Bispecific fusion proteins targeting TNF-α and IL-17A and uses thereof

Abstract

The present invention relates to a bispecific fusion protein targeting TNF-α and IL-17A, a polynucleotide encoding the same, a method for preparing the same, and uses thereof. The bispecific fusion protein targeting TNF-α and IL-17A is a dimer with a symmetric structure and contains, from the N-terminus to the C-terminus, three structural functional regions: a soluble TNF receptor or a portion thereof, a human IgG Fc fragment, and a functional domain that competitively binds to IL-17A or an anti-IL-17A domain. The fusion protein can effectively bind to both TNF-α and IL-17A and has the effect of blocking their signaling pathways. The fusion protein has good stability, specificity, and biological activity.

Description

[Technical Field]

[0001] This application is in the field of medical biotechnology. Specifically, this application relates to bispecific fusion proteins targeting TNF-α and IL-17A, polynucleotides encoding same, formulations and pharmaceutical compositions containing same, methods for their preparation, and uses thereof. [Background technology]

[0002] Tumor necrosis factor-α (TNF-α) is an important inflammatory factor primarily produced by cells such as macrophages, monocytes, T cells, NK cells, mast cells, and neutrophils. TNF-α is a transmembrane protein approximately 26 kDa in size exposed on the cell surface. It can be processed to activated soluble TNF-α, approximately 17 kDa in size, through protease cleavage by tumor necrosis factor converting enzyme (TACE). Soluble TNF-α can form trimers and activate TNFRs. TNF-α has two receptors, TNFR1 and TNFR2, and the functions mediated by the two receptors exhibit certain differences. TNFR1 (p55), 55 kDa, is expressed in all cells, while TNFR2 (p75) is primarily expressed in immune system cells and endothelial cells. Both receptors can also be cleaved from the cell membrane to form soluble forms. These soluble receptors can function as antagonists by binding to soluble TNF-α and inhibiting the binding of TNF-α to cell surface TNFRs.

[0003] In the synovial membrane of rheumatoid arthritis (RA) patients, activated macrophages can produce TNF-α, which can induce the synthesis of various other pro-inflammatory cytokines and chemokines, such as IL-1β, IL-6, and IL-8. These factors may reactivate macrophages in the joint, resulting in their persistent cytokine production, abnormal proliferation of fibroblast-like synoviocytes (FLS), increased expression of adhesion factors, and neovascularization. Additionally, TNF-α may induce MMP synthesis, resulting in the destruction of cartilage and bone tissue. TNF-α may also stimulate chondrocytes to secrete the Wnt inhibitor DKK-1, inhibiting cartilage and bone formation in the tissue. As an important pro-inflammatory factor, TNF-α may also interact with various other cytokines in the development and progression of RA, potentially producing synergistic effects and amplifying the inflammatory response in vivo. TNF-α also inhibits Treg cell differentiation by inhibiting Foxp3 transcription, reducing the body's Treg cell content and causing abnormal immune regulation, which may worsen the condition of RA. In recent years, the role of TNF-α in the pathology and physiology of RA has been deeply investigated. Furthermore, current biopharmaceuticals targeting TNF-α for the treatment of rheumatoid arthritis (e.g., infliximab, adalimumab, golimumab, and certolizumab) have been widely used and achieved good efficacy. Despite the clear clinical benefits of anti-TNF-α agents, there are still problems such as non-responsiveness, secondary loss of response, and intolerance to anti-TNF agents.

[0004] Interleukin-17A (IL-17A) was first described in 1995 as a proinflammatory cytokine produced by T cells. IL-17A, also commonly known as IL-17, is one of six members of the IL-17 family. Subsequent studies have revealed that Th17 cells are the primary source of IL-17, and that T cells are induced by IL-6, IL-1β, and TGF-β to produce mature Th17 cells with IL-23. Th17 cells are a type of highly inflammatory T cell. IL-17A secreted by Th17 cells can have multiple effects on immune cells, stimulating them to produce various other inflammatory factors, such as IL-1β, TNF-α, IL-6, and CXC chemokines. Compared to healthy individuals, a higher number of Th17 cells with higher IL-17A concentrations can be found in the peripheral blood of RA patients. In addition, the synovial fluid of RA patients contains a higher number of Th17 cells. Studies have shown that Th17 cells secrete large amounts of IL-17A at synovial sites, while the transcriptional levels of IL-17A in synovial tissues positively correlate with the progression of joint damage.

[0005] DMARDs (e.g., methotrexate) remain the mainstream drugs for treating diseases such as rheumatoid arthritis. In recent years, a series of biologics, such as TNF-α inhibitors, have brought new hope to patients with poor therapeutic efficacy of DMARDs. However, clinical trials have shown that half of rheumatoid arthritis patients treated with TNF-α inhibitors fail to achieve satisfactory results. In addition, for some rheumatoid arthritis patients treated with single-target biologics for a certain period of time, the efficacy of the drug may gradually decrease over time, necessitating switching to an alternative drug.

[0006] In previous studies (see Chinese Patent Application Publication No. 106892982), the inventors have demonstrated that fusion proteins targeting both TNF-α antigen and IL-17A antigen can achieve good efficacy in vivo and in vitro. However, in the production process of the fusion proteins and bispecific antibodies, the inventors found several major problems, such as the production of aggregates, low stability, and low expression, which require further research and exploration. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Chinese Patent Application Publication No. 106892982 [Patent Document 2] Chinese Patent Application Publication No. 109796534 Summary of the Invention [Problem to be solved by the invention]

[0008] Similar to bispecific antibodies, bispecific fusion proteins consistently face challenges including low expression levels, poor stability, complex processing, and a tendency to form aggregates. Therefore, there is a need to develop bispecific fusion proteins for the treatment of inflammation-related diseases that have advantages such as better specificity, superior therapeutic efficacy, and improved stability compared to existing fusion proteins. [Means for solving the problem]

[0009] Through research, the applicant has developed a bispecific fusion protein targeting TNF-α and IL-17A that is different from the prior art, which is a dimer with a symmetrical structure and containing three functional domains, and therefore provides a bispecific fusion protein for inflammatory factors that is structurally stable, highly specific, and easy to prepare. The fusion protein can effectively bind to both TNF-α and IL-17A, exerting the function of blocking their signal pathways and exhibiting good biological activity.

[0010] In one aspect, the present application provides, in order from N-terminus to C-terminus: a soluble TNF receptor or a portion thereof; a human IgG Fc fragment; The present invention provides a bispecific fusion protein that targets TNF-α and IL-17A, comprising a functional domain that competitively binds to IL-17A or an anti-IL-17A functional domain.

[0011] In another aspect, the present application provides a polynucleotide encoding the bispecific fusion protein.

[0012] In another aspect, the present application provides a recombinant vector comprising the above polynucleotide.

[0013] In another aspect, the present application provides a host cell comprising the above polynucleotide or recombinant vector.

[0014] In another aspect, the present application provides a method for preparing a bispecific fusion protein of the present disclosure, comprising: a) preparing a polynucleotide encoding the bispecific fusion protein; b) constructing a recombinant vector using the polynucleotide and an expression vector; c) transferring the recombinant vector into host cells and culturing the transformed cells to obtain a cell culture; d) purifying and isolating the cell culture to obtain the bispecific fusion protein.

[0015] In another aspect, the present application provides use of the bispecific fusion protein in the preparation of a medicament for treating an inflammation-related disease. Alternatively, the present application provides use of the bispecific fusion protein in the preparation of a reagent for binding and inhibiting TNF-α and IL-17A.

[0016] In another aspect, the present application provides a formulation comprising the bispecific fusion protein described above, further comprising a buffer system, a pharmaceutically acceptable excipient, and a surfactant, wherein the buffer system is selected from a solution of acetic acid and sodium acetate, a solution of citric acid and sodium citrate, or a solution of histidine and histidine hydrochloride.

[0017] In another aspect, the present application provides a pharmaceutical composition comprising the bispecific fusion protein. [Effects of the Invention]

[0018] (Beneficial effect) The bispecific fusion protein targeting TNF-α and IL-17A in the present application is a dimer with a symmetrical structure and three structural functional domains. The fusion protein can effectively bind to both TNF-α and IL-17A and exert the function of blocking their signaling pathways, particularly inhibiting the synergistic effect caused by these two signaling pathways. The fusion protein has good stability, specificity, and physiological activity, and can be used in the individual treatment, prevention, and / or diagnosis of related diseases.

[0019] Compared with the prior art (e.g., Chinese Patent Publication No. 109796534), the present application improves the stability of the protein and reduces the formation of aggregates through structural optimization of the fusion protein, while exhibiting good affinity and other effects. Furthermore, it explores the formulation of the formulation to make the fusion protein more stable, produce less aggregates, and have better pharmaceutical suitability in the formulation. [Brief explanation of the drawings]

[0020] [Figure 1] 1 shows the structures of eight exemplary proteins designed in Example 1 of the present application. [Figure 2] 1 shows the results of SDS-PAGE analysis of eight exemplary purified proteins designed in Example 1 of the present application. [Figure 3] SEC-HPLC analysis results of eight exemplary purified proteins designed in Example 1 of the present application are shown. [Figure 4] The binding response values ​​of each of the eight exemplary proteins designed in Example 1 of the present application to two antigens are shown, and the binding ratio of each protein to the two antigens is theoretically 1:2. [Figure 5] Reducing and non-reducing SDS-PAGE analysis of fusion proteins Nos. 5, 6, and 8 before storage and after storage at -80°C is shown. [Figure 6] Schematic diagram of the structure of No. 5 protein. [Figure 7] 1 shows information on purified No. 5 protein through transient expression in CHO cells. [Figure 8] The bone and joint scores of mice in each group after treatment with hIL-17A gene-transduced rheumatoid arthritis are shown. [Figure 9] Images of the paws of mice from each group after treatment are shown. [Figure 10] MicroCT results of the paws (left front paw, right front paw, left hind paw, right hind paw) of a representative mouse from each group are shown. [Figure 11] MicroCT bone damage scores for mice in each group are shown (three individuals were scored consecutively and the average value was taken). DETAILED DESCRIPTION OF THE INVENTION

[0021] The present application will be further described below in combination with specific embodiments, but those skilled in the art should understand that these embodiments are merely examples and do not limit the scope of the present application. Those skilled in the art may make various modifications, changes, substitutions or combinations to the technical solutions of the present application without departing from the spirit and scope of the present application, and the solutions obtained in this way fall within the scope of protection of the present application.

[0022] Unless otherwise defined, technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., J. Wiley & Sons (New York, NY, 1994) and Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Spring Harbor, NY, 1989).

[0023] Unless otherwise specified, the term "vector" in this disclosure refers to a self-replicating DNA molecule that is used to introduce a target gene (e.g., a homologous or heterologous foreign gene) into a recipient cell.

[0024] Unless otherwise specified, the term "linker peptide" in this disclosure may refer to a peptide sequence used to link together moieties in a fusion protein in this disclosure.

[0025] Unless otherwise specified, the terms "polynucleotide," "nucleic acid," "nucleotide sequence," and "gene" are used interchangeably in this disclosure and are all chemical in nature.

[0026] Unless otherwise specified, the term "treatment" in this disclosure means to cure, alleviate, ameliorate, slow, relieve or improve a disease or related symptoms, or to prevent, delay, arrest, interrupt or inhibit the onset or further progression of a disease or related symptoms in a statistically significant manner.

[0027] As used in this disclosure, the term "antibody" refers to a binding protein having at least one antigen-binding domain. Antibodies and antigen-binding fragments thereof herein can be intact antibodies or any antigen-binding fragment thereof. Thus, antibodies and antigen-binding fragments thereof herein include monoclonal antibodies or antigen-binding fragments thereof, antibody variants or antigen-binding fragments thereof, and immunoconjugates. Examples of antibody fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, isolated CDR regions, single-chain Fv molecules (scFv), single-domain antibodies (sdAbs), Fd fragments, and other antigen-binding fragments known in the art.

[0028] The term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for contact with human and animal tissues without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0029] Unless otherwise specified, the terms "comprises," "including," and "comprising," or their equivalents, in this disclosure are open-ended, meaning that in addition to the listed components, elements, and steps, other unspecified components, elements, and steps may also be included.

[0030] The percentage of identity (degree of homology) between sequences herein can be determined by aligning two or more sequences using computer programs commonly used for such purposes, which are freely available on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

[0031] Unless otherwise specified, all numbers used herein to express ingredient amounts, measurements, or test conditions shall be understood to be modified in all instances by the term "about." When relating to percentages, the term "about" may represent, for example, ±1%, preferably ±0.5%, and more preferably ±0.1%. Unless clearly indicated otherwise, singular terms herein include plural referents, and vice versa. Unless clearly indicated otherwise, the word "or" herein is intended to include "and."

[0032] In one embodiment, the present application provides, in order from N-terminus to C-terminus: a soluble TNF receptor or a portion thereof; a human IgG Fc fragment; and a functional domain that competitively binds to IL-17A or is anti-IL-17A.

[0033] In some embodiments, the soluble TNF receptor or a portion thereof may be soluble TNF receptor type 1 (abbreviated as sTNFRI or sTNFR1) or a portion thereof, or soluble TNF receptor type 2 (abbreviated as sTNFRII or sTNFR2) or a portion thereof. In some preferred embodiments, from the standpoint of achieving higher expression and better affinity for the target protein, the soluble TNF receptor or a portion thereof may preferably be soluble TNF receptor type 1 or a portion thereof. In a more preferred embodiment, the soluble TNF receptor or a portion thereof may be an extracellular fragment of soluble TNF receptor type 1.

[0034] In some embodiments, the soluble TNF receptor type 1 extracellular fragment comprises the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, or a conservatively modified (e.g., insertion, deletion, substitution, etc.) variant thereof. REKRDSVCPQGKYIHPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCTCHAGFFLRENECVSCSNCKKSLECTKLCL (SEQ ID NO: 1).

[0035] Additionally, the extracellular fragment of the soluble TNF receptor type 1 or a variant thereof described herein retains the ability to specifically recognize and bind to TNFα.

[0036] Conservative modifications herein refer to amino acid modifications that do not significantly affect or alter antibody-binding properties. For example, conservative amino acid substitutions can refer to substitutions with other amino acids of the same class (having similar chemical properties or functions). As an example, amino acids can be grouped according to their side chain properties into: (1) nonpolar amino acids Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar amino acids Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic amino acids Asp (D) and Glu (E); and (4) basic amino acids Lys (K), Arg (R), and His (H). Alternatively, amino acids can be grouped based on common side chain properties: (1) hydrophobic amino acids Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic amino acids Cys, Ser, Thr, Asn, Gln; (3) acidic amino acids Asp, Glu; (4) basic amino acids His, Lys, Arg; (5) amino acids that affect chain orientation Gly, Pro; and (6) aromatic amino acids Trp, Tyr, Phe.

[0037] In the present disclosure, a human IgG Fc fragment is included in the fusion protein to extend half-life. In some embodiments, the human IgG Fc fragment is a human IgG1 Fc or IgG4 Fc fragment.

[0038] In some embodiments, the human IgG Fc fragment is a hinge-CH2-CH3 fragment of IgG4, more preferably a human IgG4 Fc fragment with an S228P mutation.

[0039] In some embodiments, the IgG4 Fc fragment comprises the amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (Sequence ID number: 2).

[0040] In some embodiments, the functional domain that competitively binds to IL-17A or the anti-IL-17A may be selected from a receptor for IL-17A, a variant thereof or a portion thereof, or an anti-IL-17A antibody or antigen-binding fragment thereof.

[0041] In some embodiments, antigen-binding fragments of anti-IL-17A antibodies include, but are not limited to, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a single-chain variable region fragment (scFv), a single-domain antibody (sdAb), an isolated CDR region, or an Fd fragment derived from the antibody.

[0042] In some embodiments, the functional domain that competitively binds to IL-17A or anti-IL-17A can be an anti-IL-17A single chain variable region fragment.

[0043] In some embodiments, the anti-IL-17A single chain variable region fragment comprises: a heavy chain CDR1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 5; a heavy chain CDR2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 6; a heavy chain CDR3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 7; a light chain CDR1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 8; a light chain CDR2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 9; and a light chain CDR3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 10.

[0044] In some embodiments, the anti-IL-17A single-chain variable region fragment comprises a heavy chain CDR1 (HCDR1) described in SEQ ID NO: 5, a heavy chain CDR2 (HCDR2) described in SEQ ID NO: 6, and a heavy chain CDR3 (HCDR3) described in SEQ ID NO: 7, and a light chain CDR1 (LCDR1) described in SEQ ID NO: 8, a light chain CDR2 (LCDR2) described in SEQ ID NO: 9, and a light chain CDR3 (LCDR3) described in SEQ ID NO: 10. GYSFTDYHIH (SEQ ID NO: 5); VINPMYGTTDYNQRFKG (SEQ ID NO: 6); YDYFTGTGVY (SEQ ID NO: 7); RSSRSLVHSRGNTYLH (SEQ ID NO: 8); KVSNRFI (SEQ ID NO: 9); SQSTHLPFT (sequence ID number 10).

[0045] The above CDR ranges are defined according to the Kabat numbering scheme (Kabat et al., Ann. NY Acad. Sci. 190:382-93 (1971) and Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242 (1991)), but it will be understood by those skilled in the art that CDR sequences numbered according to any one or more numbering schemes, such as the Chothia numbering scheme, the ImMunoGenTics (IMGT) numbering scheme, the AbM numbering scheme, and the Contact numbering scheme (based on analysis of available complex crystal structures), also fall within the scope of protection of the present application.

[0046] In some embodiments, the anti-IL-17A single chain variable region fragment comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAPGQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSS (SEQ ID NO: 11); DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWYLQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEIKRT (SEQ ID NO: 12).

[0047] In some embodiments, the anti-IL-17A single chain variable region fragment comprises the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto. QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAPGQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWYLQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEIKRT (Sequence ID number: 3).

[0048] Furthermore, variants of the anti-IL-17A single-chain variable region fragments of the present disclosure retain the ability to specifically recognize and bind to IL-17A.

[0049] In some embodiments, a bispecific fusion protein of the present disclosure comprises, from N-terminus to C-terminus, (1) a soluble TNF receptor type 1 or 2 or an extracellular fragment thereof, (2) a human IgG4 Fc fragment, and (3) an anti-IL-17A single chain variable region fragment.

[0050] In a preferred embodiment, a bispecific fusion protein of the present disclosure comprises, from N-terminus to C-terminus, (1) a soluble TNF receptor type 1 or an extracellular fragment thereof, (2) a human IgG4 Fc fragment, and (3) an anti-IL-17A single-chain variable region fragment.

[0051] In a preferred embodiment, the bispecific fusion protein of the present disclosure comprises, in order from N-terminus to C-terminus, (1) an extracellular fragment of a soluble TNF receptor type 1; (2) a human IgG4 Fc fragment; and (3) an anti-IL-17A single-chain variable region fragment comprising a heavy chain CDR1 (HCDR1) set forth in SEQ ID NO:5, a heavy chain CDR2 (HCDR2) set forth in SEQ ID NO:6, and a heavy chain CDR3 (HCDR3) set forth in SEQ ID NO:7, and a light chain CDR1 (LCDR1) set forth in SEQ ID NO:8, a light chain CDR2 (LCDR2) set forth in SEQ ID NO:9, and a light chain CDR3 (LCDR3) set forth in SEQ ID NO:10.

[0052] In a preferred embodiment, a bispecific fusion protein of the present disclosure comprises, from N-terminus to C-terminus, (1) an extracellular fragment of soluble TNF receptor type 1 as set forth in SEQ ID NO:1; (2) a human IgG4 Fc fragment as set forth in SEQ ID NO:2; and (3) an anti-IL-17A single-chain variable region fragment comprising a heavy chain CDR1 (HCDR1) as set forth in SEQ ID NO:5, a heavy chain CDR2 (HCDR2) as set forth in SEQ ID NO:6, and a heavy chain CDR3 (HCDR3) as set forth in SEQ ID NO:7, and a light chain CDR1 (LCDR1) as set forth in SEQ ID NO:8, a light chain CDR2 (LCDR2) as set forth in SEQ ID NO:9, and a light chain CDR3 (LCDR3) as set forth in SEQ ID NO:10.

[0053] In a preferred embodiment, a bispecific fusion protein of the present disclosure comprises, from N-terminus to C-terminus, (1) an extracellular fragment of a soluble TNF receptor type 1 as set forth in SEQ ID NO:1; (2) a human IgG4 Fc fragment as set forth in SEQ ID NO:2; and (3) an anti-IL-17A single chain variable region fragment comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:11 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:12.

[0054] In a preferred embodiment, a bispecific fusion protein of the present disclosure comprises, from N-terminus to C-terminus, (1) an extracellular fragment of a soluble TNF receptor type 1 as set forth in SEQ ID NO:1, (2) a human IgG4 Fc fragment as set forth in SEQ ID NO:2, and (3) an anti-IL-17A single chain variable region fragment as set forth in SEQ ID NO:3.

[0055] In some embodiments, the human IgG Fc fragment competitively binds to IL-17A or is linked to an anti-IL-17A functional domain via a linker peptide.

[0056] In some embodiments, the linker peptide can be a flexible linker peptide or a rigid linker peptide, hi some embodiments, the linker peptide is a GS flexible linker peptide (e.g., a flexible peptide containing GSG repeating units, a flexible peptide containing G4S repeating units, or a flexible peptide containing two thereof), more preferably (G4S)4 (i.e., GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 4)).

[0057] In some embodiments, the bispecific fusion protein comprises, from N-terminus to C-terminus, a soluble TNF receptor type 1 or an extracellular fragment thereof, a human IgG4 Fc fragment, a GS flexible linker peptide, and an anti-IL-17A single chain variable region fragment.

[0058] In a preferred embodiment, a bispecific fusion protein of the present disclosure comprises, from N-terminus to C-terminus, an extracellular fragment of a soluble TNF receptor type 1, a human IgG4 Fc fragment, a GS flexible linker peptide, and an anti-IL-17A single chain variable region fragment comprising a heavy chain CDR1 (HCDR1) set forth in SEQ ID NO:5, a heavy chain CDR2 (HCDR2) set forth in SEQ ID NO:6, and a heavy chain CDR3 (HCDR3) set forth in SEQ ID NO:7, and a light chain CDR1 (LCDR1) set forth in SEQ ID NO:8, a light chain CDR2 (LCDR2) set forth in SEQ ID NO:9, and a light chain CDR3 (LCDR3) set forth in SEQ ID NO:10.

[0059] In a preferred embodiment, a bispecific fusion protein of the present disclosure comprises, from N-terminus to C-terminus, an extracellular fragment of soluble TNF receptor type 1 as set forth in SEQ ID NO:1, a human IgG4 Fc fragment as set forth in SEQ ID NO:2, a GS flexible linker peptide as set forth in SEQ ID NO:4, an anti-IL-17A single chain variable region fragment comprising a heavy chain CDR1 (HCDR1) as set forth in SEQ ID NO:5, a heavy chain CDR2 (HCDR2) as set forth in SEQ ID NO:6, and a heavy chain CDR3 (HCDR3) as set forth in SEQ ID NO:7, and a light chain CDR1 (LCDR1) as set forth in SEQ ID NO:8, a light chain CDR2 (LCDR2) as set forth in SEQ ID NO:9, and a light chain CDR3 (LCDR3) as set forth in SEQ ID NO:10.

[0060] In a preferred embodiment, a bispecific fusion protein of the present disclosure comprises, from N-terminus to C-terminus, an extracellular fragment of soluble TNF receptor type 1 as set forth in SEQ ID NO:1, a human IgG4 Fc fragment as set forth in SEQ ID NO:2, a GS flexible linker peptide as set forth in SEQ ID NO:4, an anti-IL-17A single chain variable region fragment comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:11, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:12.

[0061] Preferably, the bispecific fusion protein comprises, from N-terminus to C-terminus, an extracellular fragment of soluble TNF receptor type 1 as set forth in SEQ ID NO: 1, a human IgG4 Fc fragment as set forth in SEQ ID NO: 2, a GS flexible linker peptide as set forth in SEQ ID NO: 4, and an anti-IL-17A single chain variable region fragment as set forth in SEQ ID NO: 3. The bispecific fusion protein can specifically recognize and bind to IL-17A and TNFα.

[0062] In some preferred embodiments, the polypeptide chains of the fusion protein form homodimers through disulfide bonds between the Fc fragments therein. In the present disclosure, a soluble TNF receptor or a portion thereof is linked to the hinge region of an IgG Fc fragment, and the function of the soluble TNF receptor or a portion thereof is ensured through the flexibility of the hinge region, and the fusion protein is ensured to form a dimer through the disulfide bonds generated in the hinge region.

[0063] In some preferred embodiments, the fusion protein is a symmetric dimer.

[0064] In some embodiments, the fusion proteins described herein have a potency of 9×10 for IL-17A. -12 M or less, preferably 7 x 10 -12 M or less, preferably 6.5 × 10 -12 It has an affinity constant (KD) of 4 × 10 for TNFα -11 M or less, preferably 3.8 × 10 -11 M or less, preferably 3.6 × 10 -11 It has an affinity constant (KD) of less than or equal to M.

[0065] The fusion proteins of the present application can be formulated into any dosage form suitable for administration to a subject, for example, the fusion proteins can be administered intravenously, intramuscularly, parenterally, orally, subcutaneously, intrathecally, intracerebroventricularly, intraspinally, intraperitoneally, intrauterinely, intranasally, etc. In some preferred embodiments, the fusion proteins are in the form of a lyophilized powder or an injectable solution.

[0066] In one embodiment, the present application provides a polynucleotide capable of encoding the bispecific fusion protein.

[0067] In one embodiment, the present application provides a recombinant vector comprising a polynucleotide capable of encoding a fusion protein.

[0068] In the present disclosure, any expression vector known in the art can be used to construct a recombinant vector with the polynucleotide encoding the fusion protein. The expression vector can be a pcDNA series vector (pcDNA3.1 vector, pcDNA3.2 vector, pcDNA3.3 vector, pcDNA3.4 vector (e.g., pcDNA3.4-TOPO TA vector)), pBK-CMV vector, pEGFP-N1 vector, or pGenHT1.0-DGV vector.

[0069] In one embodiment, the present application provides a host cell that contains the above polynucleotide or recombinant vector and thereby expresses the bispecific fusion protein described herein.

[0070] In a preferred embodiment, the host cell expressing the fusion protein is a prokaryotic or eukaryotic cell, more preferably a mammalian cell such as an HEK293 cell (e.g., a 293T cell, a 293F cell, a 293H cell, or a 293S cell), a CHO cell (e.g., a CHOK1-GenS cell), a BHK cell, or an Sp2 / 0 cell.

[0071] The above polynucleotides or recombinant vectors can be transferred into host cells by any suitable technique known in the art for introducing exogenous genes into host cells (e.g., electroporation, transduction, transfection), such that the resulting host cells are capable of efficiently expressing the bispecific fusion proteins of the present application.

[0072] In the present disclosure, culturing of cells into which exogenous genes have been introduced can be performed by those skilled in the art by selecting conventional media and culture conditions based on the type of cells (e.g., "Cell Culture (3rd Edition)," editor-in-chief Liu Bin, World Book Publishing Company, January 2018; "Cell Culture Technology," editors-in-chief Lan Rong and Zhou Zhenhui, Chemical Industry Publishing House, August 2007; and "Tissue and Cell Culture Technology (3rd Edition)," editor-in-chief Zhang Jingbo, People's Health Publishing House, June 2014).

[0073] In one embodiment, the present application provides a method for preparing a bispecific fusion protein, comprising: a) preparing a polynucleotide encoding the bispecific fusion protein; b) constructing a recombinant vector using the polynucleotide and an expression vector; c) transferring the recombinant vector into host cells and culturing the transformed cells to obtain a cell culture; d) purifying and isolating the cell culture to obtain the bispecific fusion protein.

[0074] In the present disclosure, polynucleotides can be synthesized according to the amino acid sequence of the corresponding fusion protein by techniques such as conventional total gene synthesis or enzymatic synthesis.

[0075] In the present disclosure, recombinant vectors can be constructed from polynucleotides and expression vectors through conventional enzymatic cleavage and ligation.

[0076] As described above, the transformed cells can be cultured by those skilled in the art by selecting conventional media and culture conditions based on the cell type. In some embodiments, the cells are cultured in a fed-batch or perfusion culture mode, and more preferably, the cells are cultured in a high-density fed-batch or perfusion culture mode.

[0077] Herein, the resulting cell culture may be purified by conventional protein purification methods, including, but not limited to, electrophoresis, ultracentrifugation, dialysis, ultrafiltration, precipitation, chromatography (e.g., gel filtration chromatography (e.g., size exclusion chromatography (SEC)), ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, reversed-phase chromatography, high-performance liquid chromatography, and mixed-mode chromatography (combined layering)). In some preferred embodiments, purification is performed using affinity chromatography and mixed-mode chromatography; for example, purification may be performed using a Protein A affinity column. In a further preferred embodiment, aggregates of the fusion protein are removed using a hydroxyapatite (CHT) composite packing.

[0078] In one embodiment, the present application provides a formulation comprising the bispecific fusion protein described above, further comprising a buffer system, a pharmaceutically acceptable excipient, and a surfactant, wherein the buffer system is selected from a solution of acetic acid and sodium acetate, a solution of citric acid and sodium citrate, or a solution of histidine and histidine hydrochloride.

[0079] In some embodiments, the formulation has a pH value of 3.0 to 7.5, preferably a pH value of 3.5 to 6.5, and more preferably a pH value of 4.0 to 5.0.

[0080] In some embodiments, the formulation comprises 5 mg / mL to 100 mg / mL of bispecific fusion protein, 5 mM to 50 mM (e.g., 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, or 50 mM) of a buffer system, 50 mg / mL to 100 mg / mL of a pharmaceutically acceptable excipient, and 0.01% to 0.05% (w / v) of a surfactant.

[0081] In some embodiments, the buffer system is selected from a 5 mM to 20 mM (e.g., 10 mM) solution of acetic acid and sodium acetate, a 5 mM to 20 mM (e.g., 10 mM) solution of citric acid and sodium citrate, and a 5 mM to 20 mM (e.g., 10 mM) solution of histidine and histidine hydrochloride, more preferably a 10 mM solution of acetic acid and sodium acetate.

[0082] In some embodiments, the pharmaceutically acceptable excipient is at least one of sucrose or trehalose, more preferably trehalose.

[0083] In some embodiments, the surfactant is a non-ionic surfactant, such as a Span surfactant, a polysorbate surfactant (eg, polysorbate-20, polysorbate-60, polysorbate-80).

[0084] In one embodiment, the present application provides a pharmaceutical composition comprising the bispecific fusion protein.

[0085] In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be selected from, but is not limited to, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, disintegrants, fillers, lubricants, wetting agents, osmotic pressure adjusters, stabilizers, fluidizers, flavoring agents, preservatives, suspending agents, antioxidants, penetration enhancers, pH adjusters, surfactants, diluents, etc. Other available pharmaceutically acceptable pharmaceutical excipients can be found, for example, in "Handbook of Pharmaceutical Excipients" (4th Edition) edited by RC Rowe et al. (translated by Zheng Zemin, 2005, Chemical Industry Press).

[0086] In one embodiment, the present application provides use of the bispecific fusion protein, or the formulation or pharmaceutical composition comprising the same, in the preparation of a medicament for treating an inflammation-related disease. Alternatively, the present application provides use of the bispecific fusion protein, or the formulation or pharmaceutical composition comprising the same, in the preparation of a reagent for binding and inhibiting TNF-α and IL-17A.

[0087] In some preferred embodiments, the reagents are used to block the TNF and IL-17 signaling pathways, including inhibiting the synergistic effects caused by these two pathways.

[0088] In another preferred embodiment, the agent is used to block the interaction of TNF with the TNFR complex and / or the interaction of IL-17 with the IL-17 receptor complex.

[0089] In one embodiment, the present application relates to a method for treating an inflammation-related disease, comprising administering the bispecific fusion protein, or the formulation or pharmaceutical composition comprising same, to a subject in need thereof. Alternatively, the present application provides the bispecific fusion protein, formulation, or pharmaceutical composition for use in treating an inflammation-related disease. Alternatively, the present application provides a method for binding and inhibiting TNF-α and IL-17A, comprising administering the bispecific fusion protein, or the formulation or pharmaceutical composition comprising same, to a subject in need thereof. Alternatively, the present application provides the bispecific fusion protein, or the formulation or pharmaceutical composition comprising same, for use as a reagent for binding and inhibiting TNF-α and IL-17A.

[0090] In some embodiments, the subject is a mammal. In preferred embodiments, the mammal is a human.

[0091] In some embodiments, the inflammation-related disease is an autoimmune disease or cytokine release syndrome, more preferably rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, ulcerative colitis, a viral infection, or cytokine release syndrome caused by an immunomodulatory drug.

[0092] The dosage, frequency of administration, and route of administration of the bispecific fusion protein or the formulation or pharmaceutical composition containing it can be determined by a clinician according to the patient's weight, sex, age, medical condition, physical health condition, etc.

[0093] The exemplary technical solutions of the present application can be explained through the contents of the following numbered paragraphs.

[0094] 1.From the N-terminus to the C-terminus, a soluble TNF receptor or a portion thereof; a human IgG Fc fragment; A bispecific fusion protein comprising a functional domain that competitively binds to IL-17A or an anti-IL-17A functional domain.

[0095] 2. The bispecific fusion protein of paragraph 1, wherein the soluble TNF receptor or portion thereof is a soluble TNF receptor type 1 or portion thereof, or a soluble TNF receptor type 2 or portion thereof.

[0096] 3. The bispecific fusion protein of paragraph 2, wherein the soluble TNF receptor or portion thereof is a soluble TNF receptor type 1 or portion thereof.

[0097] 4. The bispecific fusion protein of any one of paragraphs 1 to 3, wherein the soluble TNF receptor or portion thereof is an extracellular fragment of the soluble TNF receptor type 1.

[0098] 5. The bispecific fusion protein of paragraph 4, wherein the extracellular fragment of the soluble TNF receptor type 1 comprises the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 95% identity thereto, or a conservatively modified variant thereof.

[0099] 6. The bispecific fusion protein of any one of paragraphs 1 to 5, wherein the human IgG Fc fragment is a human IgG1 Fc or a human IgG4 Fc fragment.

[0100] 7. The bispecific fusion protein of any one of paragraphs 1 to 6, wherein the human IgG Fc fragment is the hinge-CH2-CH3 fragment of IgG4.

[0101] 8. The bispecific fusion protein of any one of paragraphs 1 to 7, wherein the human IgG Fc fragment is a human IgG4 Fc fragment with an S228P mutation.

[0102] 9. The bispecific fusion protein of any one of paragraphs 1 to 8, which competitively binds to IL-17A or wherein the anti-IL-17A functional domain is selected from a receptor for IL-17A, a variant thereof or part thereof, or an anti-IL-17A antibody or antigen-binding fragment thereof.

[0103] 10. The bispecific fusion protein of paragraph 9, wherein the antigen-binding fragment of the anti-IL-17A antibody comprises a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a single-chain variable region fragment, a single domain antibody, an isolated CDR region, or an Fd fragment of the anti-IL-17A antibody.

[0104] 11. The bispecific fusion protein of any one of paragraphs 1 to 9, which competitively binds to IL-17A or wherein the anti-IL-17A functional domain is an anti-IL-17A single chain variable region fragment.

[0105] 12. A bispecific fusion protein described in paragraph 11, wherein the anti-IL-17A single chain variable region fragment comprises an HCDR1 described in SEQ ID NO:5, an HCDR2 described in SEQ ID NO:6, and an HCDR3 described in SEQ ID NO:7, and an LCDR1 described in SEQ ID NO:8, an LCDR2 described in SEQ ID NO:9, and an LCDR3 described in SEQ ID NO:10.

[0106] 13. The bispecific fusion protein of paragraph 11 or 12, wherein the anti-IL-17A single chain variable region fragment comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11, or an amino acid sequence having at least 80% identity thereto, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12, or an amino acid sequence having at least 80% identity thereto.

[0107] 14. The bispecific fusion protein of any one of paragraphs 11 to 13, wherein the anti-IL-17A single chain variable region fragment comprises the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 80% identity thereto.

[0108] 15. The bispecific fusion protein of paragraph 1 or 2, comprising, in order from N-terminus to C-terminus, (1) a soluble TNF receptor type 1 or 2 or an extracellular fragment thereof, (2) a human IgG4 Fc fragment, and (3) an anti-IL-17A single chain variable region fragment.

[0109] 16. The bispecific fusion protein of paragraph 15, comprising, in order from N-terminus to C-terminus, (1) a soluble TNF receptor type 1 or an extracellular fragment thereof, (2) a human IgG4 Fc fragment, and (3) an anti-IL-17A single chain variable region fragment.

[0110] 17. A bispecific fusion protein according to paragraph 16, comprising, in order from N-terminus to C-terminus, (1) an extracellular fragment of soluble TNF receptor type 1 as set forth in SEQ ID NO: 1; (2) a human IgG4 Fc fragment as set forth in SEQ ID NO: 2; and (3) an anti-IL-17A single chain variable region fragment comprising an HCDR1 as set forth in SEQ ID NO: 5, an HCDR2 as set forth in SEQ ID NO: 6, and an HCDR3 as set forth in SEQ ID NO: 7, and an LCDR1 as set forth in SEQ ID NO: 8, an LCDR2 as set forth in SEQ ID NO: 9, and an LCDR3 as set forth in SEQ ID NO: 10.

[0111] 18. The bispecific fusion protein of paragraph 17, comprising, in order from N-terminus to C-terminus, (1) an extracellular fragment of a soluble TNF receptor type 1 as set forth in SEQ ID NO: 1; (2) a human IgG4 Fc fragment as set forth in SEQ ID NO: 2; and (3) an anti-IL-17A single chain variable region fragment comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12.

[0112] 19. The bispecific fusion protein of paragraph 18, comprising, in order from N-terminus to C-terminus, (1) an extracellular fragment of a soluble TNF receptor type 1 as set forth in SEQ ID NO: 1, (2) a human IgG4 Fc fragment as set forth in SEQ ID NO: 2, and (3) an anti-IL-17A single chain variable region fragment as set forth in SEQ ID NO: 3.

[0113] 20. The bispecific fusion protein of any one of paragraphs 1 to 9, wherein the human IgG Fc fragment competitively binds to IL-17A or is linked to an anti-IL-17A functional domain via a linker peptide.

[0114] 21. The bispecific fusion protein of paragraph 20, wherein the linker peptide is a GS flexible linker peptide.

[0115] 22. A bispecific fusion protein according to any one of paragraphs 1 to 3, comprising, in order from N-terminus to C-terminus, a soluble TNF receptor type 1 or an extracellular fragment thereof, a human IgG4 Fc fragment, a GS flexible linker peptide, and an anti-IL-17A single chain variable region fragment.

[0116] 23. A bispecific fusion protein according to paragraph 22, comprising, in order from N-terminus to C-terminus, an extracellular fragment of soluble TNF receptor type 1 as set forth in SEQ ID NO: 1, a human IgG4 Fc fragment as set forth in SEQ ID NO: 2, a GS flexible linker peptide as set forth in SEQ ID NO: 4, an anti-IL-17A single chain variable region fragment comprising an HCDR1 as set forth in SEQ ID NO: 5, an HCDR2 as set forth in SEQ ID NO: 6, and an HCDR3 as set forth in SEQ ID NO: 7, and an LCDR1 as set forth in SEQ ID NO: 8, an LCDR2 as set forth in SEQ ID NO: 9, and an LCDR3 as set forth in SEQ ID NO: 10.

[0117] 24. A bispecific fusion protein according to paragraph 23, comprising, in order from N-terminus to C-terminus, an extracellular fragment of soluble TNF receptor type 1 as set forth in SEQ ID NO: 1, a human IgG4 Fc fragment as set forth in SEQ ID NO: 2, a GS flexible linker peptide as set forth in SEQ ID NO: 4, a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12.

[0118] 25. A bispecific fusion protein according to paragraph 24, comprising, in order from N-terminus to C-terminus, an extracellular fragment of soluble TNF receptor type 1 as set forth in SEQ ID NO: 1, a human IgG4 Fc fragment as set forth in SEQ ID NO: 2, a GS flexible linker peptide as set forth in SEQ ID NO: 4, and an anti-IL-17A single chain variable region fragment as set forth in SEQ ID NO: 3.

[0119] 26. The fusion protein inhibited IL-17A by 9 × 10 -12 Affinity constant KD of ≤ M and 4 × 10 for TNFα -11 26. The bispecific fusion protein of any one of paragraphs 1 to 25, having an affinity constant KD of less than or equal to M.

[0120] 27. A polynucleotide encoding a bispecific fusion protein according to any one of paragraphs 1 to 26.

[0121] 28. A recombinant vector comprising a polynucleotide according to paragraph 27.

[0122] 29. A host cell comprising a polynucleotide according to paragraph 27 or a recombinant vector according to paragraph 28.

[0123] 30. A method for preparing a bispecific fusion protein according to any one of paragraphs 1 to 26, comprising: a) preparing a polynucleotide encoding a bispecific fusion protein; b) constructing a recombinant vector using the polynucleotide and an expression vector; c) transferring the recombinant vector into host cells and culturing the transformed cells to obtain a cell culture; d) purifying and isolating the cell culture to obtain the bispecific fusion protein.

[0124] 31. A formulation comprising the bispecific fusion protein of any one of paragraphs 1 to 26, further comprising a buffer system, a pharmaceutically acceptable excipient, and a surfactant, wherein the buffer system is selected from a solution of acetic acid and sodium acetate, a solution of citric acid and sodium citrate, or a solution of histidine and histidine hydrochloride.

[0125] 32. The formulation according to paragraph 31, wherein the formulation has a pH value of 3.0 to 7.5.

[0126] 33. The formulation of paragraph 31 or 32, wherein the formulation comprises 5 mg / mL to 100 mg / mL of the bispecific fusion protein, 5 mM to 50 mM of a buffer system, 50 mg / mL to 100 mg / mL of a pharmaceutically acceptable excipient, and 0.01% to 0.05% (w / v) of a surfactant.

[0127] 34. The formulation of any one of paragraphs 31 to 33, wherein the buffer system is selected from a solution of 5 mM to 20 mM acetic acid and sodium acetate, a solution of 5 mM to 20 mM citric acid and sodium citrate, or a solution of 5 mM to 20 mM histidine and histidine hydrochloride.

[0128] 35. The formulation of any one of paragraphs 31 to 34, wherein the pharmaceutically acceptable excipient is at least one of sucrose or trehalose.

[0129] 36. A formulation according to any one of paragraphs 31 to 35, wherein the surfactant is a non-ionic surfactant.

[0130] 37. A pharmaceutical composition comprising a bispecific fusion protein according to any one of paragraphs 1 to 26.

[0131] 38. Use of a bispecific fusion protein according to any one of paragraphs 1 to 26, a formulation according to any one of paragraphs 31 to 36, or a pharmaceutical composition according to paragraph 37 in the preparation of a medicament for treating an inflammation-related disease.

[0132] 39. The use according to paragraph 38, wherein the inflammation-related disease is an autoimmune disease or cytokine release syndrome.

[0133] 40. The use according to paragraph 39, wherein the inflammation-related disease is rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, ulcerative colitis, a viral infection, or cytokine release syndrome caused by an immunomodulatory drug.

[0134] 41. Use of a bispecific fusion protein according to any one of paragraphs 1 to 26, a formulation according to any one of paragraphs 31 to 36, or a pharmaceutical composition according to paragraph 37 in the preparation of a reagent for binding and inhibiting TNF-α and IL-17A.

[0135] For purposes of illustration and disclosure, all patents, patent applications, and other identified publications are expressly incorporated by reference in this disclosure. These publications are provided solely because their disclosure is prior to the filing date of this application. Any statement as to the date of these documents or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates of these documents or the contents of these documents. Furthermore, any reference to these publications in this disclosure does not constitute an admission that the publications are part of the common knowledge in the art in any country. [Example]

[0136] Hereinafter, the solution of the present application will be explained in more detail with the help of examples, but it is understood by those skilled in the art that the protection scope of the present application is not limited thereto.

[0137] Unless otherwise specified, various reagents, materials, and equipment used in the following examples are commercially available. Unless otherwise specified, molecular biology techniques involved in the following examples can be found, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Spring Harbor, NY, 1989).

[0138] Example 1: Design, expression, and purification of fusion protein constructs Based on the sequences of the extracellular truncated regions of sTNFRI or sTNFRII and anti-IL-17A scFv or IL-17A Fab disclosed in the NCBI database, eight constructs were obtained by fusion via the Fc fragment (S228P) of IgG4. For ease of explanation, the proteins are numbered sequentially hereafter and referred to as proteins No. 1 to No. 8. The odd-numbered proteins (i.e., Nos. 1, 3, 5, and 7) are constructs containing sTNFRI, and the even-numbered proteins (i.e., Nos. 2, 4, 6, and 8) are constructs containing sTNFRII, which correspond to the four structures in Figure 1. The constructs of each fusion protein are shown in Figure 1, and the sequences used are as follows:

[0139] The amino acid sequence of sTNFRI is set forth in SEQ ID NO:1. The amino acid sequence of sTNFRII is as follows: LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGD (Sequence ID number 13). The amino acid sequence of the anti-IL-17A scFv is set forth in SEQ ID NO:3. The amino acid sequence of the anti-IL-17A Fab is as follows: Heavy chain: QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAPGQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV (SEQ ID NO: 14). Light chain: DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWYLQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID number 15). The amino acid sequence of the IgG4 Fc fragment (S228P) is set forth in SEQ ID NO:2.

[0140] The eight proteins were transiently expressed using the Expi293 Expression System (purchased from Thermo Fisher Scientific) according to the manufacturer's instructions, and the expressed products were purified by Protein A affinity chromatography (Praesto Jetted A50 resin, equilibration: 50 mM Tris-HAc, 110 mM NaCl, pH 7.2, elution: 50 mM NaAc-HAc, pH 3.0). The eight proteins were analyzed by SDS-PAGE (reducing / non-reducing), and the results are shown in Figure 2.

[0141] After Protein A affinity chromatography was completed for all eight proteins, through SEC-HPLC (column: TSKgel® G3000SWXL, mobile phase A: 100 mM PB, 100 mM NaSO, pH 6.7±0.1, mobile phase B: 100% HO, flow rate: 0.7 mL / min, injection volume: 1.5 μL) analysis, it was found that, except for protein No. 7, which had higher purity, there were different proportions of aggregates and degradation products in the other proteins, as shown in Figure 3.

[0142] Example 2: Affinity evaluation of eight structural proteins To further evaluate the eight proteins obtained in Example 1, except for protein No. 7, whose purity was sufficient for affinity analysis, the remaining proteins were subjected to a second purification step using molecular exclusion chromatography (resin: Sephadex G-25, buffer: PB) to obtain proteins with further improved purity. The sample amounts, concentrations, and purities of the eight proteins after purification are shown in Table 1. [Table 1]

[0143] The antigen affinity of the above eight structural proteins was analyzed by SPR technology (Biacore T200) and compared with that of the positive control drugs Humira, etanercept, and Cosentyx.

[0144] The eight proteins and control drugs Humira, etanercept, and Cosentyx were each bound to a Biacore T200 protein A sensor chip via their Fc fragments. Different concentrations of TNF or IL-17A (200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, and 0 nM) were then used as analytes to detect the affinities of the different proteins and control drugs for these two antigens using a multi-cycle assay. The results showed that the affinity of seven proteins (proteins 1, 2, 3, 4, 5, 6, and 8) for TNF-α was one order of magnitude higher than that of Humira (anti-TNF mAb) and close to that of etanercept (TNF receptor type 2), consistent with theoretical predictions. Protein 7 was close to that of Humira, and the affinity of the eight proteins for IL-17A was one order of magnitude higher than that of Cosentyx. The results of the antigen affinity assay are shown in Table 2. [Table 2]

[0145] Because the fusion protein is a dual-target protein, it is necessary to consider whether the two targets affect the binding of the antigen (TNFα or IL-17A). Therefore, a follow-up test was conducted using a Biacore T200. Specifically, a Protein A chip was used to capture different proteins and positive control drugs. One antigen was then used to bind a desaturated protein or control drug to the antigen. Furthermore, another antigen at different concentrations (200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 0 nM) was used to detect the affinity of the protein or positive control drug for the other antigen in this situation. The results are shown in Tables 3 and 4. The results showed that when one antigen-binding domain was saturated, the affinity of the eight proteins obtained in Example 1 of this application for binding to the other antigen was not affected. [Table 3] [Table 4]

[0146] Combining the expression and purification results of the eight proteins assayed in Example 1 with the affinity evaluation results described above in Example 2, the chip response values ​​of different proteins and control drugs for binding to antigen molecules were used to estimate whether each protein or control drug could guarantee a binding ratio to TNF and IL-17A. Theoretically, the binding ratio to TNF and IL-17A is 1:2. The molar binding ratio (Ratio) of the eight fusion proteins and positive control drugs prepared in Example 1 with the same molar number of binding to the antigens TNFα and IL-17A was estimated according to the chip response values ​​in the following formula:

number

[0147] In the above formula, analyte represents the analyte to be analyzed, i.e., TNFα or IL-17A, ligand represents the protein immobilized on the chip or the positive control drug, i.e., the eight proteins and the positive control antibody, analyte MW represents the relative molecular weight of the analyte, and ligand MW represents the relative molecular weight of the protein immobilized on the chip or the positive control drug.

[0148] The response values ​​of the above eight proteins and control drugs to antigen binding are shown in Figure 4. Based on the sample amount, purity, and affinity of the proteins in Examples 1 and 2, proteins No. 5, 6, and 8 were selected for subsequent mass spectrometry detection.

[0149] Example 3: Analysis of mass spectrometry detection of No. 5, 6, and 8 proteins After freezing and thawing at -80°C, SDS-PAGE analysis revealed that numerous degradation bands appeared in proteins No. 6 and No. 8, as shown in Figure 5. Mass spectrometry detection revealed that only protein No. 5 had a complete main peak, while proteins No. 6 and No. 8 had unknown peaks, and their actual molecular weights were significantly different from the theoretical values, as shown in Table 5. Therefore, protein No. 5 was selected as a candidate molecule for further experiments. A schematic diagram of the construction of protein No. 5 is shown in Figure 6. [Table 5]

[0150] Example 4: Transient transfection expression of No. 5 protein in CHO cells The transient expression of the protein described above was performed using the Expi293 expression system, so the final product of the protein expression may differ from the product expressed by CHO cells. According to the manufacturer's instructions, the No. 5 protein was expressed and purified using the ExpiCHO expression system (purchased from Thermo Fisher Scientific). The resulting protein was used for pharmacodynamic studies and pharmaceutical suitability analysis. Table 6 shows the relevant information of the final protein, and Figure 7 shows the SEC-HPLC and SDS-PAGE results of the final protein. [Table 6]

[0151] Example 5: Pharmacodynamic evaluation of No. 5 protein The protein obtained in Example 4 was used for pharmacodynamic studies of the fusion protein. First, the affinity of the No. 5 fusion protein for mouse-derived TNF-α (mTNFα) and mouse-derived IL-17A (mIL-17A) was detected. The results showed that the No. 5 fusion protein bound to mTNFα with an affinity constant of 1.630E-10 M, but not to mIL-17A. Therefore, it was necessary to use hIL-17A transgenic mice for the pharmacodynamic studies.

[0152] hIL-17A transgenic DBA / 1 mice were used, with free access to water and food. After 3–5 days of dietary adaptation, mice were randomly divided into control and model groups according to body weight. Model group mice received a bovine type II collagen (Chondrex) emulsion containing complete Freund's adjuvant via subcutaneous injection into the base of the tail. 21 days after the primary immunization, a bovine type II collagen emulsion containing incomplete Freund's adjuvant was used for the secondary immunization. The success rate of mouse modeling reached 100% 42–56 days after the primary immunization. 30 days after the primary immunization, mice were divided into the model group (receiving PB buffer) (n=4), the blank group (n=3), the No. 5 fusion protein group (n=5), and the positive control drug Cosentyx group (n=4). To verify efficacy, the model mice with the most severe scores were divided into groups and placed in the fusion protein group, and the inhibitory effect of the fusion protein on the model mice in that group was observed.

[0153] Treatment began on the day of grouping. Mice were administered 80 μL of 1.1 mg / mL No. 5 fusion protein or 1.1 mg / mL Cosentyx once every 48 hours via intraperitoneal injection at a dose of 3 mg / kg. The degree of joint swelling in the mice was scored before each administration, and the scoring criteria are shown in Table 7 below. [Table 7]

[0154] Photographs were taken for documentation and the change curves were plotted. The treatment period was 28 days, with a total of 14 doses. The results of the joint scoring of the mice are shown in Figure 8.

[0155] In the groups administered No. 5 protein or Cosentyx, the No. 5 fusion protein had a significant inhibitory effect on rheumatoid arthritis in mice, and the inhibitory effect was better than that of the positive control drug Cosentyx. The inhibition rate of rheumatoid arthritis was calculated according to the following formula: T = final score of treatment group - initial score of treatment group C = final score of model group - initial score of model group Rheumatoid arthritis inhibition rate = (1-T / C)*100%

[0156] The inhibition rate of rheumatoid arthritis in each administration group was calculated and was as follows: Inhibition rate of No. 5 fusion protein: 74%, inhibition rate of Cosentyx: 40% (photographs of the paws of mice from each group are shown in Figure 9).

[0157] Samples were collected from the paws of mice in each group, and MicroCT analysis was performed to score the degree of bone damage in the paws of mice in each group. Three technicians scored them consecutively according to the criteria in Table 7, and the average was calculated. The MicroCT results of the paws and scores of mice in each group are shown in Figures 10 and 11. The results showed that the No. 5 fusion protein could significantly alleviate bone joint damage, and the effect of Cosentyx was also good (partly because the rheumatoid arthritis scores of mice in this treatment group were lower than those of the No. 5 protein treatment group in the early stage).

[0158] Example 6: Analysis of physicochemical properties of No. 5 fusion protein (1) Differential scanning fluorometry (DSF) Differential scanning fluorimetry is a method for assessing protein thermal stability by slowly heating a sample on a fluorescent quantitative PCR instrument and detecting the amount of fluorescent dye bound to the protein, which undergoes structural changes during the heating process. Differential scanning fluorimetry was used to assay No. 5 protein, and a 1 mg / mL concentration of No. 5 protein was detected in two wells. The average Tm was 68.6 °C. The assay results are shown in Table 8. The sample had good thermal stability, but fluorescence was observed at the beginning of detection, indicating the exposure of patches of hydrophobic groups on the protein surface or the presence of protein misfolding. [Table 8]

[0159] (2) Detection of FcRn binding ability The affinity of No. 5 protein for FcRn at pH 7.4 was detected using a Protein A chip via SPR technology. After analysis, the affinity constant was found to be 2.52E-06 nM, which is within the normal range of the affinity of IgG4 subtype antibodies for FcRn. This indicates that No. 5 protein can extend the half-life of fusion proteins in the body through Fc.

[0160] (3) Detection of plasma stability of No. 5 protein by ELISA The No. 5 protein was incubated in human plasma at 37°C for 0 days (i.e., 1 day before incubation), 1 day, 4 days, 7 days, and 14 days, and then the binding ability of the No. 5 protein to hTNF-α and hIL-17A was detected by ELISA (the No. 5 protein was used as the primary antibody, and HRP-labeled anti-human Fc was used as the secondary antibody). The results showed that the EC50 values ​​after 1, 4, 7, and 14 days of incubation were almost unchanged from the EC50 value on day 0, indicating that the No. 5 protein was stable in human plasma.

[0161] (4) Dynamic Light Scattering (DLS) Test Dynamic light scattering experiments were used to detect the distribution of proteins of different sizes in a solution of No. 5 protein at a concentration of 1.10 mg / mL in PBS buffer (pH 7.4). The solution of No. 5 protein in PBS buffer was tested twice. Throughout the test, the PD% (relative dispersion) of the solution was greater than 15%, indicating that oligomers of No. 5 protein were present in the solution. The results are shown in Table 9. [Table 9]

[0162] (5) Detection of freeze-thaw stability of No. 5 protein The No. 5 protein sample on day 0 (i.e., 1 day before incubation) was freeze-thawed once below -60°C after purification. Other samples were incubated at 40°C (for a total of 14 days of incubation and detected on days 1, 4, 7, and 14) or subjected to three freeze-thaw cycles (below -60°C). Through A280 detection, the concentrations of each sample were approximately equivalent, and the sample appeared colorless and free of particles. The presence of high molecular weight components (aggregates) was detected in all samples by SEC-HPLC (chromatography column: TSKgel® G3000SWXL; mobile phase A: 100 mM PB, 100 mM Na2SO4, pH 6.7 ± 0.1; mobile phase B: 100% HO; flow rate: 0.7 mL / min; injection volume: 1.5 μL). It is noteworthy that after incubation at 40°C, the high molecular weight components were reduced and the monomer components were increased. The freeze-thaw stability detection results are shown in Table 10. The results showed that No. 5 protein had a certain freeze-thaw stability. [Table 10]

[0163] Example 7: Pharmaceutical Formulation Screening In previous experiments, it was found that the candidate molecule may produce aggregates after freeze-thawing. Therefore, in order to further improve the freeze-thaw stability of the fusion protein in this application, further reduce the production of aggregates, and thus further improve its pharmaceutical suitability, different formulations and pH values ​​suitable for No. 5 protein were investigated in this example.

[0164] The pH range of the buffer system (4.5-7.5) was designed based on the theoretical isoelectric point of the No. 5 fusion protein (8.07). Meanwhile, the effects of different types of buffer systems (acetate, histidine, citrate, and phosphate) on protein stability were investigated. The types and amounts of protein, excipients, and surfactants added to each candidate formulation were the same. Three to six optimal formulations were screened for freeze-thaw testing by examining the appearance and visible foreign matter, as well as DLS and DSC, to compare differences in particle, colloidal, and thermal stability of the candidate molecules in the different candidate formulations. The specific protocol is shown in Table 11. [Table 11]

[0165] After detection, the results of the rapid screening study of the above formulations showed that the kD values ​​of B-1 to B-3 and B-7 to B-9 were greater than 0 in the samples of each candidate formulation, indicating that the sample molecules of each formulation were mainly repulsive to each other, with B-1 showing the best performance; B-1 to B-3 and B-7 to B-10 had higher Tagg (initial aggregation temperature) values ​​and relatively high thermal stability; B-2, B-3, B-5, and B-6 had higher Tonset (initial denaturation temperature) values ​​and relatively high thermal stability; and the appearance and particle condition of the samples of formulations B-1 and B-8 were relatively good.

[0166] In summary, B-1 to B-3 and B-7 to B-8 were more advantageous in maintaining the stability of the present fusion protein No. 5. These five formulations were finally and preferably selected as candidate formulation systems. In addition, an experimental group of "buffer system + sucrose" was added to the B-1 formulation, which showed the best performance (10 mM acetic acid / sodium acetate buffer (pH 4.5), 80 mg / mL sucrose, 0.02% polysorbate 80). The above candidate formulation systems were examined by freeze-thawing. The experimental protocol is shown in Table 12. [Table 12]

[0167] The freeze-thaw study results showed that compared with other candidate formulation systems, formulation E-1 (10 mM acetic acid / sodium acetate buffer, 87 mg / mL trehalose, and 0.02% (w / v) polysorbate 80, pH 4.5) was more advantageous for maintaining the stability of the present fusion protein and had the best overall performance.

[0168] Those skilled in the art will recognize that the scope of the present application is not limited to the various specific embodiments and examples described above, and that various modifications, substitutions, or rearrangements may be made without departing from the spirit of the present application, and such adapted embodiments will fall within the protection scope of the present application.

Claims

1. From the N-terminus to the C-terminus, a soluble TNF receptor or a portion thereof; a human IgG Fc fragment; and and a functional domain that competitively binds to IL-17A or is anti-IL-17A.

2. the soluble TNF receptor or a portion thereof is a soluble TNF receptor type 1 or a portion thereof, or a soluble TNF receptor type 2 or a portion thereof, more preferably the soluble TNF receptor or a portion thereof is a soluble TNF receptor type 1 or a portion thereof, more preferably the soluble TNF receptor or a portion thereof is an extracellular fragment of a soluble TNF receptor type 1; Preferably, the extracellular fragment of soluble TNF receptor type 1 comprises the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 95% identity thereto, or a conservatively modified variant thereof.

3. 3. The bispecific fusion protein according to claim 1 or 2, wherein the human IgG Fc fragment is a human IgG1 Fc or a human IgG4 Fc fragment, more preferably the human IgG Fc fragment is a hinge-CH2-CH3 fragment of IgG4, more preferably the human IgG Fc fragment is a human IgG4 Fc fragment having an S228P mutation.

4. competitively binds to IL-17A, or the functional domain of the anti-IL-17A is selected from a receptor of IL-17A, a variant thereof or a part thereof, or an anti-IL-17A antibody or an antigen-binding fragment thereof; Preferably, the antigen-binding fragment of the anti-IL-17A antibody comprises a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a single-chain variable region fragment, a single domain antibody, an isolated CDR region, or a Fd fragment of the anti-IL-17A antibody; Preferably, said functional domain competitively binds to IL-17A or is an anti-IL-17A single chain variable region fragment; Preferably, the anti-IL-17A single chain variable region fragment comprises an HCDR1 set forth in SEQ ID NO:5, an HCDR2 set forth in SEQ ID NO:6, and an HCDR3 set forth in SEQ ID NO:7, and an LCDR1 set forth in SEQ ID NO:8, an LCDR2 set forth in SEQ ID NO:9, and an LCDR3 set forth in SEQ ID NO:10; Preferably, the anti-IL-17A single chain variable region fragment comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto; 4. The bispecific fusion protein of claim 1, wherein the anti-IL-17A single chain variable region fragment preferably comprises the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 80% identity thereto.

5. The bispecific fusion protein comprises, in order from the N-terminus to the C-terminus, (1) a soluble TNF receptor type 1 or 2 or an extracellular fragment thereof, (2) a human IgG4 Fc fragment, and (3) an anti-IL-17A single-chain variable region fragment; Preferably, the bispecific fusion protein comprises, in order from N-terminus to C-terminus, (1) a soluble TNF receptor type 1 or an extracellular fragment thereof, (2) a human IgG4 Fc fragment, and (3) an anti-IL-17A single chain variable region fragment; Preferably, the bispecific fusion protein comprises, in order from N-terminus to C-terminus, (1) an extracellular fragment of soluble TNF receptor type 1 as set forth in SEQ ID NO:1; (2) a human IgG4 Fc fragment as set forth in SEQ ID NO:2; and (3) an anti-IL-17A single chain variable region fragment comprising an HCDR1 as set forth in SEQ ID NO:5, an HCDR2 as set forth in SEQ ID NO:6, and an HCDR3 as set forth in SEQ ID NO:7, and an LCDR1 as set forth in SEQ ID NO:8, an LCDR2 as set forth in SEQ ID NO:9, and an LCDR3 as set forth in SEQ ID NO:10; Preferably, the bispecific fusion protein comprises, in order from N-terminus to C-terminus, (1) an extracellular fragment of a soluble TNF receptor type 1 as set forth in SEQ ID NO: 1; (2) a human IgG4 Fc fragment as set forth in SEQ ID NO: 2; and (3) an anti-IL-17A single chain variable region fragment comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12; 5. The bispecific fusion protein of any one of claims 1 to 4, preferably comprising, from N-terminus to C-terminus, (1) an extracellular fragment of soluble TNF receptor type 1 as set forth in SEQ ID NO: 1, (2) a human IgG4 Fc fragment as set forth in SEQ ID NO: 2, and (3) an anti-IL-17A single chain variable region fragment as set forth in SEQ ID NO:

3.

6. the human IgG Fc fragment competitively binds to IL-17A or is linked to the functional domain of anti-IL-17A via a linker peptide; The bispecific fusion protein according to any one of claims 1 to 5, wherein the linker peptide is preferably a GS flexible linker peptide.

7. the bispecific fusion protein comprises, in order from N-terminus to C-terminus, a soluble TNF receptor type 1 or an extracellular fragment thereof, a human IgG4 Fc fragment, a GS flexible linker peptide, and an anti-IL-17A single-chain variable region fragment; Preferably, the bispecific fusion protein comprises, from N-terminus to C-terminus, an extracellular fragment of soluble TNF receptor type 1 as set forth in SEQ ID NO:1, a human IgG4 Fc fragment as set forth in SEQ ID NO:2, a GS flexible linker peptide as set forth in SEQ ID NO:4, an anti-IL-17A single chain variable region fragment comprising an HCDR1 as set forth in SEQ ID NO:5, an HCDR2 as set forth in SEQ ID NO:6, and an HCDR3 as set forth in SEQ ID NO:7, and an LCDR1 as set forth in SEQ ID NO:8, an LCDR2 as set forth in SEQ ID NO:9, and an LCDR3 as set forth in SEQ ID NO:10; Preferably, the bispecific fusion protein comprises, from N-terminus to C-terminus, an extracellular fragment of soluble TNF receptor type 1 as set forth in SEQ ID NO: 1, a human IgG4 Fc fragment as set forth in SEQ ID NO: 2, a GS flexible linker peptide as set forth in SEQ ID NO: 4, and an anti-IL-17A single chain variable region fragment comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12; Preferably, the bispecific fusion protein comprises, from N-terminus to C-terminus, an extracellular fragment of soluble TNF receptor type 1 as set forth in SEQ ID NO: 1, a human IgG4 Fc fragment as set forth in SEQ ID NO: 2, a GS flexible linker peptide as set forth in SEQ ID NO: 4, and an anti-IL-17A single chain variable region fragment as set forth in SEQ ID NO: 3; Preferably, the fusion protein has a 9x10 －１２ Affinity constant K less than or equal to M Ｄ , and 4 × 10 for TNFα. －１１ Affinity constant K less than or equal to M Ｄ The bispecific fusion protein according to any one of claims 1 to 6, comprising:

8. A polynucleotide encoding the bispecific fusion protein of any one of claims 1 to 7.

9. A recombinant vector comprising the polynucleotide of claim 8.

10. A host cell comprising the polynucleotide of claim 8 or the recombinant vector of claim 9.

11. A method for preparing a bispecific fusion protein according to any one of claims 1 to 7, comprising: a) preparing a polynucleotide encoding said bispecific fusion protein; b) constructing a recombinant vector using the polynucleotide and an expression vector; c) transferring the recombinant vector into host cells and culturing the transformed cells to obtain a cell culture; d) purifying and isolating said cell culture to obtain said bispecific fusion protein.

12. 8. A formulation comprising the bispecific fusion protein of any one of claims 1 to 7, said formulation further comprising a buffer system, a pharmaceutically acceptable excipient, and a surfactant, said buffer system being selected from a solution of acetic acid and sodium acetate, a solution of citric acid and sodium citrate, or a solution of histidine and histidine hydrochloride; Preferably, the formulation has a pH value of 3.0 to 7.5; Preferably, the formulation comprises 5 mg / mL to 100 mg / mL of the bispecific fusion protein, 5 mM to 50 mM of a buffer system, 50 mg / mL to 100 mg / mL of a pharmaceutically acceptable excipient, and 0.01% to 0.05% (w / v) of a surfactant; Preferably, the buffer system is selected from a solution of 5 mM to 20 mM acetic acid and sodium acetate, a solution of 5 mM to 20 mM citric acid and sodium citrate, or a solution of 5 mM to 20 mM histidine and histidine hydrochloride; Preferably, the pharmaceutically acceptable excipient is at least one of sucrose or trehalose; Preferably, the surfactant is a non-ionic surfactant.

13. A pharmaceutical composition comprising the bispecific fusion protein of any one of claims 1 to 7.

14. An inflammation-related disease, Preferably, the inflammation-related disease is an autoimmune disease or cytokine release syndrome; More preferably, the inflammation-related disease is rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, ulcerative colitis, viral infection, or cytokine release syndrome induced by immunomodulatory drugs.

14. A bispecific fusion protein according to any one of claims 1 to 7, a formulation according to claim 12, or a pharmaceutical composition according to claim 13, for use in treating an inflammation-related disease.

15. A bispecific fusion protein according to any one of claims 1 to 7, a formulation according to claim 12, or a pharmaceutical composition according to claim 13 for use as a reagent for binding and inhibiting TNF-α and IL-17A.

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