furthermore, mutants with better activation were selected, and the enhancement of inverse bond formation was measured using optical tweezers. This involved expressing the ligand protein Fc and the receptor protein FcγRIIA on the surface of SKW-3 cells, and then measuring the force-time curves of Fc versus SKW-3-FcγRIIA+ using optical tweezers to reflect the formation of inverse bonds.

[0067] Example 1: Screening of interaction sites between human IgG1-Fc and human FcγRIIA complex (PDB: 3RY6)

[0068] The sequence of human IgG1-Fc between T223 and K447 is shown in SEQ ID NO.1:

[0069] THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO.1)

[0070] Structural analysis was performed in ChimeraX to analyze the interaction between the two chains. The interaction sites and mutation sites within the site are shown in Table 1 below.

[0071] Table 1 is less than Interacting sites:

[0072]

[0073] Example 2: Wild-type / mutant protein expression of IgG1-Fc and construction of FcγRIIA reporter cell lines

[0074] Using the gene sequences corresponding to the IgG1-Fc amino acid series (nucleotide sequences shown in SEQ ID NO. 2–5), primers for single-site histidine mutations were designed (mutant primers are shown in Table 2 below), amplified, and cloned into the pD649 plasmid. Subsequently, the pD649 plasmid was transfected into the Expi293F cell line using PEI. The desired protein was then purified for further experiments.

[0075] L234H mutant sequence (uppercase letters indicate the mutation site)

[0076] actcacacatgcccaccgtgcccagcacctgaaCATctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgat

[0077] ctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggagg

[0078] tgcataatgccaagacaaagccgcggggagagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctg

[0079] aatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgag

[0080] aaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcg

[0081] acatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctct

[0082] acagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa(SEQ ID NO.2)

[0083] L235H mutant sequence (the mutated site is in uppercase)

[0084] actcacacatgcccaccgtgcccagcacctgaactcCATgggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa(SEQ ID NO.3)

[0085] G236H mutant sequence (the mutated site is in uppercase)

[0086] actcacacatgcccaccgtgcccagcacctgaactcctgCATggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa(SEQ ID NO.4)

[0087] Nucleotide sequence of wild-type IgG1-Fc

[0088] Actcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa(SEQ ID NO.5)

[0089] Table 2 Mutation primer design:

[0090]

[0091]

[0092] Primers were designed using the known FcγRIIA gene sequence (NM_001136219.3) to specifically amplify the FcγRIIA sequence from the cDNA of the K562 cell line. This sequence was cloned into the pHR plasmid backbone, and the corresponding FcγRIIA mutant was constructed through further cloning. Lentiviral cells were packaged using the pHR, Pspax2, and pMD2G three-plasmid system. The viral solution was then used to infect the JRT3T3.5 cell line carrying the NFAT-GFP fluorescent reporter system. After appropriate culture time, flow cytometry was performed to enrich positive cells.

[0093] Wild-type FcγRIIA nucleotide sequence

[0094] atgactatggagacccaaatgtctcagaatgtatgtcccagaaacctgtggctgcttcaaccattgacagttttgctgctgctggcttctgcagacagtcaagctgcagctcccccaaaggctgtgctgaaacttgagcccccgtggatcaacgtgctccaggaggactctgtgactctgacatgccagggggctcgcagccctgagagcgactccattcagtggttccacaatgggaatctcattcccacccacacgcagcccagctacaggttcaaggccaacaacaatgacagcggggagtacacgtgccagactggccagaccagcctcagcgaccctgtgcatctgactgtgctttccgaatggctggtgctccagacccctcacctggagttccaggagggagaaaccatcatgctgaggtgccacagctggaaggacaagcctctggtcaaggtcacattcttccagaatggaaaatcccagaaattctcccatttggatcccaccttctccatcccacaagcaaaccacagtcacagtggtgattaccactgcacaggaaacataggctacacgctgttctcatccaagcctgtgaccatcactgtccaagtgcccagcatgggcagctcttcaccaatggggatcattgtggctgtggtcattgcgactgctgtagcagccattgttgctgctgtagtggccttgatctactgcaggaaaaagcggatttcagccaattccactgatcctgtgaaggctgcccaatttgagccacctggacgtcaaatgattgccatcagaaagagacaacttgaagaaaccaacaatgactatgaaacagctgacggcggctacatgactctgaaccccagggcacctactgacgatgataaaaacatctacctgactcttcctcccaacgaccatgtcaacagtaataactaa(SEQ ID NO.6)

[0095] Wild-type FcγRIIA amino acid sequence

[0096] MTMETQMSQNVCPRNLWLLQPLTVLLLLASADSQAAAPPKAVLKLEPPWINVLQEDSVTLTCQGARSPESDSIQWFHNGNLIPTHTQPSYRFKANNNDSGEYTCQTGQTSLSDPVHLTVLSEWLVLQTPHLEFQEGETIMLRCHSWKDKPLVKVTFFQNGK SQKFSHLDPTFSIPQANHSHSGDYHCTGNIGYTLFSSKPVTITVQVPSMGSSSPMGIIVAVVIATAVAAIVAAVVALIYCRKKRISANSTDPVKAAQFEPPGRQMIAIRKRQLEETNNDYETADGGYMTLNPRAPTDDDKNIYLTLPPNDHVNSNN*(SEQ ID NO.7)

[0097] Example 3: Incubation and activation detection of Fc protein with NFAT-GFP reporter cell lines

[0098] Wild-type and mutant Fc proteins were coated into 96-well plates at a concentration gradient. Specifically, Fc protein solutions of corresponding concentrations were prepared using PBS, and 100 μl of the protein solution was added to each well of the 96-well plate. The plates were incubated at 37°C for 2–2.5 h, then the supernatant was discarded. NFAT-GFP reporter cell lines were added, and the plates were incubated at 37°C with 5% CO2 for 12 h. The intensity of the GFP fluorescent protein in the reporter cell lines was detected by flow cytometry, and activation curves were fitted to measure the activation effect. Results are as follows: Figure 1 As shown, during incubation of Fc with reporter cell lines, Fc-L234H / L235H / G236H exhibited relatively strong post-modification energizing signals, indicating the success of histidine mutation and Fc mutation modification.

[0099] Example 4: Measurement with optical tweezers

[0100] First, a biotinylation site was added to the N-terminus of the Fc region of IgG1, and the resulting structure was cloned into the pd649 plasmid. This plasmid was then transfected with Expi293F to generate the Fc protein. After purification with Ni-NTA, the protein was biotinylated using birA, followed by purification via molecular sieve chromatography (Superdex 200 increase, Cytiva) to obtain purified biotinylated Fc protein. Subsequently, the force-time relationship was analyzed using M-Crap assays on cells expressing FcγRIIA and the Fc protein, and an inverse-locked curve was fitted.

[0101] Example 5: Incubation and activation detection of reporter cell lines expressing FcγRIIA wild-type and mutant Fc cells.

[0102] like Figure 2 As shown, in the incubation of wild-type Fc cells and reporter cell lines expressing several FcγRIIA variants, FcγRIIA-L168H / V152H / K153H / Y193H exhibited a relatively strong post-modification activation signal, indicating the success of histidine mutation and the successful modification of FcγRIIA.

[0103] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. An Fc signaling protein, characterized in that, The Fc signaling protein is the Fc segment of an engineered IgG antibody. The Fc segment of the engineered IgG antibody is obtained by mutating the Fc segment of a wild-type IgG antibody. When the Fc segment of the engineered IgG antibody binds to FcγRIIA, the reverse locking force formed is stronger than that formed when the wild-type IgG antibody binds to FcγRIIA.

2. The Fc signaling protein according to claim 1, characterized in that, The amino acid sequence from position 223 to position 447 of the Fc segment of the wild-type IgG antibody is shown in SEQ ID NO.

1. The Fc segment of the engineered IgG antibody is obtained by mutating one or more of the following sites in the Fc segment of the wild-type IgG antibody to histidine: L234, L235, G236, G237, S239, V264, D265, V266, S267, N297, S298, A327, L328, P329. Preferably, the Fc segment of the engineered IgG antibody is an Fc segment that can bind to wild-type FcγRIIA or engineered FcγRIIA.

3. The Fc signaling protein according to claim 1, characterized in that, The Fc signaling protein is engineered FcγRIIA, which is obtained by mutating wild-type FcγRIIA. When engineered FcγRIIA binds to the Fc segment of IgG antibody, the reverse locking force formed is stronger than that formed when wild-type FcγRIIA binds to the Fc segment of IgG antibody.

4. The Fc signaling protein according to claim 1, characterized in that, The amino acid sequence of wild-type FcγRIIA is shown in SEQ ID NO.

6. The engineered FcγRIIA is obtained by mutating one or more of the following sites in wild-type FcγRIIA to histidine: W123, W146, P150, V152, K153, F165, L168, Y193. Preferably, the engineered FcγRIIA is an FcγRIIA that can bind to the Fc segment of wild-type IgG antibody or the Fc segment of engineered IgG antibody.

5. An isolated polynucleotide, characterized in that, Encodes the Fc signaling protein as described in any one of claims 1 to 4.

6. A nucleic acid construct, characterized in that, It contains the isolated polynucleotide as described in claim 5.

7. A cell, characterized in that, The cell contains the nucleic acid construct of claim 6 or integrates an exogenous polynucleotide of claim 5 into its genome or expresses the Fc signaling protein of any one of claims 1 to 4.

8. The use of the Fc signaling protein according to any one of claims 1 to 4, the isolated polynucleotide according to claim 5, the nucleic acid construct according to claim 6, and the cell according to claim 7 in the preparation of therapeutic drugs or diagnostic drugs.

9. A drug, characterized in that, The drug comprises the Fc segment of an engineered IgG antibody in any of the Fc signaling proteins according to claims 1 to 4, wherein the drug is any one or more of engineered IgG antibodies, Fc fusion proteins, and bispecific antibodies.

10. The medicament according to claim 9, characterized in that, The engineered IgG antibody comprises the Fc segment and Fab fragment of the engineered IgG antibody according to any one of claims 1 to 4, wherein the Fc segment and Fab fragment are linked by disulfide bonds; or, the Fc fusion protein comprises the Fc segment of the engineered IgG antibody in the Fc signaling protein according to any one of claims 1 to 4 and a polypeptide linked thereto. Preferably, the Fc segment in the Fc fusion protein is used to prolong the half-life of the polypeptide or to bind to FcR to enhance ADCC and / or ADCP.

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