Fusion proteins comprising TACI polypeptides and uses thereof

Abstract

The present disclosure provides fusion proteins comprising TACI polypeptides and uses thereof, in particular for the prevention or treatment of autoimmune diseases.

Description

Fusion protein containing TACI polypeptide and its use

[0001] The present disclosure claims priority to the following patent application: Chinese patent application filed on August 29, 2022, with application number CN202211041405.0 and invention name “Fusion protein comprising TACI polypeptide and its use”; the entire contents of the above patent application are incorporated into the present disclosure by reference. Technical Field

[0002] The present disclosure relates to the field of biomedicine, and more specifically, to a fusion protein comprising a TACI polypeptide and its use in treating diseases, especially autoimmune diseases. Background Art

[0003] Systemic lupus erythematosus (SLE) is an autoimmune disease that affects multiple tissues and organs, such as the facial skin and kidneys. Its defining characteristic is the production of antibodies against self-tissue antigens, such as antinuclear antibodies (ANA). SLE has a complex etiology involving genetics, the environment, hormones, and autoantibodies. The disease course is long, and clinical symptoms vary widely from person to person, demonstrating high heterogeneity. Furthermore, SLE has a high incidence rate, exceeding one million in China alone. The global prevalence is approximately 30 to 50 per 100,000 people, with the prevalence in women being ten times higher than in men.

[0004] Activation of the type I interferon pathway plays a key role in the pathogenesis of systemic lupus erythematosus (SLE), promoting the activation of multiple immune cell types closely associated with SLE, including plasmacytoid dendritic cells (pDCs) and B cells. High expression of type I interferon is characteristic of 60% to 80% of SLE patients, and type I interferon levels are positively correlated with the Systemic Lupus Encephalopathy Disease Activity Index (SLEDAI) score. Anifrolumab, developed by AstraZeneca, is a monoclonal antibody that targets the type I interferon (IFN) receptor subunit 1 (IFNAR1), antagonizing the activity of all type I interferons (IFN-α, IFN-β, and IFN-ω), thereby controlling SLE disease progression. In the Phase III TULIP-2 clinical trial, 47.8% of patients receiving anifrolumab experienced improvement in the Comprehensive Lupus Assessment Index (BICLA), compared to 31.5% of patients receiving placebo. This improvement in disease activity across all affected organs was associated with a reduction in glucocorticoid use. However, in another Phase III clinical trial (TULIP-1) using the SRI-4 lupus response index as the endpoint, no significant improvement in disease scores was seen (36% vs 40%). This result demonstrates the potential of targeting the type I interferon pathway for the treatment of SLE, but targeting IFNAR1 alone may not be sufficient.

[0005] B lymphocyte stimulator (BLyS, also known as BAFF) and proliferation-inducing ligand (APRIL) are key factors in B lymphocyte differentiation and maturation and in promoting plasma cell survival. They belong to the tumor necrosis factor (TNF) ligand family. Overexpression of these two factors is a key driver of various B lymphocyte-related autoimmune diseases, including systemic lupus erythematosus (SLE). TNF ligand family members are typically synthesized as transmembrane proteins, and membrane-anchored proteins are often observed to be released from the cell surface after proteolysis. BAFF is a type II transmembrane protein that exists in both membrane-bound and soluble forms. Membrane-bound BAFF is cleaved by furin convertase, releasing the biologically active trimeric BAFF. BAFF is expressed on various cell types, including monocytes, dendritic cells, and bone marrow stromal cells. APRIL exists in a form different from that of TNF ligand family members. It is processed by furin convertase in the Golgi apparatus and then directly secreted into the extracellular space as a soluble trimer.

[0006] BAFF and APRIL have been found to have three receptors: B cell maturation antigen (BCMA), transmembrane activator and CAMEL interactor (TACI), and BAFF receptor (BAFF-R, also known as Br3). All belong to the TNF receptor family. The extracellular region of the TNF receptor contains multiple cysteine-rich domains (CRDs). Each CRD has six cysteines forming three pairs of disulfide bonds, and the CRD region plays a role in ligand binding. BAFF and APRIL have different binding abilities to these three receptors. BAFF binds more strongly to TACI than to BCMA, and APRIL binds more strongly to BCMA than to TACI. BAFF-R is a high-affinity receptor unique to BAFF and does not bind to APRIL.

[0007] TACI is a type III transmembrane protein. Human TACI is a 293-amino acid polypeptide consisting of an N-terminal region (amino acid residues 1-165), a transmembrane region (amino acid residues 166-186), and an intracellular region (amino acid residues 187-293). The extracellular region contains two CRDs (CRD1 and CRD2), with CRD1 having a weaker affinity for BAFF and APRIL than CRD2. Studies have found that only in human TACI is exon 2 skipped to produce a short form of TACI lacking the CRD1 domain. This short form of TACI can bind to BAFF and APRIL just as effectively as the long form. Because TACI has a high affinity for BAFF and APRIL, the use of the soluble portion of the extracellular TACI region fused to the Fc fragment of IgG can block the binding of BAFF and APRIL to the TACI, BCMA, and BAFFR receptors on the cell membrane surface, inhibiting the biological activity of BAFF and APRIL and thereby treating autoimmune diseases. Atacicept is a BAFF / APRIL trap developed by ZymoGenetics. Its molecular form is a fusion of the natural TACI extracellular domain with an IgG1-Fc fragment (deficient in FcγR binding activity), or TACI-Fc. Telitacicept (RCT-18) is a TACI-Fc developed by Rongchang Biopharma. Both telitacicept and atacicept simultaneously target both BAFF and APRIL cytokines. Compared to belimumab, which inhibits only BAFF, TACI-Fc, which inhibits both BAFF and APRIL, is more effective in reducing the immune response and achieving the goal of treating SLE. A Phase II clinical study demonstrated that the high-dose telitacicept group achieved a significantly higher SRI-4 (79.2% vs. 32.0%) at 48 weeks compared to the placebo group, meeting the primary endpoint and suggesting that APRIL inhibition may be crucial for the treatment of SLE.

[0008] BCMA is a type III transmembrane protein. Human BCMA is a 184-amino acid polypeptide consisting of an N-terminal region (amino acid residues 1-50), a transmembrane region (amino acid residues 51-93), and an intracellular region (amino acid residues 94-184). The extracellular region of BCMA contains a CRD that binds more strongly to APRIL than to TACI. BCMA can promote the survival of plasma cells and negatively impact autoimmune diseases. BCMA is also involved in the development of humoral immunity (such as antibody production) and immune-related diseases. No BCMA extracellular region or its fusion protein has yet entered the clinic.

[0009] Conventional treatment for SLE primarily includes antimalarial drugs, glucocorticoids, and immunosuppressants. Several major types of hormonal therapy are foundational medications. Clinicians adjust hormonal dosage based on the disease activity of SLE patients and, on this basis, add immunosuppressants, biologics, or other treatments. Compared with rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis, there are very few new therapies approved for SLE, especially biologics. Traditional hormonal therapy, as a widely used foundational treatment, has poor selectivity and is associated with side effects with long-term use. Belimumab is the only new targeted therapy approved in my country in nearly 50 years. Therefore, there is an urgent need to develop new, safe, and effective targeted drugs to meet the enormous clinical needs of SLE.

[0010] Because overexpression of BAFF / APRIL is closely associated with autoimmune diseases, blocking its activity by targeting it as a target molecule can reduce the incidence of the disease and alleviate the symptoms of the disease, thereby achieving the purpose of alleviating and treating autoimmune diseases. Receptors that block BAFF / APRIL signals can specifically act on target cells and are expected to become new biological agents for treating autoimmune diseases. Therefore, there is an urgent need in the art to develop new compounds that can effectively inhibit or block the BAFF / APRIL pathway. The present disclosure provides a fusion protein containing a TACI polypeptide, which has the potential to become a clinically safe and effective drug for the treatment of autoimmune diseases (such as SLE).

[0011] Summary of the Invention

[0012] The present disclosure provides a fusion protein containing a novel structure of a TACI polypeptide, its encoding nucleic acid, vector, host cell, pharmaceutical composition, method for treating or ameliorating a disease (eg, B cell disorder or autoimmune disease), and pharmaceutical use thereof.

[0013] Fusion protein

[0014] The present disclosure provides a fusion protein, wherein the fusion protein is selected from the following structures:

[0015] (1) comprising a TACI polypeptide;

[0016] (2) containing BCMA polypeptide;

[0017] (3) comprising a TACI polypeptide and a BCMA polypeptide;

[0018] (4) comprising a TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody);

[0019] (5) comprising a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody);

[0020] (6) comprising a TACI polypeptide, a BCMA polypeptide, and an antibody (e.g., an anti-IFNAR1 antibody).

[0021] About TACI peptide in fusion protein:

[0022] In some embodiments, the TACI polypeptide comprises CRD1 and / or CRD2 of the TACI extracellular region, for example, comprises CRD2 of the TACI extracellular region.

[0023] In some embodiments, the TACI polypeptide comprises a polypeptide selected from any one of the following (1)-(6):

[0024] (1) Amino acid residues 68-105 of SEQ ID NO: 1 or variants thereof

[0025] (2) SEQ ID NO: 1 or a polypeptide having at least 90% sequence identity thereto;

[0026] (3) amino acid residues 33-67 of SEQ ID NO: 1;

[0027] (4) amino acid residues 70-104 of SEQ ID NO: 1 or a variant thereof;

[0028] (5) amino acid residues 30-110, 69-111, 69-112, 13-118, or 33-104 of SEQ ID NO: 1, or a variant thereof;

[0029] (6) Amino acid residues 68-106, 68-107 or 68-108 of SEQ ID NO: 1 or variants thereof.

[0030] In some embodiments, the variant has one or more amino acid mutations selected from positions 69, 72, 73, 77, 85, 102, and 103.

[0031] In some embodiments, the variant has one or more amino acid substitutions selected from 69T or 69R, 72S, 73E or 73Q, 77E, 85T or 85A, 102A or 102R, 103Y.

[0032] In some specific embodiments, the variant has an amino acid substitution or combination of substitutions selected from any of the following: 69T; 72S; 73E; 73Q; 77E; 69R / 85T; 69R / 85A; 102A; 69R / 85T / 102R; 73E / 77E; 72S / 73E / 77E; 69T / 102A; 69T / 103Y; 69T / 102A, 103Y; 69T / 73E / 77E / 102A.

[0033] In some specific embodiments, the disclosure uses "Mutation 1 / Mutation 2" to indicate that Mutation 1 and Mutation 2 are present together in the mutant. For example, "69T / 102A" indicates that the mutant contains amino acid mutations 69T and 102A, and "69T / 73E / 77E / 102A" indicates that the mutant contains amino acid mutations 69T, 73E, 77E, and 102A.

[0034] In some specific embodiments, the variant has an amino acid substitution combination of 69T / 73E / 77E / 102A, for example, an amino acid substitution combination of L69T / K73E / K77E / Y102A.

[0035] The site of the amino acid mutation is the amino acid residue site numbered relative to the natural sequence of the TACI extracellular region (SEQ ID NO: 1). For example, "the variant has an amino acid mutation selected from position 69" means that the amino acid residue corresponding to position 69 of SEQ ID NO: 1 has a mutation.

[0036] In some embodiments, the amino acid sequence of the TACI polypeptide comprises an amino acid sequence selected from any one of SEQ ID NOs: 1, 2, 6-29.

[0037] In the present disclosure, "at least 90% (sequence) identity" encompasses at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity; "at least 80% (sequence) identity" encompasses at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity.

[0038] Regarding the BCMA peptide in the fusion protein:

[0039] In some embodiments, the BCMA polypeptide comprises the extracellular region of BCMA CRD1.

[0040] In some embodiments, the BCMA polypeptide comprises amino acid residues 7-41 of SEQ ID NO:30.

[0041] In some embodiments, the BCMA polypeptide comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 30, 67, and 68, or an amino acid sequence having at least 90% sequence identity thereto.

[0042] Regarding antibodies in fusion proteins:

[0043] In some embodiments, the antibody in the fusion protein is an anti-IFNAR1 antibody or an antigen-binding fragment thereof.

[0044] In some embodiments, the anti-IFNAR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 45-47, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 48-50, respectively.

[0045] In some embodiments, the amino acid sequence of the heavy chain variable region of the anti-IFNAR1 antibody is as shown in SEQ ID NO: 43 or has at least 90% sequence identity thereto, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 44 or has at least 90% sequence identity thereto.

[0046] In some embodiments, the amino acid sequence of the heavy chain of the anti-IFNAR1 antibody is as shown in SEQ ID NO: 55 or has at least 80% sequence identity thereto, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 54 or has at least 80% sequence identity thereto.

[0047] In some embodiments, the amino acid sequence of the heavy chain of the anti-IFNAR1 antibody is as shown in SEQ ID NO: 61 or has at least 80% sequence identity thereto, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 54 or has at least 80% sequence identity thereto.

[0048] In some embodiments, the present disclosure introduces Anifrolumab, sifalimumab, and the entire text of WO20062002177A, WO2009100309A (e.g., 3F11, 4G5, 11E2, 9D4), WO2020156474A (e.g., 7G4, 10C5, etc.), WO2020057541A (e.g., 8G11H and 485G1 0H), CN201610634601.7 (e.g., H19B7+L16C11, H19B7+L8C3, H15D10+L16C11, H15D10+L8C3 or anti-IFNAR1-C1), CN201510685200.X, and the anti-IFNAR1 antibodies or antigen-binding fragments thereof in WO2012162367A as the anti-IFNAR1 antibodies of the present disclosure.

[0049] About fusion proteins:

[0050] As described above, the present disclosure provides fusion proteins comprising a TACI polypeptide and a BCMA polypeptide, fusion proteins comprising a TACI polypeptide and an anti-IFNAR1 antibody (or an antigen-binding fragment thereof), and fusion proteins comprising a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody (or an antigen-binding fragment thereof).

[0051] In some embodiments, the fusion protein further comprises an immunoglobulin heavy chain constant region (Fc region). In some specific embodiments, the Fc region comprises two subunits capable of associating. In some embodiments, the two subunits are the same or different first subunit and second subunit. In some embodiments, the Fc region is the Fc region of IgG, IgA, IgM, or IgD; in some embodiments, the Fc region is the Fc region of human IgG1, IgG2, IgG3, or IgG4, such as the Fc region of human IgG1, and for example, the Fc region shown in SEQ ID NO: 3.

[0052] In some embodiments, the Fc region in the fusion protein comprises one or more amino acid substitutions compared to the wild-type Fc region, and the amino acid substitutions can reduce its binding to the Fc receptor (FcR); in some embodiments, the amino acid substitutions can reduce its binding to the Fcγ receptor (FcγR); in some embodiments, the Fc region has a YTE mutation (M252Y, S254T and T256E), S228P, L234F, L235E, L234F / L235E, L234A / L235A or L234F / L235E / P331S mutation, and the mutation sites are numbered according to the EU index.

[0053] In some embodiments, the Fc region in the fusion protein comprises a first subunit and a second subunit that can associate with each other, and the first subunit and the second subunit have one or more amino acid substitutions that reduce homodimerization. In some embodiments, the first subunit has a protrusion structure according to the pestle-and-mortar technique, and the second subunit has a hole structure according to the pestle-and-mortar technique, or the first subunit has a hole structure according to the pestle-and-mortar technique, and the second subunit has a protrusion structure according to the pestle-and-mortar technique. In some embodiments, the amino acid residue substitutions of the first subunit include one or more amino acid substitutions in the sites selected from 354, 356, 358, and 366, and the amino acid residues of the second subunit include one or more amino acid substitutions in the sites selected from 349, 356, 358, 366, 368, and 407. In some embodiments, the first subunit includes one or more amino acid substitutions selected from 354C, 356E, 358M, and 366W, and the second subunit includes one or more amino acid substitutions selected from 349C, 356E, 358M, 366S, 368A, and 407V. In some embodiments, the first subunit comprises amino acid substitutions of 354C, 356E, 358M, and 366W, and the second subunit comprises amino acid substitutions of 349C, 356E, 358M, 366S, 368A, and 407V.

[0054] First, regarding a fusion protein comprising a TACI polypeptide and a BCMA polypeptide:

[0055] In some embodiments, the TACI polypeptide is any one of the TACI polypeptides provided herein, and the BCMA polypeptide is any one of the BCMA polypeptides provided herein.

[0056] In some embodiments, the fusion protein comprises one or more (e.g., 2, 3, 4, 5, 6) TACI polypeptides and one or more (e.g., 2, 3, 4, 5, 6) BCMA polypeptides. In some embodiments, the fusion protein comprises 1 TACI polypeptide and 1 BCMA polypeptide, or 2 TACI polypeptides and 2 BCMA polypeptides, or 3 TACI polypeptides and 3 BCMA polypeptides, or 4 TACI polypeptides and 4 BCMA polypeptides, or 2 TACI polypeptides and 1 BCMA polypeptide, or 1 TACI polypeptide and 2 BCMA polypeptides, or 3 TACI polypeptides and 1 BCMA polypeptide, or 1 TACI polypeptide and 3 BCMA polypeptides; when comprising 2 or more TACI polypeptides, the TACI polypeptides may be TACI polypeptides of the same or different sequences as provided herein; when comprising 2 or more BCMA polypeptides, the BCMA polypeptides may be BCMA polypeptides of the same or different sequences as provided herein.

[0057] In some embodiments, in the fusion protein, the TACI polypeptide and a subunit of the Fc region are linked in any order. Optionally, the TACI polypeptide is linked to the first or second subunit of the Fc region via a linker or directly. In some embodiments, as in any of the preceding fusion proteins, the BCMA polypeptide and a subunit of the Fc region are linked in any order. Optionally, the BCMA polypeptide is linked to the first or second subunit of the Fc region via a linker or directly. In some embodiments, the C-terminus of the TACI polypeptide is linked to the N-terminus of the first or second subunit of the Fc region via a linker or directly; or the N-terminus of the TACI polypeptide is linked to the C-terminus of the first or second subunit of the Fc region via a linker or directly. In some embodiments, the C-terminus of the BCMA polypeptide is linked to the N-terminus of the first or second subunit of the Fc region via a linker or directly; or the N-terminus of the BCMA polypeptide is linked to the C-terminus of the first or second subunit of the Fc region via a linker or directly.

[0058] In some embodiments, the fusion protein containing a TACI polypeptide and a BCMA polypeptide comprises a polypeptide chain as shown in any one of the following (I)-(VIII):

[0059] (I) [TACI polypeptide]-[Linker 1]a-[BCMA polypeptide]-[Linker 2]b-[Fc region]e;

[0060] (II) [BCMA polypeptide]-[Linker 1]a-[TACI polypeptide]-[Linker 2]b-[Fc region]e;

[0061] (III) [Fc region] e-[Linker 3] c-[TACI polypeptide]-[Linker 4] d-[BCMA polypeptide];

[0062] (IV) [Fc region] e-[Linker 3] c-[BCMA polypeptide]-[Linker 4] d-[TACI polypeptide];

[0063] (V) [TACI polypeptide 1]-[Linker 1]a-[BCMA polypeptide 1]-[Linker 2]b-[Fc region]e-[Linker 3]c-[TACI polypeptide 2]-[Linker 4]d-[BCMA polypeptide 2];

[0064] (VI) [BCMA polypeptide 1]-[Linker 1]a-[TACI polypeptide 1]-[Linker 2]b-[Fc region]e-[Linker 3]c-[BCMA polypeptide 2]-[Linker 4]d-[TACI polypeptide 2];

[0065] (VII) [TACI polypeptide 1]-[Linker 1]a-[BCMA polypeptide 1]-[Linker 2]b-[Fc region]e-[Linker 3]c-[BCMA polypeptide 2]-[Linker 4]d-[TACI polypeptide 2];

[0066] (VIII) [BCMA polypeptide 1]-[Linker 1]a-[TACI polypeptide 1]-[Linker 2]b-[Fc region]e-[Linker 3]c-[TACI polypeptide 2]-[Linker 4]d-[BCMA polypeptide 2];

[0067] wherein - represents a peptide bond, a linker is a polypeptide capable of achieving a linking function, and linker 1, linker 2, linker 3, and linker 4 may be the same or different; TACI polypeptide 1 and TACI polypeptide 2 are selected from the TACI polypeptides provided herein, and may be the same or different; BCMA polypeptide 1 and BCMA polypeptide 2 are selected from the BCMA polypeptides provided herein, and may be the same or different; a, b, c, d, and e are each independently 0 or 1, for example, in scheme (I) or (II), a and b are both 1, or a is 1 and b is 0, or a is 0 and b is 1, and e is 0 or 1; For example, in Scheme (III) or (IV), c and d are all 1, or c is 1 and d is 0, or c is 0 and d is 1, and e is 0 or 1; for example, in Scheme (V), (VI), (VII) or (VIII), a, b, c, d are all 1, or a, b, c are all 1 and d is 0, or a, b are 1 and c, d are 0, or a is 1 and b, c, d are all 0, or a is 0 and b, c, d are all 1, or a, b are 0 and c, d are 1, or a, b, c are 0 and d is 1, or a, c are 1 and b, d are 0, or a, c are 0 and b, d are 1, and e is 0 or 1.

[0068] In some embodiments, the linker is (G m S n ) h or (GGNGT) h (SEQ ID NO: 75) or (YGNGT) h (SEQ ID NO: 76) or (EPKSS) h (SEQ ID NO: 77), wherein m and n are each independently selected from an integer of 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and h is independently selected from an integer of 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, the linker is (GxS) y A linker, wherein x is selected from an integer of 1-5, and y is selected from an integer of 1-6, such as a linker as shown in any one of SEQ ID NOs: 39-41. For example, the linker has (G4S) y (SEQ ID NO: 78), wherein y is independently selected from integers of 1-6 (e.g., y is 1, 2, 3, 4, 5, 6).

[0069] In some embodiments, a fusion protein comprising a TACI polypeptide and a BCMA polypeptide is provided, comprising a polypeptide as set forth in any one of SEQ ID NOs: 31-38, 69, and 70, or a polypeptide having at least 90% sequence identity thereto.

[0070] In some embodiments, the fusion protein is a multimer, such as a dimer (eg, a homodimer or a heterodimer).

[0071] In some embodiments, a fusion protein containing a TACI polypeptide and a BCMA polypeptide is provided, and the TACI polypeptide may be a variant, and the variant has 1 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations; in some embodiments, a fusion protein containing a TACI polypeptide and a BCMA polypeptide is provided, and the BCMA polypeptide may be a variant, and the variant has 1 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations; the amino acid mutations may be conservative replacements, substitutions or modifications, and / or deletions or additions that do not affect the function.

[0072] In some embodiments, a protein or molecule is provided that competes with the fusion protein containing a TACI polypeptide and a BCMA polypeptide, the TACI polypeptide, or the BCMA polypeptide for binding to BAFF and / or APRIL, or blocks the binding of the fusion protein containing a TACI polypeptide and a BCMA polypeptide, the TACI polypeptide, or the BCMA polypeptide to BAFF and / or APRIL.

[0073] Second, regarding a fusion protein comprising a TACI polypeptide and an anti-IFNAR1 antibody:

[0074] In some embodiments, the TACI polypeptide is any one of the TACI polypeptides provided herein, and the anti-IFNAR1 antibody is any one of the anti-IFNAR1 antibodies provided herein.

[0075] In some embodiments, the fusion protein comprising a TACI polypeptide and an anti-IFNAR1 antibody comprises one or more (e.g., 2, 3, 4, 5, 6) TACI polypeptides. In some embodiments, the fusion protein comprising a TACI polypeptide and an anti-IFNAR1 antibody comprises one or more (e.g., 2, 3, 4) anti-IFNAR1 antibodies.

[0076] In some embodiments, the fusion protein containing the TACI polypeptide and the anti-IFNAR1 antibody comprises a polypeptide chain as shown in any one of the following (I)-(VIII):

[0077] (I) a first polypeptide chain, which, from N-terminus to C-terminus, consists of: [TACI polypeptide]-[Linker 1]a-[antibody heavy chain], and a second polypeptide chain, which is an antibody light chain;

[0078] (II) a first polypeptide chain, which comprises, from N-terminus to C-terminus: [antibody heavy chain]-[linker 2]b-[TACI polypeptide], and a second polypeptide chain, which is an antibody light chain;

[0079] (III) a first polypeptide chain, which is an antibody heavy chain, and a second polypeptide chain, which, from N-terminus to C-terminus, consists of: [TACI polypeptide]-[Linker 3]c-[Antibody light chain];

[0080] (IV) a first polypeptide chain, which is an antibody heavy chain, and a second polypeptide chain, which, from N-terminus to C-terminus, consists of: [antibody light chain]-[Linker 4]d-[TACI polypeptide];

[0081] (V) a first polypeptide chain comprising, from N-terminus to C-terminus, [TACI polypeptide 1]-[Linker 1]a-[Antibody heavy chain], and a second polypeptide chain comprising, from N-terminus to C-terminus, [Antibody light chain]-[Linker 4]d-[TACI polypeptide 2];

[0082] (VI) a first polypeptide chain comprising, from N-terminus to C-terminus, [antibody heavy chain]-[linker 2]b-[TACI polypeptide 1], and a second polypeptide chain comprising, from N-terminus to C-terminus, [TACI polypeptide 2]-[linker 3]c-[antibody light chain];

[0083] (VII) a first polypeptide chain, which, from N-terminus to C-terminus, consists of: [TACI polypeptide 1]-[Linker 1]a-[Antibody heavy chain]-[Linker 2]b-[TACI polypeptide 2], and a second polypeptide chain, which is an antibody light chain;

[0084] (VIII) a first polypeptide chain, which is an antibody heavy chain, and a second polypeptide chain, which, from N-terminus to C-terminus, consists of: [TACI polypeptide 1]-[Linker 3]c-[Antibody light chain]-[Linker 4]d-[TACI polypeptide 2];

[0085] Wherein, - represents a peptide bond, a linker is a polypeptide capable of achieving a linking function, Linker 1, Linker 2, Linker 3, and Linker 4 may be the same or different; TACI polypeptide 1 and TACI polypeptide 2 are selected from any of the aforementioned TACI polypeptides disclosed herein, TACI polypeptide 1 and TACI polypeptide 2 may be the same or different, and a, b, c, and d are each independently 0 or 1. In Scheme (I), a is 1 or 0. In Scheme (II), b is 1 or 0. In Scheme (III), c is 1 or 0. In Scheme (IV), d is 1 or 0. In scheme (V), a and d can be 0 or 1 at the same time, or a is 0 and d is 1, or a is 1 and d is 0; in scheme (VI), b and c can be 0 or 1 at the same time, or b is 0 and c is 1, or b is 1 and c is 0; in scheme (VII), a and b can be 0 or 1 at the same time, or a is 0 and b is 1, or a is 1 and b is 0; in scheme (VIII), c and d can be 0 or 1 at the same time, or c is 0 and d is 1, or c is 1 and d is 0.

[0086] In some embodiments, the linker is (G m S n ) h or (GGNGT) h or (YGNGT) h or (EPKSS) h The amino acid sequence shown, wherein m and n are each independently selected from an integer of 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and h is independently selected from an integer of 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, the linker is (GxS) yA linker, wherein x is selected from an integer of 1-5, and y is selected from an integer of 1-6, such as a linker as shown in any one of SEQ ID NOs: 39-41. For example, the linker has (G4S) y In the amino acid sequence shown, y is independently selected from an integer of 1-6 (eg, y is 1, 2, 3, 4, 5, 6).

[0087] In some embodiments, a fusion protein comprising a TACI polypeptide and an anti-IFNAR1 antibody is provided, comprising a first polypeptide chain as set forth in any one of SEQ ID NOs: 51-53, 62, or having at least 80% or at least 90% sequence identity thereto, and a second polypeptide chain as set forth in SEQ ID NO: 54, or having at least 80% or at least 90% sequence identity thereto;

[0088] comprising a first polypeptide chain as shown in SEQ ID NO: 55, or having at least 80%, at least 90% sequence identity thereto, and a second polypeptide chain as shown in any one of SEQ ID NOs: 56-60, or having at least 80%, at least 90% sequence identity thereto; or

[0089] The invention also comprises a first polypeptide chain as shown in SEQ ID NO: 61 or having at least 80%, at least 90% sequence identity thereto, and a second polypeptide chain as shown in any one of SEQ ID NOs: 56-60 or having at least 80%, at least 90% sequence identity thereto.

[0090] In some embodiments, the aforementioned fusion protein comprises two identical first polypeptide chains and two identical second polypeptide chains.

[0091] In some embodiments, the present disclosure provides a fusion protein comprising a TACI polypeptide and an anti-IFNAR1 antibody, wherein the TACI polypeptide may be a variant having 1 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations; the amino acid mutations may be conservative replacements, substitutions or modifications, and / or deletions or additions that do not affect the function.

[0092] In some embodiments, a protein or molecule is provided, which competes with the aforementioned fusion protein containing a TACI polypeptide and an anti-IFNAR1 antibody for binding to BAFF and / or APRIL, or blocks its binding to BAFF and / or APRIL.

[0093] The third aspect is regarding a fusion protein comprising a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody:

[0094] In some embodiments, the TACI polypeptide is any TACI polypeptide provided herein, the BCMA polypeptide is any BCMA polypeptide provided herein, and the anti-IFNAR1 antibody is any anti-IFNAR1 antibody provided herein.

[0095] In some embodiments, the antibody fusion protein comprising a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody comprises one or more (e.g., 2, 3, 4, 5, 6) TACI polypeptides. In some embodiments, the antibody fusion protein comprising a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody comprises one or more (e.g., 2, 3, 4, 5, 6) BCMA polypeptides. In some embodiments, the antibody fusion protein comprising a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody comprises one or more (e.g., 2, 3, 4) anti-IFNAR1 antibodies. In some embodiments, the aforementioned antibody fusion protein comprising a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody comprises one aforementioned TACI polypeptide and one aforementioned BCMA polypeptide, or two aforementioned TACI polypeptides and two aforementioned BCMA polypeptides, or three aforementioned TACI polypeptides and three aforementioned BCMA polypeptides, or four aforementioned TACI polypeptides and four aforementioned BCMA polypeptides, or two aforementioned TACI polypeptides and one aforementioned BCMA polypeptide, or one aforementioned TACI polypeptide and two aforementioned BCMA polypeptides, or three aforementioned TACI polypeptides and one aforementioned BCMA polypeptide, or one aforementioned TACI polypeptide and three aforementioned BCMA polypeptides, and an anti-IFNAR1 antibody; when comprising two or more aforementioned TACI polypeptides, the TACI polypeptides may be TACI polypeptides having the same or different sequences as provided herein; when comprising two or more aforementioned BCMA polypeptides, the BCMA polypeptides may be BCMA polypeptides having the same or different sequences as provided herein.

[0096] In some embodiments, the fusion protein containing a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody as described in any of the preceding items comprises a polypeptide chain as shown in any one of the following (I)-(VIII):

[0097] (I) a first polypeptide chain, which consists, from N-terminus to C-terminus, of: [TACI polypeptide and BCMA polypeptide domains]-[Linker 1]a-[antibody heavy chain], and a second polypeptide chain, which is an antibody light chain;

[0098] (II) a first polypeptide chain, which comprises, from N-terminus to C-terminus: [antibody heavy chain]-[Linker 2]b-[TACI polypeptide and BCMA polypeptide domain], and a second polypeptide chain, which is an antibody light chain;

[0099] (III) a first polypeptide chain, which is an antibody heavy chain, and a second polypeptide chain, which, from N-terminus to C-terminus, consists of: [TACI polypeptide and BCMA polypeptide domains]-[Linker 3]c-[antibody light chain];

[0100] (IV) a first polypeptide chain, which is an antibody heavy chain, and a second polypeptide chain, which, from N-terminus to C-terminus, consists of: [antibody light chain]-[Linker 4]d-[TACI polypeptide and BCMA polypeptide domains];

[0101] (V) a first polypeptide chain comprising, from N-terminus to C-terminus, the following sequence: [TACI polypeptide and BCMA polypeptide domain 1]-[Linker 1]a-[antibody heavy chain], and a second polypeptide chain comprising, from N-terminus to C-terminus, the following sequence: [antibody light chain]-[Linker 4]d-[TACI polypeptide and BCMA polypeptide domain 2];

[0102] (VI) a first polypeptide chain comprising, from N-terminus to C-terminus, the following sequence: [antibody heavy chain]-[linker 2]b-[TACI polypeptide and BCMA polypeptide domain 1], and a second polypeptide chain comprising, from N-terminus to C-terminus, the following sequence: [TACI polypeptide and BCMA polypeptide domain 2]-[linker 3]c-[antibody light chain];

[0103] (VII) a first polypeptide chain, which, from N-terminus to C-terminus, consists of: [TACI polypeptide and BCMA polypeptide domain 1]-[Linker 1]a-[antibody heavy chain]-[Linker 2]b-[TACI polypeptide and BCMA polypeptide domain 2], and a second polypeptide chain, which is an antibody light chain;

[0104] (VIII) a first polypeptide chain, which is an antibody heavy chain, and a second polypeptide chain, which, from N-terminus to C-terminus, consists of: [TACI polypeptide and BCMA polypeptide domain 1]-[Linker 3]c-[Antibody light chain]-[Linker 4]d-[TACI polypeptide and BCMA polypeptide domain 2];

[0105] Wherein, the structures of "[TACI polypeptide and BCMA polypeptide domain]", "[TACI polypeptide and BCMA polypeptide domain 1]", and "[TACI polypeptide and BCMA polypeptide domain 2]" from N-terminus to C-terminus are: [TACI polypeptide]-[Linker 5]e-[BCMA polypeptide], or: [BCMA polypeptide]-[Linker 5]e-[TACI polypeptide], and the TACI in "TACI polypeptide and BCMA polypeptide domain 1" and "TACI polypeptide and BCMA polypeptide domain 2" may be the same or different, and the BCMA may be the same or different;

[0106] Wherein, - represents a peptide bond, a linker is a polypeptide capable of achieving a linking function, and linkers 1, 2, 3, 4, and 5 may be the same or different; a, b, c, d, and e are each independently 0 or 1. a, b, c, and d are each independently 0 or 1. In scheme (I), a is 1 or 0. In scheme (II), b is 1 or 0. In scheme (III), c is 1 or 0. In scheme (IV), d is 1 or 0. In scheme (V), a and d can be 0 or 1 at the same time, or a is 0 and d is 1, or a is 1 and d is 0; in scheme (VI), b and c can be 0 or 1 at the same time, or b is 0 and c is 1, or b is 1 and c is 0; in scheme (VII), a and b can be 0 or 1 at the same time, or a is 0 and b is 1, or a is 1 and b is 0; in scheme (VIII), c and d can be 0 or 1 at the same time, or c is 0 and d is 1, or c is 1 and d is 0; in the above schemes, e can independently be 0 or 1.

[0107] In some embodiments, the linker is (G m S n ) h or (GGNGT) h or (YGNGT) h or (EPKSS) h The amino acid sequence shown, wherein m and n are each independently selected from an integer of 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and h is independently selected from an integer of 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, the linker is (GxS) y A linker, wherein x is selected from an integer of 1-5, and y is selected from an integer of 1-6, such as a linker as shown in any one of SEQ ID NOs: 39-41. For example, the linker has (G4S) y In the amino acid sequence shown, y is independently selected from an integer of 1-6 (eg, y is 1, 2, 3, 4, 5, 6).

[0108] In some embodiments, a fusion protein comprising a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody is provided, comprising a first polypeptide chain as set forth in SEQ ID NO: 55, or having at least 80% or at least 90% sequence identity thereto, and a second polypeptide chain as set forth in SEQ ID NOs: 63-66, 71, or 72, or having at least 80% or at least 90% sequence identity thereto;

[0109] A polypeptide comprising a first polypeptide chain as shown in SEQ ID NO: 61, or having at least 80%, at least 90% sequence identity thereto, and a second polypeptide chain as shown in any one of SEQ ID NOs: 63-66, 71, 72, or having at least 80%, at least 90% sequence identity thereto; or

[0110] The invention also comprises a first polypeptide chain as set forth in SEQ ID NO: 73 or 74, or having at least 80%, at least 90% sequence identity thereto, and a second polypeptide chain as set forth in SEQ ID NO: 54, or having at least 80%, at least 90% sequence identity thereto.

[0111] In some embodiments, the aforementioned fusion protein comprises two identical first polypeptide chains and two identical second polypeptide chains.

[0112] In some embodiments, the present disclosure provides a fusion protein comprising a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody, wherein the TACI polypeptide may be a variant having 1 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations; the amino acid mutations may be conservative replacements, substitutions, or modifications, and / or deletions or additions that do not affect the function.

[0113] In some embodiments, a protein or molecule is provided that competes with the aforementioned fusion protein containing a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody for binding to BAFF and / or APRIL, or blocks its binding to BAFF and / or APRIL.

[0114] Regarding the fusion proteins of the first to third aspects:

[0115] In some embodiments, the fusion protein comprising a TACI polypeptide and a BCMA polypeptide, the fusion protein comprising a TACI polypeptide and an IFNAR1 antibody, or the fusion protein comprising a TACI polypeptide, a BCMA polypeptide, and an IFNAR1 antibody has one or more of the following properties or functions:

[0116] (a) Activity of binding to BAFF;

[0117] (b) activity in binding to APRIL;

[0118] (c) reducing the concentration of serum immunoglobulins (such as IgE or IgM);

[0119] (d) decreased spleen weight;

[0120] (e) inhibiting or blocking the BAFF / APRIL pathway;

[0121] (f) Reduce the number of B cells.

[0122] In some embodiments, the protein comprising a TACI polypeptide provided by the present disclosure (e.g., TACI-Fc) has one or more of the following functional activities:

[0123] a. TACI polypeptide fragments that are not susceptible to cleavage; in some embodiments, the cleavage can be detected by mass spectrometry, such as the TACI cleavage analysis method in Example 2;

[0124] b. Blocking BAFF from binding to BAFF-R; In some embodiments, TACI-Fc blocks IC binding of BAFF to BAFF-R 50 value is less than 23 nM, less than 10 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM or less; the IC 50 The value is detected by ELISA method; in some embodiments, the IC 50 The value test method is shown in Example 1;

[0125] c. Ability to bind to BAFF; In some embodiments, the TACI-Fc binds to the EC of BAFF 50 The value is less than 5 nM, 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or less, the EC 50 The value is detected by ELISA method, such as the ELISA binding experiment (coated antigen protein) in Example 2;

[0126] d. Inhibit BAFF-induced B cell proliferation; In some embodiments, TACI-Fc inhibits BAFF-induced B cell proliferation IC 50 The IC value is less than 0.3 nM, less than 0.2 nM, less than 0.1 nM, 0.06 nM, less than 0.05 nM, less than 0.01 nM or less. 50 The value is detected by ELISA method, such as the ELISA experiment in Example 17;

[0127] e. Inhibit APRIL-induced B cell proliferation; In some embodiments, TACI-Fc inhibits APRIL-induced B cell proliferation IC 50 value is less than 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, 0.2 nM, less than 0.1 nM, less than 0.05 or less; the IC 50 The values ​​were detected by ELISA method;

[0128] f. binds to human BAFF and / or human APRIL with high affinity; in some embodiments, the affinity is detected by Biacore; in some embodiments, the KD value of the TACI-Fc fusion protein binding to human BAFF is less than 1E-10M, less than 9E-11M, 8E-11M or less; in some embodiments, the KD value of the TACI-Fc fusion protein binding to human APRIL is less than 2.3E-11M, less than 2.0E-11M, 1.9E-11M, 1.3E-11M or less;

[0129] g. Good in vivo pharmacokinetics; in some embodiments, the half-life of TACI-Fc in rats is greater than 4 days; and / or

[0130] h. Good stability; in some embodiments, the TACI-Fc fusion protein can maintain a purity of more than 94% (SEC%) after being stored at a constant temperature of 40°C for four weeks; in some embodiments, the TACI-Fc has a pI value of less than 9, less than 8, less than 7, less than 6, or even less, as determined by "18cProt / TrEMBL" system analysis.

[0131] In some embodiments, the fusion protein containing a TACI polypeptide and a BCMA polypeptide provided by the present disclosure has one or more of the following functional activities:

[0132] a. TACI polypeptide fragments that are not susceptible to cleavage; in some embodiments, the cleavage can be detected by mass spectrometry, such as the TACI cleavage analysis method in Example 2;

[0133] b. Ability to bind to BAFF; In some embodiments, the fusion protein binds to BAFF EC 50 The value is less than 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or less, the EC 50 The value is detected by ELISA method, such as the ELISA experiment in Example 5;

[0134] c. Ability to bind to APRIL; In some embodiments, the fusion protein binds to the EC of APRIL 50 The value is less than 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or less, the EC 50 The value is detected by ELISA method, such as the ELISA experiment in Example 5;

[0135] d. Good pharmacokinetic properties in vivo;

[0136] e.Good stability.

[0137] In some embodiments, the antibody fusion protein provided by the present disclosure comprising a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody has one or more of the following functional activities:

[0138] a. TACI polypeptide fragments that are not susceptible to cleavage; in some embodiments, the cleavage can be detected by mass spectrometry, such as the TACI cleavage analysis method in Example 2;

[0139] b. Inhibition of type I interferon activity; in some embodiments, the fusion protein has an inhibition rate on IFN activity that is consistent with or similar to that of an anti-IFNAR1 antibody (e.g., Anifrolumab), and the inhibition rate is detected in vitro by an IFN-α / β reporter gene method, such as the experiment in Example 10; the inhibition rate is detected in vivo by detecting the mRNA expression of IFN-α downstream genes ISG in PBMCs, such as the experiment in Example 21;

[0140] c. Ability to bind to BAFF; In some embodiments, the fusion protein binds to the EC of BAFF 50 The value is less than 1.5 nM, 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or less, the EC 50 The value is detected by ELISA method, such as the ELISA experiment in Example 11;

[0141] d. Ability to bind to APRIL; In some embodiments, the fusion protein binds to the EC of APRIL 50 The value is less than 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or less, the EC 50 The value is detected by ELISA method, such as the ELISA experiment in Example 11;

[0142] e. Inhibit IFN-α secretion; In some embodiments, the fusion protein has an inhibitory ability on IFN-α cytokine secretion by plasmacytoid dendritic cells (pDCs) that is consistent or similar to that of an anti-IFNAR1 antibody (e.g., Anifrolumab), and the inhibitory ability is detected using, for example, the detection method in Example 15; In some embodiments, the fusion protein has an ability to inhibit IFN-α-induced B cell differentiation into plasma cells in vitro that is consistent or similar to that of an anti-IFNAR1 antibody (e.g., Anifrolumab), and the inhibitory ability is detected using, for example, the detection method in Example 16;

[0143] f. Inhibiting the differentiation of plasma cells and inhibiting BAFF-induced B cell proliferation; in some embodiments, the fusion protein inhibits BAFF-induced B cell proliferation by an inhibition rate of at least 50%, at least 40%, at least 30%, at least 20%, or at least 10%, wherein the inhibition rate is the ratio of the number of B cells after addition of the antibody fusion protein to the number of B cells after addition of the same concentration of IgG1, and is detected by FACS, such as the method in Example 17;

[0144] g. Inhibiting APRIL-induced B cell proliferation; in some embodiments, the fusion protein inhibits APRIL-induced B cell proliferation by an inhibition rate of at least 50%, at least 40%, at least 30%, at least 20%, or at least 10%, wherein the inhibition rate is the ratio of the number of B cells after addition of the antibody fusion protein to the number of B cells after addition of the same concentration of IgG1, and the inhibition rate is detected by FACS, for example, as described in Example 18;

[0145] h. inhibiting BAFF- and APRIL-induced plasma cell production; in some embodiments, the fusion protein inhibits BAFF- and APRIL-induced plasma cell production by at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, or at least 20%, wherein the inhibition rate is the ratio of the number of B cells after addition of the antibody fusion protein to the number of B cells after addition of the same concentration of IgG1, and the inhibition is detected by FACS, such as the method in Example 19;

[0146] i. Inhibits the production of IgA in vivo; in some embodiments, the fusion protein inhibits the production of IgA in vivo by at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, or at least 30%, compared to an equivalent concentration of PBS, and the inhibition is detected by ELISA, such as the method in Example 22;

[0147] j. Good pharmacokinetic properties in vivo;

[0148] k.Good stability.

[0149] In addition, in some embodiments, a product or combination is provided, comprising any of the aforementioned fusion proteins containing a TACI polypeptide and a BCMA polypeptide as disclosed herein, and further comprising an antibody. In some embodiments, a product or combination is provided, comprising any of the aforementioned TACI polypeptides as disclosed herein, and further comprising an antibody. In some embodiments, the antibody is an anti-IFNAR1 antibody, such as anifrolumab.

[0150] In some embodiments, a complex is provided comprising any of the aforementioned TACI polypeptides disclosed herein and any of the aforementioned BCMA polypeptides disclosed herein. Optionally, the TACI polypeptide and BCMA polypeptide may be operably linked directly or via any of the aforementioned linkers disclosed herein, or may be unlinked.

[0151] In some embodiments, a complex is provided, comprising any of the aforementioned TACI polypeptides disclosed herein and any of the aforementioned BCMA polypeptides disclosed herein; or any of the aforementioned TACI polypeptides disclosed herein and any of the aforementioned anti-IFNAR1 antibodies disclosed herein; or any of the aforementioned BCMA polypeptides disclosed herein and any of the aforementioned anti-IFNAR1 antibodies disclosed herein; or any of the aforementioned TACI polypeptides disclosed herein and any of the aforementioned anti-IFNAR1 antibodies disclosed herein and any of the aforementioned BCMA polypeptides disclosed herein. Optionally, the TACI polypeptide, BCMA polypeptide, and anti-IFNAR1 antibody may be operably linked directly or via a linker disclosed herein, or may be unlinked.

[0152] Polynucleotides and vectors

[0153] The present disclosure provides (isolated) polynucleotides encoding any of the aforementioned TACI polypeptides, BCMA polypeptides, fusion proteins comprising a TACI polypeptide and a BCMA polypeptide, fusion proteins comprising a TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), fusion proteins comprising a TACI polypeptide and a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), products, combinations, or complexes. The polynucleotides may be DNA or RNA (e.g., mRNA).

[0154] The nucleic acids disclosed herein may also be in the form of, present in, and / or part of, a vector, such as a plasmid, cosmid, YAC, or viral vector. The vector may particularly be an expression vector, i.e., a vector that provides for expression of a CD40-binding molecule in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). The expression vector typically comprises at least one nucleic acid disclosed herein, operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). The selection of such elements and their sequences for expression in a particular host is within the skill of the art. Regulatory and other elements useful or necessary for expression of the TACI polypeptides, BCMA polypeptides, fusion proteins comprising a TACI polypeptide and a BCMA polypeptide, fusion proteins comprising a TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), fusion proteins comprising a TACI polypeptide and a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), products, combinations, or complexes disclosed herein include, for example, promoters, enhancers, terminators, integration factors, selection markers, leader sequences, and reporter genes.

[0155] The nucleic acids of the present disclosure can be prepared or obtained by known means (eg, by automated DNA synthesis and / or recombinant DNA technology) based on the information of the amino acid sequence of the polypeptides of the present disclosure, and / or can be isolated from suitable natural sources.

[0156] host cells

[0157] The present disclosure provides recombinant host cells that express or are capable of expressing one or more TACI polypeptides, BCMA polypeptides, fusion proteins comprising a TACI polypeptide and a BCMA polypeptide, fusion proteins comprising a TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), fusion proteins comprising a TACI polypeptide and a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), products, combinations or complexes of the present disclosure, and / or contain a nucleic acid or vector of the present disclosure.

[0158] In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0159] Examples of bacterial cells include cells of gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0160] Exemplary fungal cells include cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or cells of species of the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0161] Examples of mammalian cells include HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0162] However, the present disclosure may also be used with amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins.

[0163] The cells of the present disclosure are incapable of developing into complete plants or animals.

[0164] Preparation method

[0165] The present disclosure provides a method for preparing a TACI polypeptide, a BCMA polypeptide, a fusion protein comprising a TACI polypeptide and a BCMA polypeptide, a fusion protein comprising a TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), a fusion protein comprising a TACI polypeptide and a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), a product, a combination, or a complex, comprising: expressing the aforementioned polypeptide, fusion protein, product, combination, or complex in a host cell as described above, and isolating the polypeptide, fusion protein, product, combination, or complex from the host cell. Optionally, a purification step may also be included. Optionally, a filtration or concentration step may also be included. Soluble mixtures and polymers may also be removed by conventional methods, such as molecular sieves and ion exchange. The obtained product must be immediately frozen, such as at -70°C, or lyophilized.

[0166] The polypeptide, fusion protein, product, combination or complex can be prepared and purified by conventional methods. For example, the cDNA sequence encoding the fusion protein can be cloned and recombined into an expression vector. The recombinant expression vector can stably transfect CHO cells. Mammalian expression systems can lead to glycosylation of the protein (for example, at the highly conserved N-terminus of the Fc region). The positive clones obtained are expanded in the culture medium of a bioreactor to produce the protein. The culture fluid that secretes the protein can be purified, collected, filtered and concentrated using conventional techniques. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. However, the polypeptide, fusion protein, product, combination or complex of the present disclosure can also be obtained by other methods of producing proteins known in the art, such as chemical synthesis, including solid phase or liquid phase synthesis.

[0167] Composition

[0168] The present disclosure provides compositions, e.g., pharmaceutical compositions, containing a therapeutically effective amount of a TACI polypeptide, a BCMA polypeptide, a fusion protein comprising a TACI polypeptide and a BCMA polypeptide, a fusion protein comprising a TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), a fusion protein comprising a TACI polypeptide and a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), a product, combination, complex, or polynucleotide, and one or more pharmaceutically acceptable excipients, diluents, or carriers.

[0169] In some embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of any of the aforementioned polypeptides, fusion proteins, products, combinations, complexes, or polynucleotides per unit dose. In some embodiments, the pharmaceutical composition may contain 0.1 to 2000 mg, and in some embodiments, 1 to 1000 mg, of any of the aforementioned polypeptides, fusion proteins, products, combinations, complexes, or polynucleotides per unit dose.

[0170] Reagent test kit

[0171] The present disclosure provides kits comprising the TACI polypeptides, BCMA polypeptides, fusion proteins comprising a TACI polypeptide and a BCMA polypeptide, fusion proteins comprising a TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), fusion proteins comprising a TACI polypeptide and a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), products, combinations, complexes, or polynucleotides of the present disclosure. In some embodiments, diagnostic reagents comprising the above-mentioned polynucleotides are also provided, as well as related diagnostic uses.

[0172] Methods for treating diseases and pharmaceutical uses

[0173] In some embodiments, the present disclosure provides a method for treating or ameliorating a disease or condition, comprising administering to a subject in need thereof a therapeutically effective amount of any of the aforementioned TACI polypeptides, BCMA polypeptides, fusion proteins comprising a TACI polypeptide and a BCMA polypeptide, fusion proteins comprising a TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), fusion proteins comprising a TACI polypeptide and a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), products, combinations, complexes, polynucleotides, or pharmaceutical compositions of the present disclosure. In some embodiments, the disease or condition is a disease or condition associated with TACI, BCMA, and / or IFN expression.

[0174] In some embodiments, methods or pharmaceutical uses of any of the aforementioned TACI polypeptides, BCMA polypeptides, and anti-IFNAR1 antibodies disclosed herein are provided for treating diseases or conditions in combination. In some embodiments, methods or pharmaceutical uses of any of the aforementioned TACI polypeptides and anti-IFNAR1 antibodies disclosed herein are provided for treating diseases or conditions in combination. In some specific embodiments, the methods or pharmaceutical uses further include combining with a BCMA polypeptide. In some embodiments, methods or pharmaceutical uses of any of the aforementioned fusion proteins containing a BCMA polypeptide and an anti-IFNAR1 antibody disclosed herein are provided for treating diseases or conditions in combination. In some specific embodiments, the methods or pharmaceutical uses further include combining with a TACI polypeptide. The anti-IFNAR1 antibody is, for example, anifrolumab.

[0175] In some embodiments, a method for treating or ameliorating a B cell disorder or an autoimmune disease is provided, comprising administering to a subject in need thereof a therapeutically effective amount of a TACI polypeptide, a BCMA polypeptide, a fusion protein comprising a TACI polypeptide and a BCMA polypeptide, a fusion protein comprising a TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), a fusion protein comprising a TACI polypeptide and a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), a product, a combination, a complex, a polynucleotide, or a pharmaceutical composition of the present disclosure.

[0176] In some embodiments, the present invention provides a TACI polypeptide, a BCMA polypeptide, a fusion protein comprising a TACI polypeptide and a BCMA polypeptide, a fusion protein comprising a TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), a fusion protein comprising a TACI polypeptide and a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), a product, a combination, a complex, a polynucleotide, or a pharmaceutical composition for use in the preparation of a medicament for treating or preventing a disease.

[0177] In some embodiments, the disease or condition is a B cell disorder or an autoimmune disease.

[0178] In some embodiments, the disease or condition is associated with TACI expression, or BCMA expression, or IFN expression, or any two or three of TACI, BCMA, and IFN expression, such as overexpression or abnormal expression.

[0179] In some embodiments, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, insulin-dependent diabetes mellitus, Crohn's disease, rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, and psoriatic arthritis. In some embodiments, the B cell disorder is selected from the group consisting of cancer, chronic leukocytic leukemia, multiple myeloma, non-Hodgkin's lymphoma, post-transplant lymphoproliferative disorder, and light chain gammopathy. In some embodiments, the autoimmune disease is systemic lupus erythematosus.

[0180] Definition of terms

[0181] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise explicitly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0182] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0183] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0184] Unless the context clearly requires otherwise, in the patent specification and claims, the words "comprising," "having," "including," and the like should be construed in the sense of "including but not limited to," rather than in an exclusive or exhaustive sense.

[0185] The term "cytokine" is a general term for proteins released by one cell population that act as intercellular mediators on other cells. Examples of such cytokines include lymphokines, monokines, chemokines, and traditional polypeptide hormones. Exemplary cytokines include IL-2, IFN-γ, IL-6, TNFα, IL-17, and IL-5.

[0186] The TACI (transmembrane activator and calcium modulator and cyclophilinligand-interactor) described in the present disclosure is a membrane-bound receptor. Human TACI belongs to the TNFR superfamily and is a 293-amino acid polypeptide comprising an N-terminal region (amino acid residues 1-165, see SEQ ID NO: 1 of the present disclosure), a transmembrane region (amino acid residues 166-186), and an intracellular region (amino acid residues 187-293). The extracellular region contains two cysteine-rich pseudo-repeats (CRD1 and CRD2). The "CRD1" of TACI in the present disclosure encompasses wild-type human TACI CRD1 (e.g., amino acids 33-67 of SEQ ID NO: 1) and variants thereof. In the present disclosure, "CRD2" of TACI encompasses wild-type human TACI CRD2 (e.g., amino acids 70-104, or 69-104, of SEQ ID NO: 1) and variants thereof (including but not limited to variants of one or more amino acids at positions 69, 72, 73, 77, 85, 102, and 103 of SEQ ID NO: 1 of the present disclosure, such as variants set forth in SEQ ID NOs: 6-29). The CRD1, CRD2, and variants thereof of TACI are all capable of binding to APRIL and / or BAFF, for example, simultaneously binding to APRIL and BAFF. Methods for detecting such binding are well known in the art, and can be found, for example, in the Examples of the present disclosure.

[0187] The BCMA (B cell maturation antigen) described in the present disclosure is a membrane-bound receptor. The human BCMA protein is a 184-amino acid polypeptide, comprising an N-terminal region of the protein (amino acid residues 1-50, see amino acids 1-50 of SEQ ID NO: 30 of the present disclosure), a transmembrane region (amino acid residues 51-93) and an intracellular region (amino acid residues 94-184). The extracellular region of BCMA contains a CRD (or CRD1). The "CRD1" of BCMA in the present disclosure encompasses wild-type human BCMA CRD1 (e.g., amino acids 7-41 of SEQ ID NO: 30 of the present disclosure) and variants thereof. The BCMA CRD1 or its variants are capable of binding to APRIL and / or BAFF, for example, simultaneously binding to APRIL and BAFF. The detection method of the binding is well known in the art, for example, as provided in the examples of the present disclosure.

[0188] In the present disclosure, “TACI extracellular region” and “TACI extracellular domain” are interchangeable, and “BCMA extracellular region” and “BCMA extracellular domain” are interchangeable.

[0189] The terms "interferon alpha", "IFNα", "IFNa", "IFNA" and "IFN alpha" are used interchangeably and shall refer to an IFNα protein encoded by a functional gene of the interferon alpha locus that has 75% or more sequence identity with IFNα1 (protein encoded by GenBank Accession No. NP_076918 or GenBank Accession No. NM_024013). Examples of IFNα subtypes include IFNα1, α2a, α2b, α4, α4bα5, α6, α7, α8, α10, α13, α14, α16, α17 and α21. "Interferon alpha" and "IFNα" shall include recombinant forms of the various IFNα subtypes, as well as naturally occurring preparations comprising IFNα proteins, such as leukocyte IFN and lymphoblastoid IFN.

[0190] The terms "interferon alpha receptor-1," "IFNAR1," "IFNAR-1," and "IFNAR-1 antigen" are used interchangeably and include variants, isoforms, germline homologs of human IFNAR-1, and analogs that share at least one common epitope with IFNAR-1. Thus, the human antibodies of the present invention may, in some embodiments, cross-react with IFNAR-1 from species other than human, or other proteins structurally related to human IFNAR-1 (e.g., human IFNAR-1 homologs). In other embodiments, the antibodies may be completely specific for human IFNAR-1 and exhibit no species or other types of cross-reactivity. The complete cDNA sequence of human IFNAR-1 has GenBank Accession No. NM_000629.

[0191] The term "and / or" is intended to include both "and" and "or". For example, the phrase "A, B and / or C" is intended to include each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0192] The term "antibody" is used in the broadest sense to encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. An antibody may refer to an immunoglobulin, a tetrapeptide chain structure composed of two heavy chains and two light chains connected by interchain disulfide bonds. Immunoglobulins differ in their antigenicity due to the amino acid composition and arrangement of their heavy chain constant regions. Consequently, immunoglobulins can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE, corresponding to μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, there are further subclasses based on the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either kappa or lambda chains based on differences in their constant regions. Each of the five classes of Ig can have either kappa or lambda chains. The approximately 110 amino acids near the N-terminus of both the heavy and light chains of antibodies vary greatly in sequence and constitute the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region (C region). The variable region consists of three hypervariable regions (CDRs) and four framework regions (FRs) whose sequences are relatively conserved. These three hypervariable regions determine the specificity of the antibody and are also known as complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3. "Antibody" in the present disclosure encompasses "antigen-binding fragments", which include: single-chain antibodies (i.e., full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g., VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody and epitope-binding fragments of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217).

[0193] For the determination or definition of CDRs, the definitive delineation of CDRs and the identification of residues comprising the binding site of the antibody can be accomplished by resolving the structure of the antibody and / or resolving the structure of the antibody-ligand complex. This can be accomplished by any of the various techniques known to those skilled in the art, such as X-ray crystallography. A variety of analytical methods can be used to identify CDRs, including but not limited to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definitions, and conformational definitions.

[0194] The Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8). The Chothia numbering system is similar to the Kabat numbering system, but takes into account the positions of certain structural loop regions. (See, e.g., Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83). The AbM numbering system uses an integrated suite of computer programs produced by the Oxford Molecular Group for modeling antibody structure (see, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd). The AbM numbering system uses a combination of knowledge databases and ab initio methods to model the tertiary structure of antibodies from primary sequences (see Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," PROTEINS, Structure, Function and Genetics Suppl., 3: 194-198). Contact definitions are based on analysis of available complex crystal structures (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 5: 732-45). In conformational definitions, CDR positions can be identified as residues that contribute to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166). Other CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened based on predictions or experimental results that a particular residue or group of residues does not significantly affect antigen binding. As used herein, CDR may refer to a CDR defined by any method known in the art (including a combination of methods). The correspondence between various numbering systems is well known to those skilled in the art, and is exemplified by the following Table 1.

[0195] Table 1. Relationships between CDR numbering systems

[0196] Unless otherwise indicated, the variable region and CDR sequences disclosed herein are numbered using the Kabat numbering system.

[0197] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of the antibody heavy chain, including native Fc regions and reconstructed Fc regions. In some embodiments, the Fc region comprises two identical or different subunits. In some embodiments, the Fc region of the human IgG heavy chain is defined as the amino acid residue at the Cys226 position or extending from Pro230 to its carboxyl terminus. Suitable Fc regions for the antibodies described herein include the Fc regions of human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. In some embodiments, the boundaries of the Fc region can also vary, such as the C-terminal lysine in the Fc region (residue 447 according to the EU numbering system) or the C-terminal glycine and lysine in the Fc region (residues 446 and 447 according to the EU numbering system) are deleted. Unless otherwise indicated, the numbering convention of the Fc region is the EU numbering system, also known as the EU index.

[0198] The terms "homology" or "identity" refer to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same nucleotide or amino acid monomer subunit, for example, if every position in two DNA molecules is occupied by the same nucleotide, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, a comparison is made when the two sequences are aligned to achieve the maximum percent homology.

[0199] The term "amino acid mutation" includes amino acid substitutions (also known as amino acid replacements), deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions, and modifications can be performed to achieve the final construct, as long as the final construct possesses the desired properties, such as reduced binding to Fc receptors. Amino acid sequence deletions and insertions include deletions and insertions at the amino and / or carboxyl termini of the polypeptide chain. Specific amino acid mutations can be amino acid substitutions. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid having different structural and / or chemical properties. Amino acid substitutions include replacement with non-naturally occurring amino acids or with derivatives of the 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like. It is anticipated that methods other than genetic engineering to alter amino acid side chain groups, such as chemical modification, may also be useful. Various names may be used herein to refer to the same amino acid mutation. Herein, the amino acid residue at a specific position can be represented by position + amino acid residue, for example, 366W means that the amino acid residue at position 366 is W. T366W means that the amino acid residue at position 366 has mutated from T to W.

[0200] It should be understood that when the claim defines the amino acid sequence in the form of position + residue, the amino acid before the mutation at that site does not constitute a limitation to the technical solution.

[0201] The term "conservative substitution" refers to a substitution with another amino acid residue having properties similar to the original amino acid residue. For example, lysine, arginine, and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have non-polar side chains. In addition, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when substituting an amino acid residue in a group that exhibits similar properties as described above, it will not exhibit specific changes in properties.

[0202] The terms "polypeptide," "protein," or "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

[0203] The term "nucleic acid" is used interchangeably with the term "polynucleotide" in this article, and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or connections, which are synthetic, naturally occurring, and non-naturally occurring, with binding properties similar to reference nucleic acids, and metabolized in a manner similar to reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). "Isolated" nucleic acids refer to polynucleotides separated from the components of their natural environment. Isolation nucleic acids are included in the polynucleotides contained in the following cells, which cells typically contain the polynucleotides, but the polynucleotides are present outside the chromosome or are present at a chromosomal position different from their natural chromosomal position. The isolated nucleic acids encoding a polypeptide or fusion protein refer to one or more polynucleotides encoding a polypeptide or fusion protein, including such one or more polynucleotides in a single vector or a separate vector, and such one or more polynucleotides present in one or more positions in a host cell. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as explicitly indicated sequences. Specifically, as described in detail below, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues.

[0204] The term "effector function" refers to those biological activities attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence mutated Fc region) and varies with the antibody isotype. Examples of antibody effector functions include, but are not limited to, C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0205] The term "binding affinity" or "affinity" is used in this disclosure as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or portion thereof and an antigen, a ligand and a receptor). The binding affinity between two molecules can be quantified by determining the dissociation constant (KD). K can be determined by measuring the kinetics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) methods (Biacore). D The rate constants corresponding to the association and dissociation of a monovalent complex are called the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. D Through equation K D = kd / ka is related to ka and kd. The value of the dissociation constant can be determined directly by well-known methods and can be calculated even for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9:340-362). For example, K can be determined using a double filtration nitrocellulose filter binding assay such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90:5428-5432). D Other standard assays for assessing the binding ability of an antibody to a target antigen are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis, as well as other assays exemplified elsewhere in this disclosure. The binding kinetics and binding affinity of an antibody can also be determined by standard assays known in the art, such as surface plasmon resonance (SPR), for example, by using Biacore TM The K of each antibody / antigen complex can be compared by comparing the K D The K values ​​can be used to compare the binding affinities associated with different molecular interactions, for example, the binding affinities of different antibodies for a given antigen. Similarly, the specificity of an interaction can be determined and compared by determining and comparing the K values ​​of the interactions of interest (e.g., the specific interaction between an antibody and an antigen). D The K value is compared with the K of non-target interactions (e.g., a control antibody known not to bind the target antigen). D The value is evaluated.

[0206] The term "linker" or "linker" refers to a connecting unit that connects two polypeptide fragments, which is generally flexible and does not cause loss of the original function of the protein domain. In this article, the linkers appearing in the same structure can be the same or different. The linker can be a peptide linker, which contains one or more amino acids, typically about 1-30, 2-24, or 3-15 amino acids. The linkers used in the present disclosure can be the same or different.

[0207] The term "fused" or "linked" means that the components (e.g., TACI polypeptide and BCMA polypeptide) are linked by covalent bonds, either directly or via one or more linkers. When the linker is a peptide linker, the covalent bond is a peptide bond.

[0208] "Administer," "apply," and "treat" as applied to an animal, a human, a laboratory subject, a cell, a tissue, an organ, or a biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, a human, a subject, a cell, a tissue, an organ, or a biological fluid. "Administer," "apply," and "treat" can refer to, for example, therapeutic, pharmacokinetics, diagnostics, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "apply," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. "Treatment," as applied to a human, veterinary, or research subject, refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0209] "Treatment" means administering an internal or external therapeutic agent, such as a composition comprising any of the antibodies or antigen-binding fragments thereof disclosed herein, or a fusion protein thereof, to a subject who has, is suspected of having, or is predisposed to having one or more diseases or symptoms thereof, and for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more disease symptoms in the treated subject or population, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable degree. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether the disease symptoms have been alleviated can be evaluated by any clinical detection method commonly used by doctors or other health care professionals to evaluate the severity or progression of the symptoms. Although embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may not be effective in alleviating symptoms of the target disease in a certain subject, they should alleviate symptoms of the target disease in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0210] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular subject or veterinary subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0211] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the offspring of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and offspring derived therefrom, without regard to the number of passages. Offspring may not be identical to the parent cell in nucleic acid content, but may contain mutations. Mutant offspring having the same function or biological activity as screened or selected in the initial transformed cells are included herein. Host cells include prokaryotic and eukaryotic host cells, wherein eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include humans, mice, rats, dogs, monkeys, pigs, goats, cattle, horses, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.

[0212] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of the specified sequence may but need not be present.

[0213] The “fusion protein comprising a TACI polypeptide and a BCMA polypeptide” of the present disclosure encompasses all molecules comprising a fusion protein of a TACI polypeptide and a BCMA polypeptide of the present disclosure.

[0214] The term "fusion protein comprising a TACI polypeptide and an anti-IFNAR1 antibody" encompasses all molecules comprising a fusion protein of a TACI polypeptide of the present disclosure and an anti-IFNAR1 antibody (or an antigen-binding fragment thereof).

[0215] "A fusion protein comprising a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody" encompasses all molecules comprising a fusion protein of a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody (or an antigen-binding fragment thereof) disclosed herein. For example, the fusion protein may comprise one or more effector molecules, for example, by conjugation.

[0216] The "effector molecule" alone can have therapeutic activity (e.g., anti-tumor activity or immune activation or inhibition activity) or detection function, and can be in any form, such as biologically active proteins (e.g., enzymes), other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof, such as DNA, RNA and fragments thereof, radionuclides (particularly radioiodides), radioisotopes, chelated metals, nanoparticles, and reporter groups (e.g., fluorescent compounds), or compounds detectable by NMR or ESR spectroscopy. The conjugation of the effector molecule to the fusion protein of the present invention can be achieved by conventional methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0217] Figure 1. Schematic diagram of the fusion protein structure of TACI and Fc.

[0218] Figure 2. Schematic diagram of the interaction between the ligands BAFF and APRIL and the receptors BAFF-R, TACI, and BCMA. BAFF is a membrane-expressed protein that functions as a soluble trimer after proteolytic cleavage. BAFF can bind to BAFF-R, TACI, and BCMA. APRIL is a soluble trimeric protein that can bind to TACI and BCMA. Thicker black arrows represent stronger interactions, while thinner black arrows represent weaker interactions.

[0219] Figure 3. Schematic diagram of different TACI and BCMA protein domains, including TACI-ECD, TACI-d2, TACI-T2, TACI-T4, and BCMA-ECD.

[0220] Figure 4. Schematic diagram of the TACI-BCMA fusion protein structure, including B575701, B575702, B575703, and B575704.

[0221] Figure 5. ELISA results of TACI-BCMA fusion proteins (B575701, B575702, B575703, B575704) binding to BAFF, using Telitacicept and IgG1 isotype as controls.

[0222] Figure 6. ELISA assay results of the binding ability of TACI-BCMA fusion proteins (B575701, B575702, B575703, B575704) to APRIL, using Telitacicept and IgG1 isotype as controls.

[0223] Figure 7. Schematic diagram of the TACI-CRD2 and TACI-19-16 domains, including TACI-CRD2 and TACI-19-16.

[0224] Figure 8. Schematic diagram of the structures of anifrolumab and TACI-T4 fusion proteins (B385801, B385802, B385803, B385804, B498301, B498302), and anifrolumab and TACI-19-16 fusion proteins (B606401, B606401-LALA, B805201, B805201-LALA, the LALA mutation is the L234A / L235A mutation of Fc, the same below).

[0225] Figure 9. The results of the fusion protein of Anifrolumab and TACI-T4 inhibiting IFN-β activity. Figure 9A shows the results of B385801, B385802, B385803, and B385804 inhibiting IFN-β activity; Figure 9B shows the results of B385804, B498301, and B498302 inhibiting IFN-β activity; and Figure 9C shows the results of B606401 inhibiting IFN-β activity. Anifrolumab and IgG1 isotype were used as controls.

[0226] Figure 10. ELISA results for the binding ability of anifrolumab and TACI-T4 fusion proteins to BAFF. Figure 10A shows the BAFF binding ability of B385801, B385802, B385803, and B385804, using Atacicept, Telitacicept, and the IgG1 isotype as controls. Figure 10B shows the BAFF binding ability of B385804, B498301, and B498302; Figure 10C shows the BAFF binding ability of B606401, using Telitacicept and the IgG1 isotype as controls.

[0227] Figure 11. Schematic diagram of the structure of anifrolumab and TACI-BCMA fusion protein, including B613301, B613302, B613303, and B613304.

[0228] Figure 12. Results of the IFN-β activity inhibition assay using anifrolumab and TACI-BCMA fusion proteins (B613301, B613302, and B613303), with anifrolumab and IgG1 isotype as controls.

[0229] Figure 13. ELISA results of the binding ability of anifrolumab and TACI-BCMA fusion proteins (B613301, B613302, B613303) to BAFF, using Telitacicept and IgG1 isotype as controls.

[0230] Figure 14. ELISA results of the binding ability of Anifrolumab and TACI-BCMA fusion proteins (B613301, B613302, B613303) to APRIL, using Telitacicept and IgG1 isotype as controls.

[0231] Figure 15. Schematic diagram of the structure of anifrolumab fusion protein with TACI-19-16 and BCMA-ECD-1 (B637301), anifrolumab fusion protein with TACI-19-16 and BCMA-ECD-2 fusion protein (B637302, B637302-LALA, B746201, B746201-LALA).

[0232] Figure 16. Results of the inhibition test on IFN-β activity by fusion proteins of anifrolumab with TACI-19-16 and BCMA (B637302, B746201), and fusion proteins of anifrolumab with TACI-19-16 (B606401, B805201), using anifrolumab and IgG1 isotype as controls.

[0233] Figure 17. ELISA results of the binding ability of B637302, B746201, B606401, and B805201 to BAFF, using Telitacicept and IgG1 isotype as controls.

[0234] Figure 18. ELISA results of the binding ability of B637302, B746201, B606401, and B805201 to APRIL, using Telitacicept and IgG1 isotype as controls.

[0235] Figure 19. pDC function assay results in PBMCs. Human PBMCs cultured in vitro were stimulated with CpG-A and treated with gradient concentrations of B637302 and B606401. Anifrolumab, IgG1 isotype, was used as a control. IFN-α secretion levels in the supernatant were measured 24 hours later.

[0236] Figure 20. Results of an in vitro plasma cell differentiation experiment. B cells isolated from human PBMCs were cultured in vitro, supplemented with CpG-B and IFN-α, and treated with gradient concentrations of B637302, B746201, B606401, and B805201. Anifrolumab and IgG1 were used as controls. Flow cytometry analysis was performed after 4 days to assess the proportion of plasma cells (CD27+CD38+).

[0237] Figure 21. Results of a BAFF-induced B cell proliferation experiment in vitro. B cells were isolated from human PBMCs, labeled with CTV, and then cultured in vitro. IL-4, anti-IgM, CD40L, and IL-17 were used as basal stimulatory signals. Under these basal conditions, BAFF was added to induce B cell proliferation. Gradual concentrations of B637302, B606401, and B805201 were then added. Telitacicept, an IgG1 isotype, was used as a control. B cell proliferation signals were detected by flow cytometry on day 5. Drug concentrations ranged from 100 nM, 300 nM, and 1000 nM.

[0238] Figure 22. APRIL-induced B cell proliferation in vitro assay. B cells isolated from human PBMCs were labeled with CTV and cultured in vitro. IL-4, anti-IgM, CD40L, and IL-17 were used as basal stimulatory signals. Under these basal conditions, APRIL was added to induce B cell proliferation. B637302 and B606401 were then treated with gradient concentrations. Telitacicept and IgG1 were used as controls. B cell proliferation was detected by flow cytometry on day 5. Drug concentrations were 100 nM, 300 nM, and 1000 nM.

[0239] Figure 23. In vitro plasma cell generation induced by BAFF and APRIL. B cells isolated from human PBMCs were cultured in vitro and stimulated with CpG-B from days 1 to 4. CpG-B was removed from days 4 to 10, and culture was continued with IL-6, IL-10, and IL-21. Under these basal conditions, plasma cell generation was induced in vitro by the addition of 500 ng / mL BAFF and 50 ng / mL APRIL from days 4 to 7, and 50 ng / mL BAFF and 500 ng / mL APRIL from days 7 to 10. On days 4 to 10, B637302, B606401, B606401, and B805201 were treated at concentrations of 10, 100, and 1000 nM. Telitacicept and an IgG1 isotype were used as controls. Plasma cell numbers were assessed by flow cytometry on day 10.

[0240] Figure 24. In vitro plasma cell generation induced by the combination of IFN-α, BAFF, and APRIL. Figure 24A is a schematic diagram of the experimental process. B cells isolated from human PBMCs were cultured in vitro. CpG-B and IFN-α were added for stimulation on days 1 to 4. CpG-B was removed from days 4 to 10, and culture was continued with IL-6, IL-10, and IL-21. Under these basal conditions, 500 ng / mL BAFF and 50 ng / mL APRIL were added on days 4 to 7, and 50 ng / mL BAFF and 500 ng / mL APRIL were added on days 7 to 10 to induce plasma cell generation in vitro. On days 0 to 10, cells were treated with drugs B637302 and B606401 at different concentrations of 10, 100, and 1000 nM. Telitacicept and IgG1 isotype were used as controls, and the number of plasma cells was detected by flow cytometry on day 10. Figures B, C, and D show the detection results of drugs at 10 nM, 100 nM, and 1000 nM, respectively.

[0241] Figure 25. Results of a PD experiment in humanized mice induced by PEG-IFN-α. Figure 25A is a schematic diagram of the experimental process. Using B-NDG immunodeficient mice, PBMCs were injected into the tail vein for humanization and PEG-IFN-α was injected intraperitoneally for induction. In this model, mice were treated with different concentrations of drugs B637302 and B606401 via intraperitoneal injection. Anifrolumab and PBS were used as controls. Figure 25B is a schematic diagram of the downstream signaling pathway induced by IFN-α. Figure 25C shows the results of PBMC collection at 2 hours for the detection of pSTAT1 levels. Figures 25D, E, and F show the mRNA expression levels of IFN-α downstream genes detected by QPCR using PBMCs collected at 1, 3, and 7 days, respectively.

[0242] Figure 26. Results of a BAFF and APRIL-co-induced PD mouse model. Figure 26A is a schematic diagram of the experimental process. BAFF+APRIL was injected intraperitoneally in C57 / B6 mice to induce PD. The model was then treated with varying concentrations of B637302 and B606401 via intraperitoneal injection. Telitacicept and PBS were used as controls. Peripheral blood was collected on days 4 and 7, and plasma IgA levels were measured by ELISA. Figures 26B and C are graphs showing the results of plasma IgA analysis on days 4 and 7, respectively.

[0243] 21 to 26 , the statistical method used was Student's t-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. DETAILED DESCRIPTION

[0244] The present disclosure is further described below with reference to the following examples, but these examples are not intended to limit the scope of the present disclosure.

[0245] Experimental methods in the disclosed embodiments or test examples, where specific conditions are not specified, generally followed conventional conditions or those recommended by the raw material or commercial manufacturer. See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory; and Current Methods in Molecular Biology, Ausubel et al., Greene Publishing Associates, Wiley Interscience, NY. Reagents whose sources are not specified were commercially available.

[0246] Example 1. TACI sequence analysis and modification

[0247] Due to the presence of multiple protease cleavage sites in the TACI sequence, full-length TACI is prone to fragmentation after expression. In this experiment, TACI sequence fragments of varying lengths were designed. The C-terminus of TACI-ECD or its fragments was directly fused to the N-terminus of human IgG1 Fc (SEQ ID NO: 3) to construct TACI-Fc fusion proteins (including full-length TACI-ECD-Fc and TACI-9-Fc). These proteins were transfected and expressed in 293E (EBNA1 gene-modified human embryonic kidney) cells. The TACI-Fc fusion protein containing two identical polypeptide chains was purified (see Figure 1 for its structure). RCT-18 (Telitacicept) was used as a positive control.

[0248] The sequence looks like this:

[0249] >TACI-ECD (amino acid residues 1 to 165 of wild-type human TACI)

[0250] >TACI-9 (human TACI-ECD positions 68 to 108 by natural numbering)

[0251] >Human IgG1 Fc sequence

[0252] >Telitacicept (RCT-18)

[0253] >Atacicept

[0254] Note: In the Telitacicept (RCT-18) sequence, the ununderlined portion is the TACI sequence (positions 13 to 118 of the human TACI extracellular domain by natural numbering), and the underlined portion is the Fc sequence. In the Atacicept sequence, the ununderlined portion is the TACI sequence (positions 30 to 110 of the human TACI extracellular domain by natural numbering) and the linker EPKSS (in italics), and the underlined portion is the Fc sequence.

[0255] The obtained TACI-Fc fusion protein was subjected to mass spectrometry analysis. The experimental results showed that TACI-9-Fc had no cleavage fragments, but TACI cleavage fragments were detected in both the positive control RCT-18 and the full-length TACI-ECD-Fc (results not shown).

[0256] The ability of the TACI-Fc fusion protein to block BAFF-R binding was further tested as follows: the receptor protein was diluted to 2 μg / mL in PBS (B320, pH 7.4) and added to a 96-well microtiter plate (Corning, 3590) at a volume of 100 μL / well. The plate was incubated overnight at 4°C. After discarding the liquid, 200 μL of 1% Casein blocking solution (Thermo, 37528) was added to each well for blocking and incubation at 37°C for 2 hours. After blocking, the blocking solution was discarded and the plate was washed three times with PBST buffer (PBS, pH 7.4 containing 0.1% Tween-20) before use. A fixed concentration of biotin-labeled ligand protein was mixed with serially diluted antibody or fusion protein, pre-incubated at 37°C for 30 minutes, and then added to the blocked microtiter plate and incubated at 37°C for 1.5 hours. After incubation, wash the plate three times with PBST, add 100 μL streptavidin-HRP (Invitrogen, 434323, 1:4000 dilution) to each well, and incubate at 37°C for 1 hour. Remove the supernatant, wash the plate three times with PBST, and add 100 μL TMB colorimetric substrate (KPL, 5120-0077) to each well. Incubate at room temperature for 10-15 minutes. Add 50 μL 1M H2SO4 to each well to terminate the reaction. Read the absorbance at 450 nm using a microplate reader. Use software to fit the curve of inhibition of ligand and receptor binding, and calculate the IC 50 The sources of the ligand and receptor proteins used are as follows: BAFF (Sino biological, 10056-HNCH), BAFF-R (Sino biological, 16079-H02H).

[0257] The results showed that TACI-9-Fc and RCT-18 blocked the IC of BAFF binding to BAFF-R. 50The values ​​were 5.02 nM and 27.48 nM, respectively. The functional activity of TACI-9-Fc was significantly stronger than that of the control RCT-18. Therefore, TACI-9 is considered a potential candidate molecule.

[0258] Example 2. Analysis of TACI truncated fragments

[0259] Based on TACI-9, the sequence length of TACI was further truncated to test the functional activity of the truncated TACI fragment. The sequence of the further truncated TACI fragment is shown below:

[0260] >TACI-10 (TACI-9 C-terminus truncated with one amino acid "L") (human TACI-ECD positions 68 to 107 by natural numbering)

[0261] >TACI-11 (TACI-9 C-terminally truncated with two amino acids "KL") (human TACI-ECD positions 68 to 106 by natural numbering)

[0262] >TACI-12 (TACI-9 C-terminally truncated with 3 amino acids "NKL") (human TACI-ECD positions 68 to 105 by natural numbering)

[0263] The C-terminus of the truncated TACI fragment was fused to the N-terminus of human IgG Fc (SEQ ID NO: 3) to construct a TACI-Fc fusion protein, which was transfected and expressed in 293E cells and purified to obtain a TACI-Fc fusion protein containing two identical polypeptides (see Figure 1 for the structure).

[0264] The TACI-Fc fusion proteins constructed above were subjected to mass spectrometry analysis. The experimental results are shown in Table 2. The experimental results showed that the TACI-Fc fusion proteins constructed with TACI-9 truncated fragments did not undergo TACI polypeptide cleavage.

[0265] In addition, the activity of the constructed TACI-Fc fusion protein in binding to BAFF was detected, as well as the activity of the TACI-Fc fusion protein in blocking the binding function of BAFF and BAFF-R (see Example 1 for experimental methods).

[0266] The BAFF binding activity of TACI-Fc fusion protein was assayed as follows: BAFF (Sino Biological, 10056-HNCH) protein was diluted to 1 μg / mL in PBS (B320, pH 7.4) and added to a 96-well microtiter plate (Corning, 3590) at a volume of 100 μL / well. The plate was incubated overnight at 4°C. After discarding the liquid, 300 μL of 5% skim milk (BD, 232100) diluted in PBS was added to each well for blocking, and the plate was incubated at 37°C for 2 hours. After blocking, the blocking solution was discarded, and the plate was washed three times with PBST buffer (PBS, pH 7.4 containing 0.1% Tween-20). Then, 100 μL of serially diluted test antibody or fusion protein solution was added to each well and incubated at 37°C for 1 hour. After incubation, wash the plate three times with PBST, add 100 μL mouse anti-human IgG (H+L) (Jackson ImmunoResearch, 209-035-088, 1:8000 dilution) to each well, and incubate at 37°C for 1 hour. After washing the plate three times with PBST, add 100 μL TMB colorimetric substrate (KPL, 5120-0077) to each well and incubate at room temperature for 10-15 minutes. Add 50 μL 1M H2SO4 to each well to terminate the reaction. Read the absorbance at 450 nm with a microplate reader, and use software to fit the binding curve of the antibody and antigen to calculate the EC 50 value.

[0267] The results in Table 2 show that the TACI-10-Fc, TACI-11-Fc, and TACI-12-Fc fusion proteins constructed from the truncated fragments of TACI-9, TACI-10, TACI-11, and TACI-12, have the same binding activity to BAFF and functional activity in blocking the binding of BAFF to BAFF-R as the TACI-9-Fc fusion protein.

[0268] Table 2. TACI-Fc fusion protein functional activity and mass spectrometry fragmentation test results

[0269] Example 3. Sequence modification of TACI truncated fragments

[0270] TACI protein is prone to aggregation in neutral solutions. To further improve TACI's stability, we used MOE (Molecular Operating Environment) software to analyze the hydrophobic and ionic groups in the TACI fragment based on the TACI crystal structure (PDB ID: 1XU1). We then made amino acid substitutions at multiple amino acid residues in TACI-9, reducing the area of ​​exposed hydrophobic and ionic groups on the TACI protein, reducing TACI's aggregation potential, and improving its stability while still retaining its functional activity. The amino acid sequence of the modified TACI fragment is as follows:

[0271] >TACI-9-1 (TACI-9 with L69T replacement)

[0272] >TACI-9-2 (TACI-9 with R72S mutation)

[0273] >TACI-9-3 (TACI-9 with K73E mutation)

[0274] >TACI-9-4 (TACI-9 with K73Q mutation)

[0275] >TACI-9-5 (TACI-9 with K77E mutation)

[0276] >TACI-9-6 (TACI-9 with L69R, D85T mutations)

[0277] >TACI-9-7 (TACI-9 with L69R, D85A mutations)

[0278] >TACI-9-8 (TACI-9 with Y102A mutation)

[0279] >TACI-9-9 (TACI-9 with L69R, D85T, and Y102R mutations)

[0280] >TACI-9-10 (TACI-9 with K73E and K77E mutations)

[0281] >TACI-9-11 (TACI-9 with R72S, K73E, K77E mutations)

[0282] >TACI-9-12 (TACI-9 with L69T, Y102A mutations)

[0283] >TACI-9-13 (TACI-9 with L69T, F103Y mutations)

[0284] >TACI-9-14 (TACI-9 with L69T, Y102A, F103Y mutations)

[0285] >TACI-9-15 (TACI-9 containing L69T, K73E, K77E, and Y102A mutations)

[0286] Note: In the above sequence, the underlined part is the amino acid residue after mutation.

[0287] The C-terminus of the TACI fragment modified from TACI-9 was fused to the N-terminus of human IgG Fc (SEQ ID NO: 3) to construct a TACI-Fc fusion protein (structure shown in Figure 1). The protein was transfected and expressed in 293E cells, and then affinity purified by Protein A to obtain a TACI-Fc fusion protein containing two identical polypeptide chains.

[0288] The activity of the constructed TACI-Fc fusion protein in binding to BAFF was detected (see Example 2 for the experimental method). The experimental results are shown in Table 3:

[0289] Table 3. Binding activity of TACI-Fc fusion protein to BAFF

[0290] Note: The mutation sites in the table are amino acid residue sites (natural sequence numbering) relative to TACI-ECD (SEQ ID NO: 1). For example, "L69T" indicates that the amino acid residue at position 69 (natural sequence numbering) in SEQ ID NO: 1 is mutated from L to T.

[0291] In addition, the partially purified TACI-Fc fusion protein was replaced with a PBS solution using an ultrafiltration tube. The solution was then visually inspected for the presence of precipitation. The results showed that the TACI-Fc fusion protein constructed with the modified TACI fragment produced no precipitation, while the PBS solution of RCT-18 did produce precipitation (results not shown).

[0292] Further provided are truncated forms of TACI-9-15 (TACI-9-15a, TACI-9-15b, TACI-9-15c), the amino acid sequences of which are as follows:

[0293] >TACI-9-15a

[0294] >TACI-9-15b

[0295] >TACI-9-15c (i.e., TACI-19-16)

[0296] Example 4. Construction, expression and purification of TACI-BCMA fusion protein

[0297] As previously mentioned, both atacicept and telitacicept are BAFF / APRIL antagonists in the form of TACI-Fc molecules. As shown in Figure 2, both TACI and BCMA bind to BAFF and APRIL, with TACI binding more strongly to BAFF and less strongly to APRIL, while BCMA binds more strongly to APRIL. We designed BCMA and TACI into a fusion protein in the hope of enhancing their neutralizing activity against APRIL.

[0298] As shown in Figure 3, TACI-ECD is the TACI extracellular region containing amino acid residues 1-165, TACI-d2 is the CRD2 ligand binding domain containing amino acid residues 69-111, TACI-T2 is the CRD1 and CRD2 ligand binding domain containing amino acid residues 13-118, TACI-T4 is the CRD1 and CRD2 ligand binding domain containing amino acid residues 30-110, and BCMA-ECD is the BCMA extracellular region containing amino acid residues 1-54.

[0299] As shown in Figure 4, four forms of fusion proteins containing TACI polypeptide and BCMA polypeptide were designed. Among them, fusion proteins B575701 and B575702 are formed by connecting TACI-d2 and BCMA-ECD sequences and fusing them with human wild-type IgG1-Fc; fusion proteins B575703 and B575704 are formed by connecting TACI-T2 and BCMA-ECD sequences and fusing them with human wild-type IgG1-Fc.

[0300] The sequence of TACI-ECD is shown in SEQ ID NO: 1. The remaining sequences are as follows, where the underlined portion represents human IgG1 Fc. The last G in SEQ ID NO: 27 may be absent.

[0301] >TACI-d2 (SEQ ID NO: 27)

[0302] >TACI-T2 (SEQ ID NO: 28)

[0303] >TACI-T4 (SEQ ID NO: 29)

[0304] >BCMA-ECD (SEQ ID NO: 30)

[0305] >TACI-d2-BCMA-ECD(SEQ ID NO: 31)

[0306] >BCMA-ECD-TACI-d2(SEQ ID NO: 32)

[0307] >TACI-T2-BCMA-ECD(SEQ ID NO: 33)

[0308] >BCMA-ECD-TACI-T2(SEQ ID NO: 34)

[0309] B575701, B575702, B575703, and B575704 are human IgG1 Fc (SEQ ID NO: 3) linked to the C-terminus of TACI-d2-BCMA-ECD, BCMA-ECD-TACI-d2, TACI-T2-BCMA-ECD, and BCMA-ECD-TACI-T2, respectively. In the following sequences, the underlined Fc is shown.

[0310] >B575701 (SEQ ID NO: 35)

[0311] >B575702 (SEQ ID NO: 36)

[0312] >B575703 (SEQ ID NO: 37)

[0313] >B575704 (SEQ ID NO: 38)

[0314] The gene sequence encoding the above-mentioned protein was synthesized and subcloned into pcDNA3.4. 50 μg of expression plasmid was diluted with culture medium and mixed. 200 μL of transfection reagent was diluted with culture medium and mixed. The mixture was then incubated at 37°C for 15 minutes. The mixture was then added dropwise to HEK293 cells. After incubation at 37°C on a shaker for one week, the supernatant was centrifuged at 8000 rpm for 5 minutes and collected. A Protein A affinity chromatography column was equilibrated with 20 mL of 1× PBS at a flow rate of 1 mL / min. The protein supernatant was loaded at a flow rate of 1 mL / min. Nonspecifically bound proteins were washed away with another 20 mL of 1× PBS at a flow rate of 1 mL / min. Finally, the column was eluted with pH 3.4 citrate buffer at a flow rate of 1 mL / min. The eluted protein was transferred to a dialysis bag and dialyzed against 1× PBS, replacing the PBS storage buffer. The target protein was detected.

[0315] Example 5. In vitro binding experiment of TACI-BCMA fusion protein to BAFF and APRIL

[0316] ELISA was used to detect the binding ability of TACI-BCMA fusion proteins (B575701, B575702, B575703, B575704) to BAFF and APRIL, respectively.

[0317] The method is as follows: BAFF (PeproTech, Cat: 310-13) or ARPIL (Acro Biosystems, Cat: APL-H52D1) was diluted in PBS to 1 μg / mL and coated in 96-well plates (Costar, Cat: 3590) overnight at 4°C. The plates were washed with PBST and blocked with Blocking Buffer (PBST containing 1% BSA) at 37°C for 1 hour. The plates were tapped and the test antibody was diluted in Blocking Buffer and incubated at 37°C for 1.5 hours. The plates were washed with PBST and HRP-mouse anti-human Fc antibody (GenScript, Cat: A01854-200) was diluted in Blocking Buffer and incubated at 37°C for 40 minutes. The plate was washed with PBST, 100 μL of TMB color development solution (Biopanda, Cat: TMB-S-003) was added and color was developed at 37° C. for 3 minutes, 100 μL of ELISA stop solution (Solarbio, Cat: C1058) was added, and OD450 was detected.

[0318] The results are shown in Table 4 and Figure 5. All four fusion proteins can bind to BAFF significantly, and their binding ability is stronger than Telitacicept. Among them, B575702 and B575704 have the same ability to bind to BAFF. Compared with Telitacicept, EC 50B575701 and B575703 have the same ability to bind to BAFF, and compared with Telitacicept, EC 50 Reduced to 1 / 2 of its level.

[0319] Table 4. Binding of TACI-BCMA fusion protein to BAFF

[0320] As shown in Table 5 and Figure 6, the four fusion proteins can clearly bind to APRIL, and the binding ability is basically the same; the binding ability of the four fusion proteins to APRIL is stronger than that of Telitacicept, EC 50 Reduced to 1 / 4 to 1 / 3 of its level.

[0321] Table 5. Binding of TACI-BCMA fusion protein to APRIL

[0322] Example 6. Construction, expression and purification of anifrolumab and TACI fusion protein

[0323] Anifrolumab is an antagonist targeting IFNAR1, blocking type I interferon activation of cells through IFNAR1. It is used to treat systemic lupus erythematosus. As shown in Figure 7, TACI-CRD2 is a CRD2 ligand-binding domain containing amino acid residues 68-105, and TACI-19-16 is a TACI-CRD2 peptide with several amino acid mutations (L69T, K73E, K77E, and Y102A).

[0324] As shown in Figure 8, TACI-T4 or TACI-19-16 were connected to the N-terminus or C-terminus of the anifrolumab heavy chain or light chain, respectively, through linkers of different lengths. Specifically, B385801 connected TACI-T4 to the N-terminus of the anifrolumab heavy chain through linker 1 of (G4S)2; B385802 connected TACI-T4 to the C-terminus of the anifrolumab heavy chain through linker 1; B385803 connected TACI-T4 to the N-terminus of the anifrolumab light chain through linker 1; B385804 connected TACI-T4 to the C-terminus of the anifrolumab light chain through linker 1; B498301 connected TACI-T4 to the C-terminus of the anifrolumab light chain through linker 2 of (G4S)3; and B498302 connected TACI-T4 to the C-terminus of the anifrolumab light chain through linker 3 of (G4S)4. B606401 links TACI-19-16 to the C-terminus of the anifrolumab light chain via Linker 1; B805201 links TACI-19-16 to the C-terminus of the anifrolumab heavy chain via Linker 2. B606401-LALA links TACI-19-16 to the C-terminus of the anifrolumab light chain via Linker 1, and the Fc portion is a human IgG1 Fc with L234A and L235A mutations. B805201-LALA links TACI-19-16 to the C-terminus of the anifrolumab heavy chain via Linker 2, and the Fc portion is a human IgG1 Fc with L234A and L235A mutations.

[0325] TACI-19-16 is shown in SEQ ID NO: 26, and the other sequences are as follows:

[0326] >Linker 1 (SEQ ID NO: 39)

[0327] >Linker 2 (SEQ ID NO: 40)

[0328] >Linker 3 (SEQ ID NO: 41)

[0329] >TACI-CRD2 (SEQ ID NO: 42, same as SEQ ID NO: 8)

[0330] >VH of Anifrolumab (SEQ ID NO: 43)

[0331] >VL of Anifrolumab (SEQ ID NO: 44)

[0332] >HCDR1 of Anifrolumab (SEQ ID NO: 45)

[0333] >HCDR2 of Anifrolumab (SEQ ID NO: 46)

[0334] >HCDR3 of Anifrolumab (SEQ ID NO: 47)

[0335] >LCDR1 of Anifrolumab (SEQ ID NO: 48)

[0336] >LCDR2 of Anifrolumab (SEQ ID NO: 49)

[0337] >LCDR3 of Anifrolumab (SEQ ID NO: 50)

[0338] >Heavy chain of B385801 (SEQ ID NO: 51)

[0339] >Heavy chain of B385802 (SEQ ID NO: 52)

[0340] >Heavy chain of B805201 (SEQ ID NO: 53)

[0341] >Light chain of B385801, B385802, B805201, B805201-LALA (SEQ ID NO: 54)

[0342] >Heavy chain of B385803, B385804, B498301, B498302, B606401 (SEQ ID NO: 55)

[0343] >Light chain of B385803 (SEQ ID NO: 56)

[0344] >Light chain of B385804 (SEQ ID NO: 57)

[0345] >Light chain of B498301 (SEQ ID NO: 58)

[0346] >Light chain of B498302 (SEQ ID NO: 59)

[0347] >Light chain of B606401, B606401-LALA (SEQ ID NO: 60)

[0348] >Heavy chain of B606401-LALA (SEQ ID NO: 61)

[0349] >Heavy chain of B805201-LALA (SEQ ID NO: 62)

[0350] The Fc region of IgG is underlined in the heavy chain, CH1 is underlined in the light chain, and the linker is in italics.

[0351] The protein preparation method provided in Example 1 was used, and transient transfection and protein expression were performed. The protein was passed through a Protein A affinity chromatography column and eluted. After detection, the target protein was obtained.

[0352] Example 7. Inhibition of Type I Interferon Activity by Fusion Protein of Anifrolumab and TACI

[0353] The HEK-Blue IFN-α / β cell (InvivoGen, Cat: hkb-ifnab) kit was used to detect the inhibitory effect of the Anifrolumab and TACI fusion proteins B385801, B385802, B385803, B385804, B498301, B498302, and B606401 on the activity of IFN-α / β.

[0354] Method: Digest HEK-Blue TMIFN-α / β cells were resuspended to 2.8E5 / mL in DMEM (Gibco, Cat: 11995065) containing 10% FBS and 1% P / S and seeded into 96-well plates. The test protein was added and incubated at 37°C for 30 minutes. IFN-β (Sino biological, Cat: 10704-HNAS) was added to a final concentration of 0.01 ng / mL and incubated at 37°C for 24 hours. 20 μL of culture supernatant was mixed with 180 μL of Quanti-Blue (InvivoGen, Cat: rep-qbs) in a new 96-well plate and incubated at 37°C for 1 hour. OD655 was measured.

[0355] The results showed that all seven fusion proteins had inhibitory effects on the activity of IFN-α / β. Among them, the activities of B385801 and B385803 were weaker, followed by B385802, and the activities of B385804, B498301, B498302 and B606401 were basically equivalent to those of Anifrolumab (Figure 9A, Figure 9B, Figure 9C).

[0356] Example 8. In vitro binding experiment of the fusion protein of Anifrolumab and TACI to BAFF

[0357] ELISA was used to detect the binding ability of Anifrolumab and TACI fusion proteins B385801, B385802, B385803, B385804, B498301, B498302, and B606401 to BAFF.

[0358] Methods: BAFF (PeproTech, Cat: 310-13) was diluted to 1 μg / mL in PBS and coated in 96-well plates overnight at 4°C. The plates were washed with PBST and blocked with Blocking Buffer (PBST containing 1% BSA) at 37°C for 1 hour. The plates were tapped and the test antibody was diluted in Blocking Buffer and incubated at 37°C for 1.5 hours. The plates were washed with PBST and HRP-mouse anti-human Fc antibody (GenScript, Cat: A01854-200) was diluted in Blocking Buffer and incubated at 37°C for 40 minutes. The plates were washed with PBST and developed with 100 μL of TMB colorimetric solution (Biopanda, Cat: TMB-S-003) at 37°C for 3 minutes. 100 μL of ELISA stop solution (Solarbio, Cat: C1058) was added and the OD450 was measured.

[0359] The results, as shown in Figures 10A to 10C and Table 6, show that the seven fusion proteins significantly bind to BAFF. B385801, B385803, and B606401 have the strongest binding to BAFF, exceeding that of Telitacicept and Atacicept. B385802 has weaker binding, while B385804, B498301, and B498302 have similar binding abilities, but slightly weaker than Telitacicept. Linker length does not affect the binding of TACI to BAFF.

[0360] Table 6. Binding of Anifrolumab and TACI fusion protein to BAFF

[0361] Example 9. Construction, expression and purification of anifrolumab and TACI-BCMA fusion protein

[0362] Referring to Figure 11 , the TACI-BCMA fusion proteins described in Example 4 were linked to the C-terminus of the anifrolumab antibody light chain via Linker 1. B613301 and B613302 are fusion proteins of TACI-d2 and BCMA-ECD linked to the C-terminus of the anifrolumab antibody light chain via Linker 1; and B613303 and B613304 are fusion proteins of TACI-T2 and BCMA-ECD linked to the C-terminus of the anifrolumab antibody light chain via Linker 1.

[0363] The heavy chains of B613301, B613302, B613303, and B613304 are all the full-length heavy chains of Anifrolumab (SEQ ID NO: 55). The specific sequences of the light chains are as follows, with the light chain constant region being underlined:

[0364] >Light chain of B613301 (SEQ ID NO: 63)

[0365] >Light chain of B613302 (SEQ ID NO: 64)

[0366] >Light chain of B613303 (SEQ ID NO: 65)

[0367] >Light chain of B613304 (SEQ ID NO: 66)

[0368] The protein preparation method provided in Example 4 was used, and transient transfection and protein expression were performed. The protein was passed through a Protein A affinity chromatography column and eluted. After detection, the target protein was obtained.

[0369] Example 10. Inhibitory effect of anifrolumab and TACI-BCMA fusion protein on type I interferon activity

[0370] The HEK-Blue IFN-α / β cell kit (InvivoGen, Cat: hkb-ifnab) was used to detect the inhibitory effect of the Anifrolumab and TACI-BCMA fusion proteins B613301, B613302, and B613303 on IFN-α / β activity in Example 9.

[0371] Method: Digest HEK-Blue TM IFN-α / β cells were resuspended to 2.8E5 / mL in DMEM medium (Gibco, Cat: 11995065) containing 10% FBS and 1% P / S and seeded into 96-well plates (Costar, Cat: 3599). The test antibody was added and the cells were incubated at 37°C for 30 minutes. IFN-β (Sino biological, Cat: 10704-HNAS) was added at a final concentration of 0.01 ng / mL and incubated at 37°C for 24 hours. Twenty microliters of culture supernatant was mixed with 180 microliters of Quanti-Blue (InvivoGen, Cat: rep-qbs) in a new 96-well plate and incubated at 37°C for 1 hour. OD655 was measured.

[0372] The results are shown in Figure 12, showing that all three fusion proteins were able to inhibit IFN-α / β activity, with comparable activity and consistent with that of anifrolumab. This suggests that the fusion of TACI-BCMA to anifrolumab, particularly at the C-terminus of the light chain, has little effect on anifrolumab's activity.

[0373] Example 11. In vitro binding experiment of anifrolumab and TACI-BCMA fusion protein to BAFF and APRIL

[0374] ELISA was used to detect the binding ability of Anifrolumab and TACI-BCMA fusion proteins B613301, B613302, and B613303 to BAFF and APRIL.

[0375] Method: BAFF (PeproTech, Cat: 310-13) or ARPIL (Acro Biosystems, Cat: APL-H52D1) was diluted in PBS to 1 μg / mL and coated in 96-well plates (Costar, Cat: 3590) overnight at 4°C. The plates were washed with PBST and blocked with Blocking Buffer (PBST containing 1% BSA) at 37°C for 1 hour. The plates were then tapped and the test antibody was diluted in Blocking Buffer and incubated at 37°C for 1.5 hours. The plates were then washed with PBST and HRP-mouse anti-human Fc antibody (GenScript, Cat: A01854-200) was diluted in Blocking Buffer and incubated at 37°C for 40 minutes. The plate was washed with PBST, 100 μL of TMB color development solution (Biopanda, Cat: TMB-S-003) was added and color was developed at 37° C. for 3 minutes, 100 μL of ELISA stop solution (Solarbio, Cat: C1058) was added, and OD450 was detected.

[0376] As shown in Figure 13 and Table 7, the results show that all three fusion proteins bind to BAFF significantly. B613302 has a significantly stronger ability to bind to BAFF than Telitacicept, EC 50 The level of TACI was reduced to 1 / 8 to 1 / 7 of that of BCMA, indicating that placing TACI at the C-terminus of BCMA (such as in the case of B613302) may be more conducive to maintaining the binding ability to BAFF.

[0377] Table 7. Binding of Anifrolumab and TACI-BCMA fusion protein to BAFF

[0378] As shown in Figure 14 and Table 8, the results show that all three fusion proteins bind to APRIL significantly. B613301, B613302 and B613303 bind to APRIL in a similar manner. Compared with Telitacicept, EC 50 The results showed that the BCMA-fused fusion protein indeed enhanced the binding to APRIL.

[0379] Table 8. Binding of Anifrolumab and TACI-BCMA fusion protein to APRIL

[0380] Example 12. Construction, expression and purification of anifrolumab and TACI-19-16-BCMA fusion protein

[0381] BCMA-ECD-1 (amino acid residues 1-43) and BCMA-ECD-2 (amino acid residues 1-41) remove risk points such as glycosylation sites (amino acid residues 42-44, NAS) and deamidation sites (amino acid residues 47-48, NS) that may exist in the BCMA non-CRD domain. Removing risk points can make the fusion protein more uniform.

[0382] As shown in Figure 15, B637301 fuses TACI-19-16 to the N-terminus of BCMA-ECD-1, and then connects TACI-19-16-BCMA-ECD-1 to the C-terminus of the light chain of the anifrolumab antibody through linker 1; B637302 fuses TACI-19-16 to the C-terminus of BCMA-ECD-2, and then connects BCMA-ECD-2-TACI-19-16 to the C-terminus of the light chain of the anifrolumab antibody through linker 1; B746201 fuses TACI-19-16 to the C-terminus of BCMA-ECD-2, and then connects BCMA-ECD-2-TACI-19-16 to the C-terminus of the heavy chain of the anifrolumab antibody through linker 2. B637302-LALA fuses TACI-19-16 to the C-terminus of BCMA-ECD-2, which is then linked to the C-terminus of the anifrolumab light chain via Linker 1. The Fc portion of the anifrolumab antibody is a human IgG1 Fc with L234A and L235A mutations. B746201-LALA fuses TACI-19-16 to the C-terminus of BCMA-ECD-2, which is then linked to the C-terminus of the anifrolumab heavy chain via Linker 2. The Fc portion of the anifrolumab antibody is a human IgG1 Fc with L234A and L235A mutations (see Figure 15).

[0383] >BCMA-ECD-1 (SEQ ID NO: 67)

[0384] >BCMA-ECD-2 (SEQ ID NO: 68)

[0385] >TACI-19-16-BCMA-ECD-1(SEQ ID NO: 69)

[0386] >BCMA-ECD-2-TACI-19-16(SEQ ID NO: 70)

[0387] The heavy chains of B637301 and B637302 are both the heavy chain of Anifrolumab SEQ ID NO: 55, and the sequences of the light chains are as follows:

[0388] >Light chain of B637301 (SEQ ID NO: 71)

[0389] >Light chain of B637302, B637302-LALA (SEQ ID NO: 72)

[0390] The B637302-LALA heavy chain is SEQ ID NO: 61.

[0391] >Heavy chain of B746201 (SEQ ID NO: 73)

[0392] The light chain of B746201 and B746201-LALA is the light chain of Anifrolumab, SEQ ID NO: 54.

[0393] >Heavy chain of B746201-LALA (SEQ ID NO: 74)

[0394] The B637301, B637302, and B746201 fusion proteins were expressed and purified, and SDS-PAGE analysis revealed that the heavy and light chains of the antibodies linked to TACI-BCMA were single bands (results not shown). This result suggests that removing risky glycosylation sites in the BCMA protein sequence can make the fusion proteins more homogeneous.

[0395] Example 13. Inhibitory effect of anifrolumab and TACI-19-16-BCMA fusion protein on type I interferon activity

[0396] The HEK-Blue IFN-α / β cell kit (InvivoGen, Cat: hkb-ifnab) was used to detect the inhibitory effect of anifrolumab and TACI-19-16-BCMA fusion proteins (B606401, B637302, B746201 and B805201) on IFN-α / β activity.

[0397] Method: Digest HEK-Blue TMIFN-α / β cells were resuspended to 2.8E5 / mL in DMEM medium (Gibco, Cat: 11995065) containing 10% FBS and 1% P / S and seeded into 96-well plates (Costar, Cat: 3599). The test antibody was added and the cells were incubated at 37°C for 30 minutes. IFN-β (Sino biological, Cat: 10704-HNAS) was added at a final concentration of 0.01 ng / mL and incubated at 37°C for 24 hours. Twenty microliters of culture supernatant was mixed with 180 microliters of Quanti-Blue (InvivoGen, Cat: rep-qbs) in a new 96-well plate and incubated at 37°C for 1 hour. OD655 was measured.

[0398] Referring to FIG. 16 and Table 9 , the results showed that all four fusion proteins were able to inhibit the activity of IFN-α / β, and the activities of the four fusion proteins were comparable and consistent with those of Anifrolumab.

[0399] Table 9. Inhibition results of the fusion protein of Anifrolumab and TACI-19-16-BCMA on the activity of type I interferon

[0400] Example 14. In vitro binding experiment of the fusion protein of Anifrolumab and TACI-19-16-BCMA to BAFF and APRIL

[0401] ELISA was used to detect the binding ability of B606401, B637302, B746201 and B805201 fusion proteins to BAFF and APRIL.

[0402] The method is as follows: BAFF (PeproTech, Cat: 310-13) or ARPIL (Acro Biosystems, Cat: APL-H52D1) was diluted in PBS to 1 μg / mL and coated in 96-well plates overnight at 4°C. The plates were washed with PBST and blocked with Blocking Buffer (PBST containing 1% BSA) at 37°C for 1 hour. The plates were tapped and the test antibody was diluted in Blocking Buffer and incubated at 37°C for 1.5 hours. The plates were washed with PBST and HRP-mouse anti-human Fc antibody (GenScript, Cat: A01854-200) was diluted in Blocking Buffer and incubated at 37°C for 40 minutes. The plates were washed with PBST and developed with 100 μL of TMB colorimetric solution (Biopanda, Cat: TMB-S-003) at 37°C for 3 minutes. 100 μL of ELISA stop solution (Solarbio, Cat: C1058) was added and the OD450 was measured.

[0403] As shown in Figure 17 and Table 10, all four fusion proteins bind to BAFF significantly, and their ability to bind to BAFF is stronger than that of Telitacicept. B606401 and B637302 have similar abilities to bind to BAFF. Compared with Telitacicept, EC 50 The levels of B746201 and B805201 were both reduced to 1 / 4 to 1 / 3 of their levels; B746201 and B805201 had similar abilities to bind to BAFF, and compared with Telitacicept, EC 50 were reduced to 1 / 2 of their levels.

[0404] Table 10. Binding of Anifrolumab and TACI-19-16-BCMA Fusion Protein to BAFF

[0405] As shown in Figure 18 and Table 11, all four fusion proteins bind to APRIL significantly. The ability of B637302 to bind to APRIL is significantly higher than that of Telitacicept. 50 B746201 and B805201 have similar binding abilities to APRIL, compared to the EC of Telitacicept. 50 When the binding capacity of B606401 was reduced to 1 / 3 of that of Telitacicept, its ability to bind APRIL was comparable to that of Telitacicept.

[0406] Table 11. Binding of Anifrolumab and TACI-19-16-BCMA Fusion Protein to APRIL

[0407] Example 15. Plasmacytoid dendritic cell (pDC) function experiment

[0408] To evaluate the in vitro efficacy of anifrolumab and TACI-19-16-BCMA fusion protein, we established an in vitro assay for testing bioactive IFNAR1 antagonists. The functional assay for pDCs was as follows: freshly isolated PBMCs from healthy individuals were cultured in 1640GlutaMAX (sugar concentration = 11 mM) basal medium. PBMCs were plated at 3 x 10 6 The cells were seeded at a density of 100 μg / mL in a 96-well plate and stimulated with 0.5 μM CpG-A ODN 2216 (InvivoGen cat: tlrl-2216). Then, the cells were treated with gradient concentrations of anifrolumab, B637302, B606401, and IgG1 isotype. After 24 hours, the IFN-α cytokine secretion level in the cell culture supernatant was detected using a human IFN-α kit (Cisbio cat: 62HIFNAPEG).

[0409] The results showed that B637302, B606401 and the control antibody Anifrolumab were able to inhibit the production of IFN-α in a dose-dependent manner, and the inhibitory activities of the three were comparable ( FIG. 19 ).

[0410] Example 16. IFN-α-induced plasma cell differentiation in vitro functional experiment

[0411] To evaluate the antagonistic biological activity of anifrolumab and TACI-19-16-BCMA fusion protein against IFNAR1, we established a functional experimental method for in vitro differentiation of plasma cells. The method was as follows: human B cells were isolated from fresh healthy human PBMC using a B cell sorting kit (Stemcell cat: 17954) and cultured in vitro in 1640GlutaMAX (Gibco cat: 72400-47) basal medium. B cells were cultured at a density of 1×10 5 The cells were seeded at a density of 100 μg / mL at 400 μg / well in a 96-well plate and stimulated with 2 μg / mL CpG-B 2006 (InvivoGen cat: tlrl-2006) and 250 U / mL IFN-α (Biolegend cat: 592704). The cells were then treated with gradient concentrations of Anifrolumab, B637302, B746201, B606401, B805201, and IgG1 isotype control. After 4 days, the plasma cells (CD27 + CD38 + ) differentiation ratio.

[0412] Referring to FIG20 , the results showed that B637302, B746201, B606401, B805201 and the control antibody Anifrolumab were able to effectively inhibit the in vitro differentiation of B cells into plasma cells induced by IFN-α in a dose-dependent manner. 50 They were 10.75nM, 1.895nM, 17.45nM, 6.208nM and 12.66nM respectively, indicating that the inhibitory activity of the fusion protein was comparable to that of the control antibody Anifrolumab.

[0413] Example 17. BAFF-induced B cell proliferation function experiment

[0414] To evaluate the in vitro efficacy of anifrolumab and TACI-19-16-BCMA fusion protein, we established an in vitro assay for testing TACI-BCMA bioactive molecules. The BAFF-induced B cell proliferation assay was performed as follows: Human B cells were isolated from fresh healthy human PBMC using a B cell sorting kit (Stemcell cat: 17954) and labeled with CTV (Invitrogen cat: C34557). B cells were cultured in vitro in 1640GlutaMAX (Gibco cat: 72400-47) basal medium at a density of 1×10 5 The cells were seeded at a density of 100 μg / mL / well in a 96-well plate and stimulated with 20 μg / mL anti-human IgM antibody (Jacksonimmuno cat:109-006-129), 10 ng / mL recombinant human IL-4 (PeproTech cat:AF-200-04-20), 100 ng / mL CD40L (R&D cat:2706-CL-025 / CF), 1.5 μg / mL anti-His antibody (R&D cat:MAB050-500), 1 ng / mL recombinant human IL-17 (SinoBiological cat:12047-HNAS), and 200 ng / mL recombinant human BAFF (R&D cat:7537-BF-025 / CF). Under these conditions, gradient concentrations of Telitacicept, B637302, B606401, B80521, and IgG1 isotype were added, and B cell proliferation signals were detected by flow cytometry 5 days later.

[0415] The results showed, as shown in Table 12, that B637302, B606401, B805201, and the control, Telitacicept, all effectively inhibited BAFF-induced B cell proliferation in vitro in a dose-dependent manner. As shown in Figure 21, B637302 and B805201 exhibited significantly stronger inhibitory effects than Telitacicept. At a low drug concentration of 10 nM, Telitacicept exhibited no inhibitory activity, while B637302, B606401, and B805201 all demonstrated significant inhibitory activity against plasma cell differentiation.

[0416] Table 12. Results of BAFF-induced B cell proliferation

[0417] Example 18. APRIL-induced B cell proliferation function experiment

[0418] To evaluate the in vitro efficacy of anifrolumab and TACI-19-16-BCMA fusion protein, we established an in vitro assay for testing TACI-BCMA bioactive molecules. The APRIL-induced B cell proliferation assay was performed as follows: Human B cells were isolated from fresh healthy human PBMC using a B cell sorting kit (Stemcell cat: 17954), labeled with CTV (Invitrogen cat: C34557), and cultured in vitro in 1640GlutaMAX (Gibco cat: 72400-47) basal medium. B cells were plated at 1×10 5 The cells were seeded at a density of 100 μg / mL / well in a 96-well plate and stimulated with 20 μg / mL anti-human IgM antibody (Jacksonimmuno cat:109-006-129), 10 ng / mL recombinant human IL-4 (PeproTech cat:AF-200-04-20), 100 ng / mL CD40L (R&D cat:2706-CL-025 / CF), 1.5 μg / mL anti-His antibody (R&D cat:MAB050-500), 1 ng / mL recombinant human IL-17 (SinoBiological cat:12047-HNAS), and 20 ng / mL recombinant human ARPIL (R&D cat:5860-AP-010 / CF). Under these conditions, gradient concentrations of Telitacicept, B637302, B606401, and IgG1 isotype were added, and B cell proliferation signals were detected by flow cytometry 5 days later.

[0419] The results showed that, as shown in Table 13 and Figure 22, B637302, B606401 and the control Telitacicept were all able to effectively inhibit APRIL-induced B cell proliferation in vitro in a dose-dependent manner, and the efficacy of B637302 was significantly better than that of Telitacicept; under the condition of 1000nM drug treatment, the efficacy of B606401 was also significantly better than that of the control Telitacicept.

[0420] Table 13. Results of APRIL-induced B cell proliferation

[0421] Example 19. Experimental study on the function of BAFF+APRIL-induced plasma cell production

[0422] To evaluate the biological activity of TACI-BCMA in the fusion protein of Anifrolumab and TACI-19-16-BCMA, we established an in vitro experimental method for BAFF+APRIL-induced plasma cell generation. The specific process is as follows: human B cells were isolated from fresh healthy human PBMC using a B cell sorting kit (Stemcell cat: 17954) and cultured in vitro in 1640GlutaMAX (Gibco cat: 72400-47) basal medium. B cells were cultured at a density of 1×10 5 Cells were seeded at a density of 100 μg / well in a 96-well plate and stimulated with 2 μg / mL CpG-B 2006 (InvivoGen, cat: tlrl-2006). On day 4, the medium was replaced with 10 ng / mL recombinant human IL-6, 50 ng / mL recombinant human IL-10 (Peprotech, cat: 200-10), 50 ng / mL recombinant human IL-21 (Peprotech, cat: AF-200-21-10), 500 ng / mL recombinant human BAFF (R&D, cat: 7537-BF-025 / CF), and 50 ng / mL recombinant human ARPIL (R&D, cat: 5860-AP-010 / CF). Seven days later, half of the medium was replaced, and the concentrations of BAFF and ARPIL were adjusted to 50 ng / mL and 500 ng / mL, respectively. The concentrations of the remaining stimulatory signals remained unchanged. During this process, Telitacicept, B637302, B746201, B606401, B805201, and IgG1 isotype were added from day 4 to day 10, and plasma cells (CD27 + CD38 + )number.

[0423] As shown in Table 14, the results of the flow cytometry assay for plasma cell counts on day 10 showed that BAFF and ARPIL were able to effectively induce plasma cell generation in vitro. As shown in Figure 23, under 10 nM drug treatment conditions, Telitacicept was unable to inhibit plasma cell generation, while B637302, B746201, B606401, and B805201 were able to significantly inhibit plasma cell generation, indicating that B637302, B746201, B606401, and B805201 were more effective than Telitacicept in inhibiting BAFF and ARPIL. Under 100 nM drug treatment conditions, all four test drugs and Telitacicept significantly inhibited plasma cell generation. Under 1000 nM drug treatment conditions, B637302 had the strongest activity in inhibiting plasma cell generation, surpassing Telitacicept.

[0424] Table 14. Results of BAFF+APRIL-induced plasma cell production

[0425] Example 20. Experiment on the function of IFN-α+BAFF+APRIL in inducing plasma cell production

[0426] To evaluate the synergistic biological activity of Anifrolumab and TACI-19-16-BCMA in the fusion protein of Anifrolumab and TACI-BCMA in the differentiation and generation of B cells into plasma cells, we established an in vitro experimental method for inducing plasma cell generation by IFN-α+BAFF+APRIL. The specific process is as follows: human B cells were isolated from fresh healthy human PBMC using a B cell sorting kit (Stemcell cat: 17954) and cultured in vitro in 1640GlutaMAX (Gibco cat: 72400-47) basal medium. B cells were cultured at a density of 1x10 5 Cells were seeded at a density of 1 / well in a 96-well plate and stimulated with 2 μg / mL CpG-B 2006 (InvivoGen, cat: tlrl-2006) and 250 U / mL IFN-α (Biolegend, cat: 592704). On day 4, the medium was replaced with 10 ng / mL recombinant human IL-6, 50 ng / mL recombinant human IL-10 (Peprotech, cat: 200-10), 15 ng / mL recombinant human IL-21 (Peprotech, cat: AF-200-21-10), 500 ng / mL recombinant human BAFF (R&D, cat: 7537-BF-025 / CF), and 50 ng / mL recombinant human ARPIL (R&D, cat: 5860-AP-010 / CF). Seven days later, half of the medium was replaced, and the concentrations of BAFF and ARPIL were adjusted to 50 ng / mL and 500 ng / mL, respectively. The concentrations of the remaining stimulatory signals remained unchanged. During this process, Anifrolumab, Telitacicept, B637302, B606401, and IgG1 isotype were added at gradient concentrations from day 0 to day 10, and plasma cells (CD27 + CD38 + ) number (A in Figure 24).

[0427] In an in vitro synergistic experiment on plasma cell generation, after optimizing experimental conditions, we found that under IL-21 15 ng / mL conditions, IFN-α and BAFF+APRIL synergistically promoted plasma cell differentiation and generation, with comparable promoting effects. Therefore, we chose to compare the inhibitory activity of various protein drugs at different concentrations under these conditions. The results showed that B637302, B606401, Telitacicept, and Anifrolumab all inhibited plasma cell generation in a dose-dependent manner (see Table 15 and Figures 24B to D). At a low drug concentration of 10 nM, Telitacicept or Anifrolumab alone had no significant inhibitory activity on plasma cell generation, while B637302 and B606401 showed significant inhibitory activity (see Figure 24B).

[0428] At a drug concentration of 100 nM, anifrolumab alone had no significant inhibitory activity, while telitacicept alone had some activity, and the activity of B637302 was significantly superior to that of telitacicept, as shown in Figure 24C. At a drug concentration of 1000 nM, anifrolumab alone had some activity, while the activity of the B637302 and B606401 fusion proteins was significantly superior to that of anifrolumab, as shown in Figure 24D.

[0429] Table 15. Results of IFN-α+BAFF+APRIL-induced plasma cell production

[0430] Example 21. PEG-IFN-α induced PD experiment in PBMC humanized mice

[0431] To test the in vivo efficacy of anifrolumab and TACI-19-16-BCMA fusion protein, we used B-NDG immunodeficient mice and injected 2×10 7 PBMC cells were humanized and 0.6 μg PEG-IFN-α ( Roche) for induction. In this model, mice were treated with intraperitoneal injections of various concentrations of the drugs anifrolumab, B637302, and B606401, as well as PBS. PBMCs were collected 2 h later and pSTAT1 (#9167, Cell Signaling Technology) levels were detected by flow cytometry. Peripheral blood was collected on days 1 and 3 to isolate PBMCs, and the mRNA expression levels of IFN-α downstream genes (such as ISG-15, IFI13, MX-1, HERC5, etc.) were detected by QPCR, as shown in Figures 25A and 25B.

[0432] Referring to Figure 25C, flow cytometry analysis of pSTAT1 in PBMCs collected 2 hours after induction revealed that B637302 and B606401 exhibited comparable hIFN-α inhibitory activity as anifrolumab. Referring to Figures 25D and 25E, QPCR analysis of ISG mRNA expression levels on days 1, 3, and 7 also revealed similar results, consistent with the results of the previous in vitro reporter assays and biological activity experiments.

[0433] Example 22. BAFF+APRIL-induced PD in mice

[0434] To detect the in vivo efficacy of anifrolumab and the TACI-19-16-BCMA end of the TACI-19-16-BCMA fusion protein, we used C57 / B6 mice and induced them by intraperitoneal injection of 3 μg BAFF (BAF-H52D4-1 mg, AcroBio) and 0.5 μg APRIL (APL-H52D1-1 mg, AcroBio). In this model, mice were treated with different concentrations of drugs such as Telitacicept, B637302, and B606401, as well as PBS, by intraperitoneal injection. Peripheral blood was collected on days 4 and 7, and plasma IgA levels were detected by ELISA (Figure 26A).

[0435] The results showed that there was no significant difference in IgA between the PBS and drug-treated groups on days 0 and 2 (results not shown), while on days 4 and 7, B637302, B606401, and Telitacicept significantly inhibited IgA production compared to the PBS control group, as shown in Figures 26B and 26C. Furthermore, the results on day 7 showed that under equimolar dosing conditions, the efficacy of B637302 > B606401 > Telitacicept, as shown in Figure 26C. This is consistent with the results of previous in vitro BAFF and APRIL ELISA binding experiments and in vitro biological activity experiments.

Claims

1. A fusion protein comprising: (a) a TACI polypeptide and an anti-IFNAR1 antibody or an antigen-binding fragment thereof, or, (b) TACI polypeptide, BCMA polypeptide, and anti-IFNAR1 antibody or antigen-binding fragment thereof; in, The TACI polypeptide comprises CRD1 and / or CRD2 of the TACI extracellular region; Preferably, the BCMA polypeptide comprises CRD1 of the BCMA extracellular region.

2. The fusion protein according to claim 1, wherein The TACI polypeptide comprises a polypeptide selected from any one of the following (1)-(6): (1) amino acid residues 68-105 of SEQ ID NO: 1 or a variant thereof; (2) SEQ ID NO: 1 or a polypeptide having at least 95% sequence identity thereto; (3) amino acid residues 33-67 of SEQ ID NO: 1; (4) amino acid residues 70-104 of SEQ ID NO: 1 or a variant thereof; (5) amino acid residues 30-110, 69-111, 69-112, 13-118, or 33-104 of SEQ ID NO: 1, or a variant thereof; (6) amino acid residues 68-106, 68-107, or 68-108 of SEQ ID NO: 1, or variants thereof; Preferably, the variant has one or more amino acid mutations selected from positions 69, 72, 73, 77, 85, 102 and 103; More preferably, the variant has one or more amino acid substitutions selected from 69T or 69R, 72S, 73E or 73Q, 77E, 85T or 85A, 102A or 102R, 103Y; Most preferably, the variant has an amino acid substitution or combination of amino acid substitutions selected from any one of the following: 69T; 72S; 73E; 73Q; 77E; 69R and 85T; 69R and 85A; 102A; 69R, 85T and 102R; 73E and 77E; 72S, 73E and 77E; 69T and 102A; 69T and 103Y; 69T, 102A and 103Y; 69T, 73E, 77E and 102A; The numbering of amino acid residues is based on the natural order of the sequence shown in SEQ ID NO:

1.

3. The fusion protein according to any one of claims 1-2, wherein the TACI polypeptide comprises an amino acid sequence selected from any one of SEQ ID NOs: 1, 2, 6-29.

4. The fusion protein according to any one of claims 1 to 3, wherein the anti-IFNAR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NOs: 45-47, respectively. The light chain variable region comprises LCDR1, LCDR2 and LCDR3, the amino acid sequences of which are shown in SEQ ID NOs: 48-50, respectively.

5. The fusion protein according to claim 4, wherein: The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 43 or a sequence having at least 90% sequence identity thereto, The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 44, or an amino acid sequence having at least 90% sequence identity thereto.

6. The fusion protein according to any one of the preceding claims, which comprises an Fc region of an immunoglobulin, preferably an Fc region of IgG1, more preferably an Fc region represented by SEQ ID NO:

3.

7. The fusion protein according to claim 6, wherein the Fc region comprises one or more amino acid mutations, wherein the amino acid mutations can reduce the binding of Fc to FcR, preferably a mutation that can reduce the binding of Fc to FcγR, more preferably any one of the following amino acid mutations or a combination of amino acid mutations: 234A; 235A; 234F; 235E; 234F and 235E; 234A and 235A; 234F, 235E and 331S; Mutation sites are defined according to the EU numbering system.

8. The fusion protein of any one of claims 1 to 7, wherein the anti-IFNAR1 antibody comprises a heavy chain and a light chain, the heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 55 or 61, or having at least 90% sequence identity thereto, and the light chain comprising an amino acid sequence as shown in SEQ ID NO: 54, or having at least 90% sequence identity thereto.

9. The fusion protein according to any one of the preceding claims, comprising a polypeptide chain as shown in any one of (I) to (VIII): (I) a first polypeptide chain, which, from N-terminus to C-terminus, consists of: [TACI polypeptide]-[Linker 1]a-[antibody heavy chain], and a second polypeptide chain, which is an antibody light chain; (II) a first polypeptide chain, which comprises, from N-terminus to C-terminus: [antibody heavy chain]-[linker 2]b-[TACI polypeptide], and a second polypeptide chain, which is an antibody light chain; (III) a first polypeptide chain, which is an antibody heavy chain, and a second polypeptide chain, which, from N-terminus to C-terminus, consists of: [TACI polypeptide]-[Linker 3]c-[Antibody light chain]; (IV) a first polypeptide chain, which is an antibody heavy chain, and a second polypeptide chain, which, from N-terminus to C-terminus, consists of: [antibody light chain]-[Linker 4]d-[TACI polypeptide]; (V) a first polypeptide chain comprising, from N-terminus to C-terminus, [TACI polypeptide 1]-[Linker 1]a-[Antibody heavy chain], and a second polypeptide chain comprising, from N-terminus to C-terminus, [Antibody light chain]-[Linker 4]d-[TACI polypeptide 2]; (VI) a first polypeptide chain comprising, from N-terminus to C-terminus, [antibody heavy chain]-[linker 2]b-[TACI polypeptide 1], and a second polypeptide chain comprising, from N-terminus to C-terminus, [TACI polypeptide 2]-[linker 3]c-[antibody light chain]; (VII) The first polypeptide chain, which is sequentially composed from N-terminus to C-terminus: [TACI polypeptide 1]-[linker 1]a-[antibody heavy chain]-[Linker 2]b-[TACI polypeptide 2], and a second polypeptide chain, which is an antibody light chain; (VIII) a first polypeptide chain, which is an antibody heavy chain, and a second polypeptide chain, which, from N-terminus to C-terminus, consists of: [TACI polypeptide 1]-[Linker 3]c-[Antibody light chain]-[Linker 4]d-[TACI polypeptide 2]; in, - represents a peptide bond, Linker 1, Linker 2, Linker 3, and Linker 4 may be the same or different; The TACI polypeptide, the TACI polypeptide 1 and the TACI polypeptide 2 are each independently selected from the TACI polypeptide defined in any one of claims 1 to 3, and the TACI polypeptide 1 and the TACI polypeptide 2 may be the same or different; a, b, c, and d are each independently 0 or 1; The antibody refers to the anti-IFNAR1 antibody defined in any one of claims 4-5; Preferably, the linker 1, linker 2, linker 3, and linker 4 are each independently (GxS) y , where x is an integer from 1 to 5, and y is an integer from 1 to 6; More preferably, the amino acid sequences of linker 1, linker 2, linker 3, and linker 4 are each independently as shown in any one of SEQ ID NOs: 39-41.

10. The fusion protein according to any one of the preceding claims, comprising: Two identical first polypeptide chains as shown in any one of SEQ ID NOs: 51-53, 62, and two identical second polypeptide chains as shown in SEQ ID NO: 54; or Two identical first polypeptide chains as shown in SEQ ID NO: 55, and two identical second polypeptide chains as shown in any one of SEQ ID NOs: 56-60; or Two identical first polypeptide chains as shown in SEQ ID NO: 61, and two identical second polypeptide chains as shown in any one of SEQ ID NOs: 56-60.

11. The fusion protein of claim 1, wherein the BCMA polypeptide comprises amino acid residues 7-41 of SEQ ID NO: 30; Preferably, the amino acid sequence of the BCMA polypeptide comprises a sequence selected from any one of SEQ ID NOs: 30, 67, and 68.

12. The fusion protein according to any one of claims 1 to 11, comprising a polypeptide chain as shown in any one of (i) to (viii): (i) a first polypeptide chain, which consists of, from N-terminus to C-terminus: [TACI polypeptide and BCMA polypeptide domain]-[Linker 1]a-[antibody heavy chain], and a second polypeptide chain, which is an antibody light chain; (ii) a first polypeptide chain, which comprises, from N-terminus to C-terminus: [antibody heavy chain]-[Linker 2]b-[TACI polypeptide and BCMA polypeptide domain], and a second polypeptide chain, which is an antibody light chain; (iii) a first polypeptide chain, which is an antibody heavy chain, and a second polypeptide chain, which, from N-terminus to C-terminus, consists of: [TACI polypeptide and BCMA polypeptide domain]-[Linker 3]c-[Antibody light chain]; (iv) a first polypeptide chain, which is an antibody heavy chain, and a second polypeptide chain, which, from N-terminus to C-terminus, consists of: [antibody light chain]-[Linker 4]d-[TACI polypeptide and BCMA polypeptide domains]; (v) a first polypeptide chain comprising, from N-terminus to C-terminus, the following sequence: [TACI polypeptide and BCMA polypeptide domain 1]-[Linker 1]a-[antibody heavy chain], and a second polypeptide chain comprising, from N-terminus to C-terminus, the following sequence: [antibody light chain]-[Linker 4]d-[TACI polypeptide and BCMA polypeptide domain 2]; (vi) a first polypeptide chain comprising, from N-terminus to C-terminus, the following sequence: [antibody heavy chain]-[linker 2]b-[TACI polypeptide and BCMA polypeptide domain 1], and a second polypeptide chain comprising, from N-terminus to C-terminus, the following sequence: [TACI polypeptide and BCMA polypeptide domain 2]-[linker 3]c-[antibody light chain]; (vii) a first polypeptide chain, which, from N-terminus to C-terminus, consists of: [TACI polypeptide and BCMA polypeptide domain 1]-[Linker 1]a-[antibody heavy chain]-[Linker 2]b-[TACI polypeptide and BCMA polypeptide domain 2], and a second polypeptide chain, which is an antibody light chain; (viii) a first polypeptide chain, which is an antibody heavy chain, and a second polypeptide chain, which, from N-terminus to C-terminus, consists of: [TACI polypeptide and BCMA polypeptide domain 1]-[Linker 3]c-[Antibody light chain]-[Linker 4]d-[TACI polypeptide and BCMA polypeptide domain 2]; in, The [TACI polypeptide and BCMA polypeptide domain], the [TACI polypeptide and BCMA polypeptide domain 1], and the [TACI polypeptide and BCMA polypeptide domain 2] are sequentially: [TACI polypeptide]-[Linker 5]e-[BCMA polypeptide] from N-terminus to C-terminus, or: [BCMA polypeptide]-[Linker 5]e-[TACI polypeptide], The TACI polypeptides in the [TACI polypeptide and BCMA polypeptide domain 1] and the [TACI polypeptide and BCMA polypeptide domain 2] may be the same or different, and the BCMA polypeptide domain 1 and the BCMA polypeptide domain 2 may be the same or different; The TACI polypeptide is selected from the TACI polypeptide defined in any one of claims 1 to 3, the BCMA polypeptide is selected from the BCMA polypeptide defined in claim 1 or 11, and the antibody is selected from the anti-IFNAR1 antibody defined in any one of claims 4 to 8; in: - represents a peptide bond, The linker 1, linker 2, linker 3, linker 4, and linker 5 may be the same or different; a, b, c, d, and e are each independently 0 or 1; Preferably, the linker 1, linker 2, linker 3, linker 4, and linker 5 are each independently (GxS) y , where x is an integer from 1 to 5, and y is an integer from 1 to 6; More preferably, the amino acid sequences of linker 1, linker 2, linker 3, linker 4, and linker 5 are each independently as shown in any one of SEQ ID NOs: 39-41.

13. The fusion protein according to any one of claims 1 to 12, comprising two identical first polypeptide chains as shown in SEQ ID NO: 55, and two identical second polypeptide chains as shown in any one of SEQ ID NOs: 63 to 66, 71, and 72; It comprises two identical first polypeptide chains as shown in SEQ ID NO: 61, and two identical second polypeptide chains as shown in any one of SEQ ID NOs: 63-66, 71, and 72; or It comprises two identical first polypeptide chains as shown in SEQ ID NO: 73 or 74, and two identical second polypeptide chains as shown in any one of SEQ ID NO:

54.

14. A fusion protein comprising a TACI polypeptide and a BCMA polypeptide, wherein: The TACI polypeptide comprises CRD1 and / or CRD2 of the TACI extracellular domain, The BCMA polypeptide comprises the CRD1 extracellular domain of BCMA; Preferably, the TACI polypeptide and the BCMA polypeptide are linked directly or via a linker.

15. The fusion protein according to claim 14, wherein The TACI polypeptide comprises a polypeptide selected from any one of the following (1)-(6): (1) amino acid residues 68-105 of SEQ ID NO: 1 or a variant thereof; (2) SEQ ID NO: 1 or a polypeptide having at least 95% sequence identity thereto; (3) amino acid residues 33-67 of SEQ ID NO: 1; (4) amino acid residues 70-104 of SEQ ID NO: 1 or a variant thereof; (5) amino acid residues 30-110, 69-111, 69-112, 13-118, or 33-104 of SEQ ID NO: 1, or variants thereof; (6) amino acid residues 68-106, 68-107, or 68-108 of SEQ ID NO: 1, or variants thereof; Preferably, the variant has one or more amino acid mutations selected from positions 69, 72, 73, 77, 85, 102 and 103; More preferably, the variant has one or more amino acid substitutions selected from 69T or 69R, 72S, 73E or 73Q, 77E, 85T or 85A, 102A or 102R, 103Y; Most preferably, the variant has an amino acid substitution or combination of amino acid substitutions selected from any one of the following: 69T; 72S; 73E; 73Q; 77E; 69R and 85T; 69R and 85A; 102A; 69R, 85T and 102R; 73E and 77E; 72S, 73E and 77E; 69T and 102A; 69T and 103Y; 69T, 102A and 103Y; 69T, 73E, 77E and 102A; The numbering of amino acid residues is based on the natural order of the sequence shown in SEQ ID NO:

1.

16. The fusion protein according to claim 14 or 15, wherein the TACI polypeptide comprises an amino acid sequence selected from any one of SEQ ID NOs: 1, 2, 6-29.

17. The fusion protein according to any one of claims 14 to 16, wherein the BCMA polypeptide comprises amino acid residues 7 to 41 of SEQ ID NO:

30.

18. The fusion protein according to claim 17, wherein The amino acid sequence of the BCMA polypeptide comprises a sequence selected from any one of SEQ ID NOs: 30, 67, and 68.

19. The fusion protein according to any one of claims 14 to 18, further comprising an immunoglobulin Fc region, preferably an IgG1 Fc region, more preferably the Fc region shown in SEQ ID NO:

3.

20. The fusion protein according to claim 19, wherein the Fc region comprises one or more amino acid mutations, wherein the amino acid mutations can reduce the binding of Fc to FcR, preferably a mutation that can reduce the binding of Fc to FcγR, more preferably any one of the following amino acid mutations or a combination of amino acid mutations: 234A; 235A; 234F; 235E; 234F and 235E; 234A and 235A; 234F, 235E and 331S; Mutation sites are defined according to the EU numbering system.

21. The fusion protein according to any one of claims 14 to 20, comprising a polypeptide chain as shown in any one of (I) to (VIII): (I) [TACI polypeptide]-[Linker 1]a-[BCMA polypeptide]-[Linker 2]b-[Fc region]e; (II) [BCMA polypeptide]-[Linker 1]a-[TACI polypeptide]-[Linker 2]b-[Fc region]e; (III) [Fc region] e-[Linker 3] c-[TACI polypeptide]-[Linker 4] d-[BCMA polypeptide]; (IV) [Fc region] e-[Linker 3] c-[BCMA polypeptide]-[Linker 4] d-[TACI polypeptide]; (V) [TACI polypeptide 1]-[Linker 1]a-[BCMA polypeptide 1]-[Linker 2]b-[Fc region]e-[Linker 3]c-[TACI polypeptide 2]-[Linker 4]d-[BCMA polypeptide 2]; (VI) [BCMA polypeptide 1]-[Linker 1]a-[TACI polypeptide 1]-[Linker 2]b-[Fc region]e-[Linker 3]c-[BCMA polypeptide 2]-[Linker 4]d-[TACI polypeptide 2]; (VII) [TACI polypeptide 1]-[Linker 1]a-[BCMA polypeptide 1]-[Linker 2]b-[Fc region]e-[Linker 3]c-[BCMA polypeptide 2]-[Linker 4]d-[TACI polypeptide 2]; (VIII) [BCMA polypeptide 1]-[Linker 1]a-[TACI polypeptide 1]-[Linker 2]b-[Fc region]e-[Linker 3]c-[TACI polypeptide 2]-[Linker 4]d-[BCMA polypeptide 2]; in, - represents a peptide bond, and the linker 1, linker 2, linker 3 and linker 4 may be the same or different; The TACI polypeptide 1 and the TACI polypeptide 2 are selected from the TACI polypeptides defined in any one of claims 14 to 16, and the TACI polypeptide 1 and the TACI polypeptide 2 may be the same or different; The BCMA polypeptide 1 and the BCMA polypeptide 2 are selected from the BCMA polypeptides defined in any one of claims 14 to 18, and the BCMA polypeptide 1 and the BCMA polypeptide 2 may be the same or different; a, b, c, d, and e are each independently 0 or 1; Preferably, the linker 1, linker 2, linker 3, and linker 4 are each independently (GxS) y , where x is an integer from 1 to 5, and y is an integer from 1 to 6; More preferably, the amino groups of the linker 1, linker 2, linker 3, linker 4, and linker 5 The amino acid sequences are each independently shown in any one of SEQ ID NOs: 39-41.

22. The fusion protein according to any one of claims 14 to 21, comprising the amino acid sequence shown in any one of SEQ ID NOs: 31 to 38, 69, and 70. The fusion protein according to any one of claims 14 to 22, which is a dimer.

24. A compound comprising: The TACI polypeptide defined in any one of claims 1 to 3, the anti-IFNAR1 antibody defined in any one of claims 4 to 8, and the BCMA polypeptide defined in claim 1 or 11; or It contains the TACI polypeptide defined in any one of claims 14-23 and the BCMA polypeptide defined in any one of claims 14-23.

25. A pharmaceutical composition comprising: The fusion protein of any one of claims 1 to 23, or the complex of claim 24, and One or more pharmaceutically acceptable carriers, diluents or excipients.

26. A polynucleotide encoding: The fusion protein according to any one of claims 1 to 23, or The complex of claim 24.

27. A host cell comprising or expressing the polynucleotide of claim 26.

28. A method for treating or ameliorating a disease or condition, comprising administering to a subject in need thereof a therapeutically effective amount of the fusion protein of any one of claims 1 to 23, the complex of claim 24, the pharmaceutical composition of claim 25, or the polynucleotide of claim 26; Preferably, the disease or condition is a B cell disorder or an autoimmune disease; More preferably, the B cell disorder or autoimmune disease is a disease or condition associated with TACI and / or BCMA expression; Most preferably, the autoimmune disease is systemic lupus erythematosus.

29. A method for preparing the fusion protein of any one of claims 1-23 or the complex of claim 24, comprising culturing the host cell of claim 27 to express the fusion protein or complex; optionally, comprising isolating or purifying the fusion protein or complex.

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