Fusion proteins containing TACI polypeptides and uses thereof

Abstract

The present disclosure provides fusion proteins comprising TACI polypeptides and uses thereof, particularly for preventing or treating autoimmune diseases.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This disclosure claims priority to a Chinese patent application filed on August 29, 2022, bearing application number CN202211041405.0 and entitled "Fusion protein containing TACI polypeptide and use thereof," the entire contents of which are incorporated herein by reference. [Technical Field]

[0002] The present disclosure relates to the biopharmaceutical field, and in particular, the disclosure relates to fusion proteins comprising TACI polypeptides and their use in treating diseases, particularly autoimmune diseases. [Background technology]

[0003] Systemic lupus erythematosus (SLE) is an autoimmune disease that threatens various tissues and organs (e.g., facial skin, kidneys, etc.). Its defining feature is the production of antibodies (e.g., antinuclear antibodies (ANA)) against antigens in the patient's own tissues. The etiology of SLE is complex, linked to numerous factors, including genetic inheritance, environment, hormones, and autoantibodies. The disease course is long, and clinical symptoms vary from person to person, resulting in high heterogeneity. Furthermore, SLE has a high morbidity rate, with over one million SLE patients in China alone. The global prevalence rate is approximately 30-50 per 100,000, with women 10 times more likely to develop the disease than men.

[0004] Activation of the type I interferon pathway plays an important role in the pathogenesis of SLE, promoting the activation of multiple types of immune cells, such as plasmacytoid dendritic cells (pDCs) and B cells, which are closely related to the development of SLE. 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 SLE Disease Activity Index (SLEDAI) score. Anifrolumab, developed by AstraZeneca, is a monoclonal antibody targeting type I interferon (IFN) receptor subunit 1 (IFNAR1) that controls the progression of SLE by antagonizing the associated activities of all type I interferons (e.g., IFN-α, IFN-β, and IFN-ω). In the TULIP-2 phase III clinical trial, patients receiving anifrolumab achieved improvement in the Integrated Lupus Assessment Index (BICLA) in 47.8% of patients compared with 31.5% of placebo-treated patients, suggesting that disease activity in all involved organs improved and that patients could receive reduced glucocorticoid doses. However, in another phase III clinical trial (TULIP-1) using the Lupus Response Index (SRI-4) as an endpoint, no significant improvement in disease scores was observed (36% vs. 40%). These results suggest the potential of targeting the type I interferon pathway for the treatment of SLE, but the therapeutic effect of targeting only IFNAR1 may not be sufficient.

[0005] B lymphocyte stimulator (BLyS, also known as BAFF) and proliferation-inducing ligand (APRIL) are important factors that promote the differentiation and maturation of B lymphocytes and the survival of plasma cells. They belong to the tumor necrosis factor (TNF) ligand family, and overexpression of these two factors is an important driver of various B lymphocyte-related autoimmune diseases, such as SLE. TNF ligand family members are generally synthesized as transmembrane proteins, and membrane-anchored proteins are often released from the cell surface after protease hydrolysis. BAFF is a type II transmembrane protein that exists in two forms: membrane-bound and soluble. Membrane-bound BAFF is cleaved by furin convertase to release the trimeric form, which is biologically active. BAFF is expressed in various cell types, including monocytes, dendritic cells, and bone marrow stromal cells. APRIL differs from other members of the TNF ligand family in that it is processed by furin convertase in the Golgi and then directly secreted outside the cell in a soluble trimeric form.

[0006] Currently, BAFF and APRIL have been identified as having three receptors: B cell maturation antigen (BCMA), transmembrane activator and CAMEL interactor (TACI), and BAFF receptor (BAFF-R, also known as Br3). All of these receptors belong to the TNF receptor family. The extracellular domains of TNF receptors contain multiple cysteine-rich domains (CRDs). Each CRD contains six cysteines that form three pairs of disulfide bonds, and the CRD domains function as ligand-binding domains. BAFF and APRIL have different binding affinities to the 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 unique high-affinity receptor for BAFF that does not bind to APRIL.

[0007] TACI is a type III transmembrane protein. Human TACI is a 293-amino acid polypeptide containing amino acid residues in the N-terminal region (amino acid residues 1-165), the transmembrane region (amino acid residues 166-186), and the intracellular region (amino acid residues 187-293). The extracellular region contains two CRDs (CRD1 and CRD2), and CRD1 has a weaker affinity for BAFF and APRIL than CRD2. Studies have shown that human TACI alone can produce a short form of TACI lacking the CRD1 domain by skipping exon 2. This short form of TACI can bind BAFF and APRIL as effectively as the long form. Because TACI has a relatively high affinity for BAFF and APRIL, fusing the soluble portion of the extracellular region of TACI to the Fc fragment of IgG blocks the binding of BAFF and APRIL to the TACI, BCMA, and BAFFR receptors on the cell membrane, inhibiting their biological activity and thereby treating autoimmune diseases. Atacicept is a BAFF / APRIL Trap developed by ZymoGenetics, and its molecular form is a native TACI extracellular domain-fused IgG1-Fc fragment (lacking FcγR binding activity), i.e., TACI-Fc. Telitaciccept (RCT-18) is a TACI-Fc developed by Rongchang Biosciences. Both telitacicept and atacicept can simultaneously target two cytokines, BAFF and APRIL. Compared to belimumab, which only inhibits BAFF, TACI-Fc, which simultaneously inhibits BAFF and APRIL, can more effectively reduce the body's immune response and achieve the goal of treating SLE. A phase II clinical study revealed that the high-dose telitacicept treatment group achieved the primary endpoint of a significantly higher 48-week lupus response index (SRI-4) score than the placebo control group (79.2% vs. 32.0%), suggesting that APRIL inhibition may be very important in the treatment of lupus erythematosus.

[0008] BCMA is a type III transmembrane protein. Human BCMA is a 184-amino acid polypeptide containing amino acid residues in the N-terminal region (amino acid residues 1-50), the transmembrane region (amino acid residues 51-93), and the intracellular region (amino acid residues 94-184). BCMA contains one CRD in the extracellular region and binds to APRIL more strongly than TACI. BCMA promotes plasma cell survival and can adversely affect autoimmune diseases. BCMA is also involved in the development of humoral immunity (e.g., antibody production) and immune-related diseases. The BCMA extracellular region or its fusion proteins have not yet entered clinical use.

[0009] Conventional treatments for SLE primarily include antimalarials, glucocorticoids, and immunosuppressants. Several types of hormone therapy are the basic drug therapy, and clinicians adjust hormone dosage according to the disease activity of SLE patients and then add immunosuppressants, biologics, or other treatments accordingly. Compared with rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis, very few new treatments, especially biologics, have been approved for SLE. While traditional hormones are widely used as basic treatments, they have poor selectivity and can cause side effects with long-term use. Belimumab is the only new targeted therapy approved in China in the past 50 years. Therefore, the development of safe and effective new targeted drugs is urgently needed to address the enormous clinical needs of SLE.

[0010] Because overexpression of BAFF / APRIL is closely associated with autoimmune diseases, blocking its activity as a target molecule can reduce the onset of disease and alleviate its symptoms, thereby achieving the goal of alleviating and treating autoimmune diseases. Receptors involved in blocking BAFF / APRIL signaling are expected to be able to act specifically on target cells and become new biological agents for treating autoimmune diseases. Therefore, there is a strong need in the field for the development of novel compounds that can effectively inhibit or block the BAFF / APRIL pathway. The present disclosure provides fusion proteins containing a TACI polypeptide that could potentially be used as drugs for the safe and effective clinical treatment of autoimmune diseases (e.g., SLE). Summary of the Invention

[0011] The present disclosure provides novel fusion proteins comprising TACI polypeptides, their encoding nucleic acids, vectors, host cells, pharmaceutical compositions, methods for treating or ameliorating diseases (e.g., B cell disorders or autoimmune diseases), and pharmaceutical uses thereof.

[0012] fusion proteins The present disclosure provides: (1) a structure comprising a TACI polypeptide; (2) a structure comprising a BCMA polypeptide; (3) a structure comprising a TACI polypeptide and a BCMA polypeptide; (4) a structure comprising a TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody); (5) A structure comprising a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody); (6) A structure comprising a TACI polypeptide, a BCMA polypeptide, and an antibody (e.g., an anti-IFNAR1 antibody).

[0013] The present invention provides a fusion protein having a structure selected from the following: Regarding the TACI polypeptide in the fusion protein: In some embodiments, the TACI polypeptide comprises CRD1 and / or CRD2 of the TACI extracellular domain, for example, CRD2 of the TACI extracellular domain.

[0014] In some embodiments, the TACI polypeptide is (1) amino acid residues 68 to 105 of SEQ ID NO: 1 or a mutant thereof; (2) SEQ ID NO: 1 or a polypeptide having at least 90% sequence identity thereto; (3) amino acid residues 33 to 67 of SEQ ID NO: 1; (4) amino acid residues 70 to 104 of SEQ ID NO: 1 or a mutant thereof; (5) amino acid residues at positions 30 to 110, 69 to 111, 69 to 112, 13 to 118, or 33 to 104 of SEQ ID NO: 1, or a mutant thereof; (6) Amino acid residues at positions 68 to 106, 68 to 107, or 68 to 108 of SEQ ID NO: 1, or a mutant thereof The present invention includes any one polypeptide selected from (1) to (6).

[0015] In some specific embodiments, the mutant has one or more amino acid mutations selected from positions 69, 72, 73, 77, 85, 102, and 103.

[0016] In some specific embodiments, the variant has one or more amino acid replacements selected from 69T or 69R, 72S, 73E or 73Q, 77E, 85T or 85A, 102A or 102R, 103Y.

[0017] In some specific embodiments, the variant has any one amino acid substitution or combination of substitutions selected from 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.

[0018] In some specific embodiments, the present disclosure indicates that Mutation 1 and Mutation 2 are present together in a mutant after mutation by "Mutation 1 / Mutation 2." 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.

[0019] In some specific embodiments, the mutant has the following combination of amino acid substitutions: 69T / 73E / 77E / 102A, for example, the following combination of amino acid substitutions: L69T / K73E / K77E / Y102A.

[0020] The site of the above amino acid mutation is an amino acid residue site numbered according to the natural order of the TACI extracellular domain (sequence number 1), and for example, ``the above mutant has an amino acid mutation selected from position 69'' means that it has a mutation corresponding to the 69th amino acid residue of sequence number 1.

[0021] In some embodiments, the amino acid sequence of the TACI polypeptide comprises any one of the amino acid sequences selected from SEQ ID NOs: 1, 2, and 6-29.

[0022] In the present disclosure, "at least 90% sequence identity" covers 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, and "at least 80% sequence identity" covers 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.

[0023] Regarding the BCMA polypeptide in the fusion protein: In some embodiments, the BCMA polypeptide comprises CRD1 of the BCMA extracellular domain.

[0024] In some embodiments, the BCMA polypeptide comprises amino acid residues 7-41 of SEQ ID NO:30.

[0025] In some embodiments, the BCMA polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 30, 67, 68, or having at least 90% sequence identity thereto.

[0026] Regarding antibodies in fusion proteins: In some embodiments, the antibody in the fusion protein is an anti-IFNAR1 antibody or an antigen-binding fragment thereof.

[0027] 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, whose amino acid sequences are set forth in SEQ ID NOs: 45 to 47, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, whose amino acid sequences are set forth in SEQ ID NOs: 48 to 50, respectively.

[0028] In some embodiments, the anti-IFNAR1 antibody has a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 43 or having at least 90% sequence identity thereto, and a light chain variable region amino acid sequence set forth in SEQ ID NO: 44 or having at least 90% sequence identity thereto.

[0029] In some embodiments, the anti-IFNAR1 antibody has a heavy chain amino acid sequence set forth in SEQ ID NO: 55 or has at least 80% sequence identity thereto, and a light chain amino acid sequence set forth in SEQ ID NO: 54 or has at least 80% sequence identity thereto.

[0030] In some embodiments, the anti-IFNAR1 antibody has a heavy chain amino acid sequence set forth in SEQ ID NO: 61 or has at least 80% sequence identity thereto, and a light chain amino acid sequence set forth in SEQ ID NO: 54 or has at least 80% sequence identity thereto.

[0031] In some embodiments, the present disclosure incorporates anifrolumab and sifalimumab as anti-IFNAR1 antibodies according to the present disclosure, and also includes the antibodies disclosed in WO20062002177A, WO2009100309A (e.g., 3F11, 4G5, 11E2, 9D4), WO2020156474A (e.g., 7G4, 10C5, etc.), WO20200575A (e.g., 5G4, 5G5, 5G6, 5G7, 5G8, 5G9, 5G10, 5G11, 5G20, 5G3, 5G4, 5G5, 5G6, 5G7, 5G8, 5G9, 5G10, 5G11, 5G12, 5G13, 5G14, 5G15, 5G16, 5G17, 5G18, 5G19, 5G20, 5G21, 5G22, 5G23, 5G24, 5G25, 5G26, 5G27, 5G28, 5G29, 5G30, 5G31, 5G32, 5G33, 5G34, 5G35, 5G36, 5G37, 5G38, 5G40, 5G41, 5G42, 5G43, 5G44, 5G45, 5G46, 5G47, 5G48, 5G49, 5G51, 5G52, 5G53, 5G54, 5G55, 5G56, 5G57, 5G 41A (e.g., 8G11H and 485G10H), CN201610634601.7 (e.g., H19B7+L16C11, H19B7+L8C3, H15D10+L16C11, H15D10+L8C3 or anti-IFNAR1-C1), CN201510685200.X, or WO2012162367A, incorporating an anti-IFNAR1 antibody or an antigen-binding fragment thereof.

[0032] About the fusion protein: 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).

[0033] 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 associable subunits. In some embodiments, the two subunits are a first subunit and a second subunit, which may be the same or different. In some embodiments, the Fc region is an Fc region of IgG, IgA, IgM, or IgD, and in some embodiments, the Fc region is an Fc region of human IgG1, IgG2, IgG3, or IgG4, for example, an Fc region of human IgG1, or, for example, an Fc region set forth in SEQ ID NO: 3.

[0034] In some embodiments, the Fc region in the fusion protein comprises one or more amino acid substitutions compared to a wild-type Fc region, wherein the amino acid substitutions are capable of reducing binding to an Fc receptor (FcR); in some embodiments, the amino acid substitutions are capable of reducing binding to an Fc gamma receptor (FcγR); and 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, wherein the mutation sites are numbered according to the EU index.

[0035] In some embodiments, the Fc region of the fusion protein comprises a first subunit and a second subunit capable of associating with each other, wherein 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 knob-in-hole structure and the second subunit has a hole structure, or the first subunit has a hole structure and the second subunit has a knob-in-hole structure. In some embodiments, the amino acid residue substitutions of the first subunit include one or more amino acid substitutions selected from positions 354, 356, 358, and 366, and the amino acid residues of the second subunit include one or more amino acid substitutions selected from positions 349, 356, 358, 366, 368, and 407. In some embodiments, the first subunit comprises one or more amino acid substitutions selected from 354C, 356E, 358M, and 366W, and the second subunit comprises 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 354C, 356E, 358M, and 366W, and the second subunit comprises amino acid substitutions 349C, 356E, 358M, 366S, 368A, and 407V.

[0036] In a first embodiment, for a fusion protein comprising a TACI polypeptide and a BCMA polypeptide: 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.

[0037] In some embodiments, the fusion protein comprises one or more (e.g., 2, 3, 4, 5, 6) of the TACI polypeptides and one or more (e.g., 2, 3, 4, 5, 6) of the BCMA polypeptides. In some embodiments, the fusion protein comprises one of the TACI polypeptides and one of the BCMA polypeptides, or two of the TACI polypeptides and two of the BCMA polypeptides, or three of the TACI polypeptides and three of the BCMA polypeptides, or four of the TACI polypeptides and four of the BCMA polypeptides, or two of the TACI polypeptides and one of the BCMA polypeptides, or one of the TACI polypeptides and two of the BCMA polypeptides, or three of the TACI polypeptides and one of the BCMA polypeptides, or one of the TACI polypeptides and three of the BCMA polypeptides, and when it comprises two or more of the TACI polypeptides, the TACI polypeptides may be TACI polypeptides having the same or different sequences as provided in the present disclosure, and when it comprises two or more of the BCMA polypeptides, the BCMA polypeptides may be BCMA polypeptides having the same or different sequences as provided in the present disclosure.

[0038] In some embodiments, the fusion protein includes a TACI polypeptide linked to one subunit of the Fc region in any order, and optionally, the TACI polypeptide is linked to the first or second subunit of the Fc region via a linker or directly. In some embodiments, the fusion protein includes a BCMA polypeptide linked to one subunit of the Fc region in any order, and 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 and the N-terminus of the first or second subunit of the Fc region are linked directly or via a linker, or the N-terminus of the TACI polypeptide is linked directly or via a linker to the C-terminus of the first or second subunit of the Fc region. In some embodiments, the C-terminus of the BCMA polypeptide and the N-terminus of the first or second subunit of the Fc region are linked via a linker or directly, or the N-terminus of the BCMA polypeptide is linked via a linker or directly to the C-terminus of the first or second subunit of the Fc region.

[0039] In some embodiments, the fusion protein comprises the TACI polypeptide and the BCMA polypeptide, which comprises: (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]; The polypeptide chain is represented by any one of (I) to (VIII): wherein - represents a peptide bond, linker is a polypeptide capable of performing 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 the TACI polypeptides provided in the present disclosure and may be the same or different, BCMA polypeptide 1 and BCMA polypeptide 2 are selected from the BCMA polypeptides provided in the present disclosure and may be the same or different, and 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, b is 1, and e is 0 or 1, for example, scheme (III) Or in (IV), c and d are all 1, or c is 1 and d is 0, or c is 0, d is 1, and e is 0 or 1; for example, in scheme (V), (VI), (VII) or (VIII), a, b, c, and d are all 1, or a, b, and c are all 1 and d is 0, or a and b are 1 and c and d are 0, or a is 1 and b, c, and d are all 0, or a is 0 and b, c, and d are all 1, or a and b are all 0 and c and d are all 1, or a, b, and c are 0 and d is 1, or a and c are 1 and b and d are 0, or a and c are 0 and b and d are 1, and e is 0 or 1.

[0040] In some embodiments, the linker is 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 integers of 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and h is independently selected from integers of 1 to 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 specific embodiments, the linker is (GxS). yA linker, wherein x is selected from an integer of 1 to 5 and y is selected from an integer of 1 to 6, is, for example, a linker represented by any one of SEQ ID NOs: 39 to 41. For example, the linker is (G4S) y (SEQ ID NO: 78), and y is independently selected from integers of 1 to 6 (for example, y is 1, 2, 3, 4, 5, or 6).

[0041] In some embodiments, a fusion protein comprising a TACI polypeptide and a BCMA polypeptide is provided, which comprises a polypeptide set forth in any one of SEQ ID NOs: 31-38, 69, and 70, or having at least 90% sequence identity thereto.

[0042] In some embodiments, the fusion protein is a multimer, eg, a dimer (eg, a homodimer or heterodimer).

[0043] In some embodiments, a fusion protein comprising a TACI polypeptide and a BCMA polypeptide is provided, wherein the TACI polypeptide may be a mutant, and the mutant has 1 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations. In some embodiments, a fusion protein comprising a TACI polypeptide and a BCMA polypeptide is provided, wherein the BCMA polypeptide may be a mutant, and the mutant has 1 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations. The amino acid mutations may be conservative substitutions, substitutions or modifications, and / or deletions or additions that do not affect function.

[0044] In some embodiments, a protein or molecule is provided that binds to BAFF and / or APRIL in competition with a fusion protein comprising the TACI polypeptide and a BCMA polypeptide, the TACI polypeptide, or the BCMA polypeptide, or that blocks the binding of the fusion protein comprising the TACI polypeptide and a BCMA polypeptide, the TACI polypeptide, or the BCMA polypeptide to BAFF and / or APRIL.

[0045] In a second embodiment, for a fusion protein comprising a TACI polypeptide and an anti-IFNAR1 antibody: 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.

[0046] In some embodiments, the fusion protein comprising the 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 the TACI polypeptide and an anti-IFNAR1 antibody comprises one or more (e.g., 2, 3, 4) anti-IFNAR1 antibodies.

[0047] In some embodiments, the TACI polypeptide is a fusion protein comprising an anti-IFNAR1 antibody, which is (I) a first polypeptide chain which is, from the N-terminus to the C-terminus, [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 is, from the N-terminus to the 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 that is an antibody heavy chain, and a second polypeptide chain that is, from the N-terminus to the C-terminus, [TACI polypeptide]-[linker 3]c-[antibody light chain]; (IV) a first polypeptide chain that is an antibody heavy chain, and a second polypeptide chain that is, from the N-terminus to the C-terminus, [antibody light chain]-[linker 4]d-[TACI polypeptide]; (V) a first polypeptide chain consisting of, from the N-terminus to the C-terminus, [TACI polypeptide 1]-[linker 1]a-[antibody heavy chain]; and a second polypeptide chain consisting of, from the N-terminus to the C-terminus, [antibody light chain]-[linker 4]d-[TACI polypeptide 2]. (VI) a first polypeptide chain consisting of, from the N-terminus to the C-terminus, [antibody heavy chain]-[linker 2]b-[TACI polypeptide 1]; and a second polypeptide chain consisting of, from the N-terminus to the C-terminus, [TACI polypeptide 2]-[linker 3]c-[antibody light chain]. (VII) a first polypeptide chain which is, from the N-terminus to the 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 that is an antibody heavy chain, and a second polypeptide chain that is, from the N-terminus to the C-terminus, [TACI polypeptide 1]-[linker 3]c-[antibody light chain]-[linker 4]d-[TACI polypeptide 2]; The polypeptide chain is represented by any one of (I) to (VIII): wherein - represents a peptide bond, linker is a polypeptide capable of performing 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 one of the above-mentioned TACI polypeptides of the present disclosure, 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 may simultaneously be 0 or 1, or a may be 0 and d may be 1, or a may be 1 and d may be 0; in scheme (VI), b and c may simultaneously be 0 or 1, or b may be 0 and c may be 1, or b may be 1 and c may be 0; in scheme (VII), a and b may simultaneously be 0 or 1, or a may be 0 and b may be 1, or a may be 1 and b may be 0; and in scheme (VIII), c and d may simultaneously be 0 or 1, or c may be 0 and d may be 1, or c may be 1 and d may be 0.

[0048] In some embodiments, the linker is (G m S n ) h or (GGNGT) hor (YGNGT) h or (EPKSS) h wherein m and n are each independently selected from integers of 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and h is independently selected from integers of 1 to 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 specific embodiments, the linker is (GxS). y A linker, wherein x is selected from an integer of 1 to 5 and y is selected from an integer of 1 to 6, is, for example, a linker represented by any one of SEQ ID NOs: 39 to 41. For example, the linker is (G4S) y wherein y is independently selected from integers of 1 to 6 (for example, y is 1, 2, 3, 4, 5, or 6).

[0049] In some embodiments, a fusion protein is provided comprising a TACI polypeptide and an anti-IFNAR1 antibody, the fusion protein comprising: a first polypeptide chain set forth in any one of SEQ ID NOs: 51-53, 62, or having at least 80%, at least 90% sequence identity thereto; and a second polypeptide chain set forth in SEQ ID NO: 54, or having at least 80%, at least 90% sequence identity thereto; a first polypeptide chain set forth in SEQ ID NO: 55 or having at least 80%, at least 90% sequence identity thereto, and a second polypeptide chain set forth in any one of SEQ ID NOs: 56-60 or having at least 80%, at least 90% sequence identity thereto; or The antibody comprises a first polypeptide chain having a sequence identity of at least 80%, at least 90% thereto, as set forth in SEQ ID NO: 61, and a second polypeptide chain having a sequence identity of at least 80%, at least 90% thereto, as set forth in any one of SEQ ID NOs: 56 to 60.

[0050] In some embodiments, the fusion protein comprises two identical first polypeptide chains and two identical second polypeptide chains.

[0051] 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 mutant, and the mutant has 1 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations, and the amino acid mutations may be conservative substitutions, substitutions or modifications, and / or deletions or additions that do not affect function.

[0052] In some embodiments, a protein or molecule is provided that competes with the fusion protein comprising the TACI polypeptide and an anti-IFNAR1 antibody to bind to BAFF and / or APRIL or blocks its binding to BAFF and / or APRIL.

[0053] In a third aspect, for a fusion protein comprising a TACI polypeptide, a BCMA polypeptide and an anti-IFNAR1 antibody: In some embodiments, the TACI polypeptide is any one of the TACI polypeptides provided in the present disclosure, the BCMA polypeptide is any one of the BCMA polypeptides provided in the present disclosure, and the anti-IFNAR1 antibody is any one of the anti-IFNAR1 antibodies provided in the present disclosure.

[0054] In some embodiments, the antibody fusion protein comprising the 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 the 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 the 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 antibody fusion protein comprising the TACI polypeptide, BCMA polypeptide, and anti-IFNAR1 antibody comprises one of the TACI polypeptides and one of the BCMA polypeptides, or two of the TACI polypeptides and two of the BCMA polypeptides, or three of the TACI polypeptides and three of the BCMA polypeptides, or four of the TACI polypeptides and four of the BCMA polypeptides, or two of the TACI polypeptides and one of the BCMA polypeptides, or one of the TACI polypeptides and two of the BCMA polypeptides, or three of the TACI polypeptides and one of the BCMA polypeptides, or one of the TACI polypeptides and three of the BCMA polypeptides, and one anti-IFNAR1 antibody; when comprising two or more of the TACI polypeptides, the TACI polypeptides may be TACI polypeptides having the same or different sequences as provided in the present disclosure, and when comprising two or more of the BCMA polypeptides, the BCMA polypeptides may be BCMA polypeptides having the same or different sequences as provided in the present disclosure.

[0055] In some embodiments, a fusion protein comprising any one of the above TACI polypeptides, a BCMA polypeptide, and an anti-IFNAR1 antibody, which is (I) a first polypeptide chain which is, from the N-terminus to the 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 is, from the N-terminus to the 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 that is an antibody heavy chain, and a second polypeptide chain that is, from the N-terminus to the C-terminus, [TACI polypeptide and BCMA polypeptide domain]-[linker 3]c-[antibody light chain]; (IV) a first polypeptide chain that is an antibody heavy chain; and a second polypeptide chain that is, from the N-terminus to the C-terminus, [antibody light chain]-[linker 4]d-[TACI polypeptide and BCMA polypeptide domain]; (V) a first polypeptide chain which is, from the N-terminus to the C-terminus, [TACI polypeptide and BCMA polypeptide domain 1]-[linker 1]a-[antibody heavy chain]; and a second polypeptide chain which is, from the N-terminus to the C-terminus, [antibody light chain]-[linker 4]d-[TACI polypeptide and BCMA polypeptide domain 2]; (VI) a first polypeptide chain which is, from the N-terminus to the C-terminus, [antibody heavy chain]-[linker 2]b-[TACI polypeptide and BCMA polypeptide domain 1]; and a second polypeptide chain which is, from the N-terminus to the C-terminus, [TACI polypeptide and BCMA polypeptide domain 2]-[linker 3]c-[antibody light chain]; (VII) a first polypeptide chain which is, from the N-terminus to the C-terminus, [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 that is an antibody heavy chain, and a second polypeptide chain that is, from the N-terminus to the C-terminus, [TACI polypeptide and BCMA polypeptide domain 1]-[linker 3]c-[antibody light chain]-[linker 4]d-[TACI polypeptide and BCMA polypeptide domain 2]; The polypeptide chain is represented by any one of (I) to (VIII): Among them, "[TACI polypeptide and BCMA polypeptide domain]", "[TACI polypeptide and BCMA polypeptide domain 1]", and "[TACI polypeptide and BCMA polypeptide domain 2]" are, from the N-terminus to the C-terminus, [TACI polypeptide]-[linker 5]e-[BCMA polypeptide], or [BCMA polypeptide]-[linker 5]e-[TACI polypeptide], and the TACIs in "TACI polypeptide and BCMA polypeptide domain 1" and "TACI polypeptide and BCMA polypeptide domain 2" may be the same or different, and the BCMAs may be the same or different, wherein - represents a peptide bond, linker is a polypeptide capable of performing a linking function, linker 1, linker 2, linker 3, linker 4, and linker 5 may be the same or different, and 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 may simultaneously be 0 or 1, or a may be 0 and d may be 1, or a may be 1 and d may be 0; in scheme (VI), b and c may simultaneously be 0 or 1, or b may be 0 and c may be 1, or b may be 1 and c may be 0; in scheme (VII), a and b may simultaneously be 0 or 1, or a may be 0 and b may be 1, or a may be 1 and b may be 0; in scheme (VIII), c and d may simultaneously be 0 or 1, or c may be 0 and d may be 1, or c may be 1 and d may be 0; and in the above schemes, e may independently be 0 or 1.

[0056] In some embodiments, the linker is (G m Sn ) h or (GGNGT) h or (YGNGT) h or (EPKSS) h wherein m and n are each independently selected from integers of 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and h is independently selected from integers of 1 to 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 specific embodiments, the linker is (GxS). y A linker, wherein x is selected from an integer of 1 to 5 and y is selected from an integer of 1 to 6, is, for example, a linker represented by any one of SEQ ID NOs: 39 to 41. For example, the linker is (G4S) y wherein y is independently selected from integers of 1 to 6 (for example, y is 1, 2, 3, 4, 5, or 6).

[0057] In some embodiments, a fusion protein is provided comprising a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody, the fusion protein comprising a first polypeptide chain set forth in SEQ ID NO: 55 or having at least 80%, at least 90% sequence identity thereto, and a second polypeptide chain set forth in SEQ ID NOs: 63-66, 71, 72 or having at least 80%, at least 90% sequence identity thereto; a first polypeptide chain set forth in SEQ ID NO: 61 or having at least 80%, at least 90% sequence identity thereto, and a second polypeptide chain set forth in any one of SEQ ID NOs: 63-66, 71, 72 or having at least 80%, at least 90% sequence identity thereto; or a first polypeptide chain 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 set forth in SEQ ID NO: 54 or having at least 80%, at least 90% sequence identity thereto.

[0058] In some embodiments, the fusion protein comprises two identical first polypeptide chains and two identical second polypeptide chains.

[0059] 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 mutant, and the mutant has 1 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations, and the amino acid mutations may be conservative substitutions, substitutions or modifications, and / or deletions or additions that do not affect function.

[0060] In some embodiments, a protein or molecule is provided that binds to BAFF and / or APRIL in competition with or blocks the binding of a fusion protein comprising the above-mentioned TACI polypeptide, BCMA polypeptide, and anti-IFNAR1 antibody to BAFF and / or APRIL.

[0061] Regarding the fusion proteins according to the first to third aspects: In some embodiments, the fusion protein comprising a TACI polypeptide and a BCMA polypeptide, a TACI polypeptide and an IFNAR1 antibody, or a fusion protein comprising a TACI polypeptide, a BCMA polypeptide, and an IFNAR1 antibody has one or more of the following properties or functions: (a) BAFF-binding activity; (b) APRIL-binding activity; (c) a reduction in serum immunoglobulin (e.g., IgE or IgM) concentrations; (d) reduction in spleen weight; (e) inhibition or blocking of the BAFF / APRIL pathway; (f) Decrease in B cell numbers.

[0062] In some embodiments, a protein comprising a TACI polypeptide (e.g., TACI-Fc) provided herein has one or more of the following functional activities: a. The TACI polypeptide is not susceptible to cleavage fragments, which in some embodiments can be detected by mass spectrometry, e.g., the TACI cleavage assay method described in Example 2; b. Blocking the binding of BAFF to BAFF-R, and in some embodiments, TACI-Fc is an IC that blocks the binding of BAFF to BAFF-R. 50 values ​​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, 50 The IC value is detected by an ELISA method, and in some embodiments, 50 The test method for the value is shown in Example 1. c. Ability to bind to BAFF, and in some embodiments, the EC of the TACI-Fc that binds to BAFF. 50 The EC value is less than or equal to 5 nM, 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, and 50 The value is detected by an ELISA method, for example, the ELISA binding experiment of Example 2 (antigen protein coated), d. Inhibiting the induction of B cell proliferation by BAFF, and in some embodiments, IC20 of TACI-Fc inhibiting the induction of B cell proliferation by BAFF. 50 The IC value is less than 0.3 nM, less than 0.2 nM, less than 0.1 nM, less than 0.06 nM, less than 0.05 nM, less than 0.01 nM or less. 50 The value is detected by an ELISA method, for example, the ELISA experiment in Example 17, e. Inhibiting the induction of B cell proliferation by APRIL, and in some embodiments, IC 50 values ​​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, less than 0.2 nM, less than 0.1 nM, less than 0.05 or less, 50 The values ​​were detected by ELISA method, f. binds to human BAFF and / or human APRIL with high affinity, wherein in some embodiments, the affinity is detected by Biacore method, and in some embodiments, the K value of the TACI-Fc fusion protein binding to human BAFF is less than 1E-10 M, 9E-11 M, 8E-11 M or less, and in some embodiments, the K value of the TACI-Fc fusion protein binding to human APRIL is less than 2.3E-11 M, 2.0E-11 M, 1.9E-11 M, 1.3E-11 M or less; g. good in vivo pharmacokinetics, in some embodiments, the half-life of TACI-Fc in rats is greater than 4 days; and / or h. It has good stability, and in some embodiments, the purity of the TACI-Fc fusion protein can still maintain 94% or more (SEC%) even after storage at a constant temperature of 40°C for 4 weeks, and in some embodiments, the pI value of the TACI-Fc is less than 9, less than 8, less than 7, less than 6 or less, and the pI value is analyzed and measured using the ``18cProt / TrEMBL'' system.

[0063] In some embodiments, a fusion protein comprising a TACI polypeptide and a BCMA polypeptide provided herein has one or more of the following functional activities: a. The TACI polypeptide is not susceptible to cleavage fragments, which in some embodiments can be detected by mass spectrometry, e.g., the TACI cleavage assay method described in Example 2; b. Ability to bind to BAFF, and in some embodiments, the EC of the fusion protein that binds to BAFF. 50 The EC value is less than or equal to 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, or less. 50 The value is detected by an ELISA method, for example, the ELISA experiment of Example 5, c. Ability to bind to APRIL, and in some embodiments, the EC of the fusion protein that binds to APRIL. 50The EC value is less than or equal to 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, or less. 50 The value is detected by an ELISA method, for example, the ELISA experiment of Example 5, d. Good in vivo pharmacokinetics; e. Good stability.

[0064] In some embodiments, an antibody fusion protein comprising a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody provided herein has one or more of the following functional activities: a. The TACI polypeptide is not susceptible to cleavage fragments, which in some embodiments can be detected by mass spectrometry, e.g., the TACI cleavage assay method described in Example 2; In some embodiments, the fusion protein has an IFN activity inhibitory rate that is consistent with or similar to that of an anti-IFNAR1 antibody (e.g., anifrolumab), and the inhibitory rate is detected in vitro by detecting an IFN-α / β reporter gene, such as in the experiment in Example 10; or in vivo by detecting the mRNA expression of an IFN-α downstream gene ISG in PBMC, such as in the experiment in Example 21. c. Ability to bind to BAFF, and in some embodiments, the EC of the fusion protein that binds to BAFF. 50 The EC values ​​are less than or equal to 1.5 nM, 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, and 50 The value is detected by ELISA method, for example, the ELISA experiment of Example 11, d. Ability to bind to APRIL, and in some embodiments, the EC of the fusion protein that binds to APRIL. 50 The EC value is less than or equal to 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, or less. 50 The value is detected by ELISA method, for example, the ELISA experiment of Example 11, e. inhibiting IFN-α secretion; in some embodiments, the fusion protein has an inhibitory ability on IFN-α cytokine secretion from plasmacytoid dendritic cells (pDCs) that is consistent with or similar to that of an anti-IFNAR1 antibody (e.g., anifrolumab), as detected, for example, by the detection method in Example 15; in some embodiments, the fusion protein has an inhibitory ability on IFN-α-induced in vitro differentiation of B cells into plasma cells that is consistent with or similar to that of an anti-IFNAR1 antibody (e.g., anifrolumab), as detected, for example, by the detection method in Example 16; f. inhibiting plasma cell differentiation and inhibiting BAFF-induced B cell proliferation. In some embodiments, the fusion protein inhibits BAFF-induced B cell proliferation by at least 50%, at least 40%, at least 30%, at least 20%, or at least 10%, where 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 an equal concentration of IgG1, as detected by FACS, e.g., the method in Example 17; g. inhibiting APRIL-induced B cell proliferation, and in some embodiments, the fusion protein inhibits APRIL-induced B cell proliferation by at least 50%, at least 40%, at least 30%, at least 20%, or at least 10%, where 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 an equal concentration of IgG1, and the inhibition rate is detected by FACS, e.g., the method in Example 18; h. inhibiting BAFF- and APRIL-induced plasma cell production, and 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 is measured as the ratio of the number of B cells after addition of the antibody fusion protein to the number of B cells after addition of an equal concentration of IgG1, and wherein the inhibition is detected by FACS, e.g., the method in Example 19; i. inhibiting in vivo IgA production, and in some embodiments, the fusion protein inhibits in vivo IgA production by at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, or at least 30%, relative to an equal concentration of PBS, and the inhibition is detected by ELISA, e.g., the method in Example 22; j. Good in vivo pharmacokinetics; k. Good stability.

[0065] Furthermore, in some embodiments, there are provided products or combinations comprising any one of the fusion proteins comprising a TACI polypeptide and a BCMA polypeptide of the present disclosure, and further comprising an antibody. In some embodiments, there are provided products or combinations comprising any one of the TACI polypeptides of the present disclosure, and further comprising an antibody. In some embodiments, the antibody is an anti-IFNAR1 antibody, such as anifrolumab.

[0066] In some embodiments, a composite is provided comprising any one of the TACI polypeptides disclosed herein and any one of the BCMA polypeptides disclosed herein, optionally operably linked directly or via any one of the linkers disclosed herein, or unlinked.

[0067] In some embodiments, a composite is provided comprising any one of the TACI polypeptides disclosed herein and any one of the BCMA polypeptides disclosed herein, or any one of the TACI polypeptides disclosed herein and any one of the anti-IFNAR1 antibodies disclosed herein, or any one of the BCMA polypeptides disclosed herein and any one of the anti-IFNAR1 antibodies disclosed herein, or any one of the TACI polypeptides disclosed herein, any one of the anti-IFNAR1 antibodies disclosed herein, and any one of the BCMA polypeptides disclosed herein. Optionally, the TACI polypeptide, BCMA polypeptide, and anti-IFNAR1 antibody may or may not be operably linked directly or via any one of the linkers disclosed herein.

[0068] Polynucleotides and Vectors The present disclosure provides an (isolated) polynucleotide encoding any one 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, a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), products, combinations, or composites according to the present disclosure. The polynucleotide may be DNA or RNA (e.g., mRNA).

[0069] Nucleic acids according to the present disclosure may 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 in particular be an expression vector, i.e., a vector that allows for the 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). Such expression vectors typically contain at least one nucleic acid according to the present disclosure, operably linked to one or more suitable expression control elements (e.g., promoter, enhancer, terminator, 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 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, a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), a product, combination or composite of the present disclosure include, for example, promoters, enhancers, terminators, integration factors, selectable markers, leader sequences, and reporter genes.

[0070] Nucleic acids according to the present disclosure may be prepared or obtained by known methods (e.g., automated DNA synthesis and / or recombinant DNA techniques) based on information about the amino acid sequence of a polypeptide according to the present disclosure, and / or may be isolated from a suitable natural source.

[0071] host cell The present disclosure provides recombinant host cells that express or are capable of expressing one or more 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, a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), products, combinations or composites of the present disclosure, and / or that contain a nucleic acid or vector of the present disclosure.

[0072] In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0073] Bacterial cells include, for example, cells of Gram-negative strains (e.g., Escherichia coli, Proteus, and Pseudomonas) and Gram-positive strains (e.g., Bacillus, Streptomyces, Staphylococcus, and Lactococcus).

[0074] Fungal cells include, for example, cells of species of Trichoderma, Neurospora, and Aspergillus, or cells of species of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0075] Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0076] However, the present disclosure may also be used with amphibian cells, insect cells, plant cells and any other cells in the art for expressing heterologous proteins.

[0077] The cells of the present disclosure are incapable of developing into complete plants or animals.

[0078] Preparation method 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, a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), a product, combination, or composite, which comprises expressing the polypeptide, fusion protein, product, combination, or composite in a host cell as described above and isolating the polypeptide, fusion protein, product, combination, or composite from the host cell. Optionally, a purification step may be further included. Optionally, a filtration or concentration step may be further included. Soluble components and polymers may be removed by conventional methods such as molecular sieving or ion exchange. The resulting product should be immediately frozen, e.g., at −70° C., or lyophilized.

[0079] The polypeptides, fusion proteins, products, combinations, or composites can be prepared and purified by conventional methods. For example, a cDNA sequence encoding the fusion protein can be cloned and recombined into an expression vector. The recombinant expression vector can be stably transfected into CHO cells. Mammalian expression systems result in glycosylation of the protein (e.g., at the highly conserved N-terminus of the Fc region). Positive clones are selected and expanded in a bioreactor culture to produce the protein. The culture medium from which the protein is secreted can be purified, collected, filtered, or concentrated by conventional techniques. Soluble compounds and multimers may be removed by conventional methods such as molecular sieving or ion exchange. However, the polypeptides, fusion proteins, products, combinations, or composites of the present disclosure may also be obtained by other protein production methods known in the art, such as chemical synthesis, including solid-phase or liquid-phase synthesis.

[0080] composition The present disclosure provides compositions, such as pharmaceutical compositions, comprising 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, a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), a product, combination, composite or polynucleotide, and one or more medicament excipients, diluents or carriers.

[0081] In some specific embodiments, a unit dose of the pharmaceutical composition may contain 0.01 to 99% by weight of any one of the above polypeptides, fusion proteins, products, combinations, composites, or polynucleotides. In some specific embodiments, the content of any one of the above polypeptides, fusion proteins, products, combinations, composites, or polynucleotides in a unit dose of the pharmaceutical composition is 0.1 to 2000 mg, and in some specific embodiments, 1 to 1000 mg.

[0082] Reagent Kit The present disclosure 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, a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), a product, combination, composite, or reagent kit comprising a polynucleotide according to the present disclosure. In some embodiments, diagnostic reagents comprising the polynucleotides are also provided, and related diagnostic uses are provided.

[0083] Methods for treating diseases and pharmaceutical uses In some embodiments, the present disclosure provides a method of treating or ameliorating a disease or condition, comprising administering to a subject in need thereof a therapeutically effective amount of any one 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, a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), product, combination, composite, polynucleotide, or pharmaceutical composition disclosed herein. In some embodiments, the disease or condition is a disease or condition associated with expression of TACI, BCMA, and / or IFN.

[0084] In some embodiments, there is provided a method for combined treatment or pharmaceutical use of any one of the TACI polypeptides, BCMA polypeptides, and anti-IFNAR1 antibodies disclosed herein for treating a disease or condition. In some embodiments, there is provided a method for combined treatment or pharmaceutical use of any one of the TACI polypeptides and anti-IFNAR1 antibodies disclosed herein for treating a disease or condition. In some specific embodiments, the method further comprises combination use with a BCMA polypeptide. In some embodiments, there is provided a method for treatment or pharmaceutical use of any one of the fusion proteins comprising the BCMA polypeptide and anti-IFNAR1 antibody disclosed herein for treating a disease or condition. In some specific embodiments, the method further comprises combination use with a TACI polypeptide. The anti-IFNAR1 antibody is, for example, anifrolumab.

[0085] In some embodiments, a method for treating or ameliorating a B cell disorder or autoimmune disease is provided, the method 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, a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), product, combination, complex, polynucleotide, or pharmaceutical composition disclosed herein.

[0086] In some embodiments, there is provided use 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, a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), a product, combination, composite, polynucleotide, or pharmaceutical composition of the present disclosure in the preparation of a medicament for treating or preventing a disease.

[0087] In some embodiments, the disease or condition is a B cell disorder or an autoimmune disease.

[0088] In some embodiments, the disease or condition is associated with expression of TACI, or associated with expression of BCMA, or associated with expression of IFN, or associated with expression of any two or three of TACI, BCMA, and IFN, e.g., excessive or abnormal expression.

[0089] In some embodiments, the autoimmune disease is selected from 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 tumors, chronic leukemia, multiple myeloma, non-Hodgkin's lymphoma, post-transplant lymphoproliferative disease, and light chain gammopathy. In some embodiments, the autoimmune disease is systemic lupus erythematosus.

[0090] Definition of Terms In order that the present disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0091] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p. 3558 (1968).

[0092] As used in this disclosure, the singular forms "a," "an," and "the above" include plural referents unless the context clearly dictates otherwise.

[0093] Unless the context clearly indicates otherwise, in the patent specification and claims, the words "contain," "have," "include," and the like are to be understood in the sense of "including, but not limited to," rather than in the exclusive or exhaustive sense.

[0094] The term "cytokine" is a general term for proteins released by one cell population that act on other cells as intercellular substances. 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.

[0095] TACI (transmembrane activator, calcium modulator, and cyclophilin ligand-interactor) described in this disclosure is a membrane-bound receptor. Human TACI belongs to the TNFR superfamily and is a 293-amino acid polypeptide containing amino acid residues in the protein's N-terminal region (amino acid residues 1-165, see SEQ ID NO: 1 in this disclosure), transmembrane region (amino acid residues 166-186), and intracellular region (amino acid residues 187-293). The extracellular region contains two cysteine-rich pseudo-repeats (CRDs) (CRD1 and CRD2). In this disclosure, the term "CRD1" of TACI refers to the wild-type human TACI CRD1 (e.g., amino acids 33-67 in SEQ ID NO: 1) and its mutants. In the present disclosure, the term "CRD2" of TACI refers to the CRD2 of wild-type human TACI (e.g., amino acids 70 to 104 or 69 to 104 of SEQ ID NO: 1) and variants thereof (including, but not limited to, variants of one or more amino acids among amino acids 69, 72, 73, 77, 85, 102, and 103 of SEQ ID NO: 1 of the present disclosure, e.g., variants set forth in SEQ ID NOs: 6 to 29). The above-mentioned CRD1, CRD2, and variants thereof of TACI can all bind to APRIL and / or BAFF, for example, simultaneously to APRIL and BAFF. Methods for detecting such binding are known in the art, and may refer to those provided in the Examples of the present disclosure.

[0096] BCMA (B cell maturation antigen) as described herein is a membrane-bound receptor, and human BCMA protein is a 184-amino acid polypeptide comprising amino acid residues in the N-terminal region (amino acid residues 1-50, see amino acids 1-50 of SEQ ID NO: 30 of the present disclosure), the transmembrane region (amino acid residues 51-93), and the intracellular region (amino acid residues 94-184). The extracellular region of BCMA contains one CRD (also referred to as CRD1). In the present disclosure, the term "CRD1" of BCMA refers to the CRD1 of wild-type human BCMA (e.g., amino acids 7-41 of SEQ ID NO: 30 of the present disclosure) and variants thereof. The above-mentioned BCMA CRD1 and its variants can both bind to APRIL and / or BAFF, for example, simultaneously. Methods for detecting such binding are known in the art, see, for example, those provided in the Examples of the present disclosure.

[0097] In this disclosure, the terms "TACI extracellular region" and "TACI extracellular domain" are interchangeable, and the terms "BCMA extracellular region" and "BCMA extracellular domain" are interchangeable.

[0098] The terms "interferon α," "IFNα," "IFNa," "IFNA," and "IFN alpha" are used interchangeably and shall refer to the IFNα protein encoded by a functional gene in the interferon α locus, which shares 75% or greater sequence identity with IFNα1 (the protein encoded by GenBank Accession No. NP_076918 or GenBank Accession No. NM_024013). Examples of IFNα subtypes include IFNα1, α2a, α2b, α4, α4b, α5, α6, α7, α8, α10, α13, α14, α16, α17, and α21. "Interferon α," "IFNα," shall include recombinant forms of the various IFNα subtypes, as well as naturally occurring preparations containing the IFNα protein, such as leukocyte IFN and lymphoblastoid IFN.

[0099] The terms "interferon alpha receptor-1," "IFNAR1," "IFNAR-1," and "IFNAR-1 antigen" are used interchangeably and include variants, isoforms, germline homologs, and analogs of human IFNAR-1 that share at least one epitope with IFNAR-1. Thus, in some embodiments, the human antibodies of the invention may cross-react with IFNAR-1 from species other than human or with 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 but do not exhibit species or other types of cross-reactivity. The complete cDNA sequence of human IFNAR-1 has GenBank accession number NM_000629.

[0100] The term "and / or" is intended to be inclusive. For example, the phrase "A, B and / or C" is intended to cover each of 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).

[0101] The term "antibody" is used in the broadest sense to encompass a variety of 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). Antibodies may also refer to immunoglobulins, which have a tetrapeptide chain structure consisting of two heavy chains and two light chains linked by interchain disulfide bonds. Immunoglobulins differ in the amino acid composition and sequence of the heavy chain constant regions, resulting in different antigenicities. Therefore, immunoglobulins can be divided into five types, or immunoglobulin isotypes: IgM, IgD, IgG, IgA, and IgE, with the corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Ig of the same type can be further divided into different subclasses based on differences in 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 divided into κ chains and λ chains based on differences in the constant region. Each of the five types of Ig may have either κ chains or λ chains. In antibody heavy and light chains, the sequence of approximately 110 amino acids near the N-terminus is highly variable and forms the variable region (V region), while the remaining amino acid sequence near the C-terminus is relatively stable and forms the constant region (C region). The variable region contains three hypervariable regions (CDRs) and four framework regions (FRs) with relatively conserved sequences. The three hypervariable regions determine the specificity of the antibody and are also called complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3, and the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.In the present disclosure, "antibody" covers "antigen-binding fragments," and the above "antigen-binding fragments" 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, trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope-binding fragments of any one 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).

[0102] Determination or definition of CDRs can be achieved by solving the structure of the antibody and / or the structure of the antibody-ligand complex, thereby enabling accurate delineation of the CDRs and identification of the residues comprising the antibody's binding site. This can be accomplished by any one of a variety of techniques known to those skilled in the art, such as X-ray crystallography. Various 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.

[0103] 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 location 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 from the Oxford Molecular Group that model antibody structure (see, e.g., Martin et al., 1989, ProcNatl Acad Sci (USA), 86:9268-9272, "AbMTM, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK, Oxford Molecular, Ltd.). The AbM numbering system models the tertiary structure of antibodies from the base sequence using a combination of knowledge databases and ab initio methods (see, e.g., "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach" in Samudrala et al., 1999, 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).It should be noted that the definition of the boundaries of other CDRs may not strictly follow one of the above methods, but may be shortened or extended depending on predictions or experimental results that show that a particular residue or group of residues does not significantly affect antigen binding, while still overlapping with at least a portion of the Kabat CDRs. As used in this disclosure, CDR can refer to a CDR defined by any method (including a combination of methods) known in the art. The correspondence between each numbering system is well known to those skilled in the art and is illustratively shown in Table 1 below.

[0104] [Table 1-1] [Table 1-2]

[0105] Unless otherwise stated, both variable region and CDR sequences in this disclosure are referred to in the Kabat numbering system.

[0106] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, and includes native Fc regions and modified Fc regions. In some embodiments, the Fc region comprises two subunits, which may be the same or different. In some embodiments, the Fc region of a human IgG heavy chain is defined to extend from the amino acid residue at Cys226, or from Pro230 to its carboxy terminus. Suitable Fc regions for use in 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 may be altered, for example, by deletion of the C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) or deletion of the C-terminal glycine and lysine of the Fc region (residues 446 and 447 according to the EU numbering system). Unless otherwise specified, the numbering convention for the Fc region is the EU numbering system, also known as the EU index.

[0107] The term "homology" or "identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. If every position in 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 percentage of 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, multiplied by 100%. For example, if 6 out of 10 positions in two sequences are matched or homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, two sequences are compared when aligned to obtain the maximum percentage of homology.

[0108] The term "amino acid mutation" includes amino acid substitution (also called amino acid replacement), deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be used to achieve the final construct, provided that the final construct possesses the desired properties, such as reduced binding to Fc receptors. Deletions and insertions in the amino acid sequence include deletions and insertions at the amino and / or carboxy termini of the polypeptide chain. A specific amino acid mutation may be an amino acid substitution. 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 derivatives of the 20 naturally occurring amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated by genetic or chemical methods known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. It is anticipated that methods for modifying amino acid side groups other than genetic engineering, such as chemical modification, may also be utilized. As used herein, the same amino acid mutation may be referred to by various names. As used herein, the amino acid residue at a particular site may be designated by the format of position + amino acid residue, for example, 366W indicates that the amino acid residue at site 366 is W. T366W indicates that the amino acid residue at site 366 has been mutated from the original T to W.

[0109] When the amino acid sequence is defined in the claims in the form of position + residue, it should be understood that the amino acid at that site before mutation does not limit the technical solution.

[0110] The term "conservative substitution" refers to a substitution of an amino acid residue with another amino acid residue having similar properties 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. Furthermore, 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 nonpolar side chains. Furthermore, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it is clear to those skilled in the art that even when amino acid residues in the above-mentioned group showing similar properties are substituted, it does not show a specific change in properties.

[0111] The terms "polypeptide," "protein," or "protein" are used interchangeably herein and refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of corresponding naturally occurring amino acids, and to naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise specified, a particular polypeptide sequence also implicitly covers conservatively modified variants thereof.

[0112] The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term covers nucleic acids containing known nucleoside analogs or modified backbone residues or linkers, including synthetic, naturally occurring, and non-naturally occurring nucleic acids, which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to the reference nucleoside. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). An "isolated" nucleic acid refers to a polynucleotide that has been separated from a component of its natural environment. Isolated nucleic acid includes a polynucleotide contained in a cell, as defined below, which generally contains the polynucleotide, but where the polynucleotide is present extrachromosomally or at a chromosomal location different from its natural chromosomal location. An isolated nucleic acid encoding a polypeptide or a fusion protein refers to one or more polynucleotides encoding a polypeptide or a fusion protein, including one or more such polynucleotides in a single vector or separate vectors, and one or more such polynucleotides present in one or more locations in a host cell. Unless otherwise specified, a particular nucleic acid sequence implicitly covers conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly stated sequence. Specifically, as detailed below, degenerate codon substitutions can be obtained by substituting mixed-base and / or deoxyinosine residues in the third position of one or more selected (or all) codons to produce sequences.

[0113] The term "effector function" refers to a biological activity that can be attributed to an antibody Fc region (a native sequence Fc region or an amino acid sequence mutated Fc region) and varies depending on 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, down-regulation of cell surface receptors (e.g., B cell receptors), and activation of B cells.

[0114] The terms "binding affinity" or "affinity" are 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). For example, the KD can be determined by measuring the kinetics of complex formation and dissociation using surface plasmon resonance (SPR) methods (Biacore). D The rate constants corresponding to the association and dissociation of the monovalent complex are called the association rate constant ka (or k) and the dissociation rate constant kd (or koff), respectively. D is K D The dissociation constants ka and kd are related by the equation ka = kd / ka. The value of the dissociation constant can be determined directly by well-known methods and can also be calculated for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9:340-362). For example, the K can be calculated by double filtration nitrocellulose filter binding assays such as those 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 mentioned elsewhere in this disclosure. The binding kinetics and binding affinity of an antibody can be determined by standard assays known in the art, such as surface plasmon resonance (SPR), e.g., Biacore. TMThe K of each antibody / antigen complex may be evaluated by the KinExA system or KinExA. D By comparing K values, it is possible to compare the binding affinities associated with interactions with different molecules, for example, to compare the binding affinities of different antibodies to a given antigen. Similarly, the specificity of an interaction can be determined by comparing the K values ​​of the interaction of interest (e.g., the specific interaction between an antibody and an antigen). D value and the K of a non-target interaction (e.g., a known control antibody that does not bind to the target antigen). D It can be evaluated by determining and comparing values.

[0115] The term "linker" or "joint" refers to a linking unit that connects two polypeptide fragments, and generally has a certain degree of flexibility, so that the use of the joint does not impair the original function of the protein domain. Linkers appearing in the same structure herein may be the same or different. A linker may be a peptide linker and contain one or more amino acids, typically about 1 to 30, 2 to 24, or 3 to 15 amino acids. Linkers used in the present disclosure may be the same or different.

[0116] The terms "fused" or "linked" refer to elements (e.g., a TACI polypeptide and a BCMA polypeptide) that are covalently linked either directly or via one or more linkers. When the linker is a peptide linker, the covalent bond is a peptide bond.

[0117] "Giving," "administration," and "treatment," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to the contact of an exogenous agent, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Giving," "administration," and "treatment" can refer, for example, to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of cells includes contact of a reagent with a cell and contact of a reagent with a fluid, where the fluid contacts the cell. "Giving," "administration," and "treatment" also refer to ex vivo and in vitro treatment, e.g., of cells, with a reagent, diagnostic, binding composition, or via another cell. "Treatment," when applied to a human, veterinary, or research subject, refers to therapeutic treatment, preventative or prophylactic measures, and research and diagnostic uses.

[0118] "Treatment" refers to the administration of an internal or external therapeutic agent, such as a composition comprising any one of the antibodies or antigen-binding fragments thereof or fusion proteins thereof, to a subject suffering from, at risk of, or prone to one or more diseases or symptoms thereof, where the therapeutic agent is known to have a therapeutic effect on those symptoms. Typically, the therapeutic agent is administered to the subject or population being treated in an amount that effectively alleviates one or more disease symptoms, whether by inducing regression of such symptoms or by inhibiting such symptoms so that they do not progress to any clinically measurable extent. The amount of therapeutic agent that effectively alleviates any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on several factors, including the disease state, age and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Reduction of disease symptoms can be assessed by any clinical detection method commonly used by physicians or other professional healthcare providers to assess the severity or progression of the condition. An embodiment of the present disclosure (e.g., a method of treatment or product) may be ineffective in alleviating a target disease symptom in a subject, but it can be determined to alleviate the target disease symptom in a statistically significant number of subjects using any statistical testing method known in the art, such as Student's t-test, chi-square test, Mann and Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0119] An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or pathology of a medical condition. An effective amount also refers to an amount sufficient to permit or facilitate diagnosis. The effective amount used for a particular subject or veterinary subject can vary depending on factors such as the condition being treated, the subject's overall health, the method, route and dose of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

[0120] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," and include the original transformed cell and its derived progeny, without regard to the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. As used herein, progeny include mutants that have the same function or biological activity as screened or selected for in the initially transformed cell. Host cells include prokaryotic and eukaryotic host cells, of which eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse, 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.

[0121] The words "optionally" or "optionally" mean that the subsequently described event or circumstance may or may not occur, and the description includes both cases where the event or circumstance occurs and cases where it does not. For example, "optionally comprising 1 to 3 antibody heavy chain variable regions" means that antibody heavy chain variable regions of a particular sequence may be present, but are not necessarily present.

[0122] The term "fusion protein comprising a TACI polypeptide and a BCMA polypeptide" according to the present disclosure covers all molecules of the fusion protein comprising a TACI polypeptide and a BCMA polypeptide according to the present disclosure.

[0123] The term "fusion protein comprising a TACI polypeptide and an anti-IFNAR1 antibody" covers all molecules of a fusion protein comprising a TACI polypeptide and an anti-IFNAR1 antibody (or an antigen-binding fragment thereof) according to the present disclosure.

[0124] The term "fusion protein comprising a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody" encompasses all molecules of the fusion protein comprising a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody (or antigen-binding fragment thereof) according to the present disclosure. For example, the fusion protein may comprise, e.g., multiple, one or more effector molecules.

[0125] The "effector molecule" may have therapeutic activity alone (e.g., antitumor activity or immune stimulatory or inhibitory activity) or may have a detectable function, and may take any form, such as, for example, a biologically active protein (e.g., an enzyme), another antibody or antibody fragment, a synthetic or naturally occurring polymer, a nucleic acid and fragments thereof, e.g., DNA, RNA and fragments thereof, a radionuclide (particularly radioactive iodide), a radioisotope, a chelating metal, a nanoparticle and a reporter group (e.g., a fluorescent compound), or a compound detectable by NMR or ESR spectroscopy. Conjugation of an effector molecule to a fusion protein according to the present disclosure can be achieved by conventional methods. [Brief explanation of the drawings]

[0126] [Figure 1] FIG. 1 is a schematic diagram of the structure of a fusion protein of TACI and Fc. [Figure 2] Schematic diagram of the interactions of the ligands BAFF and APRIL with the receptors BAFF-R, TACI, and BCMA. BAFF is a membrane-expressed protein that functions in a soluble trimeric form after protease 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 indicate stronger interactions, while thinner black arrows indicate weaker interactions. [Figure 3] Schematic representation of the different TACI and BCMA protein domains, including TACI-ECD, TACI-d2, TACI-T2, TACI-T4, and BCMA-ECD. [Figure 4]Schematic diagram of the structure of the TACI-BCMA fusion protein, including B575701, B575702, B575703, and B575704. [Figure 5] ELISA measurement of the binding ability of TACI-BCMA fusion proteins (B575701, B575702, B575703, B575704) to BAFF. Telitaccept and the IgG1 isotype were used as controls. [Figure 6] ELISA measurement of the binding ability of TACI-BCMA fusion proteins (B575701, B575702, B575703, B575704) to APRIL. Telitaccept and the IgG1 isotype were used as controls. [Figure 7] Schematic diagram of the TACI-CRD2 and TACI-19-16 domains, including TACI-CRD2 and TACI-19-16. [Figure 8] Schematic diagrams of the structures of fusion proteins of anifrolumab and TACI-T4 (B385801, B385802, B385803, B385804, B498301, B498302) and fusion proteins of anifrolumab and TACI-19-16 (B606401, B606401-LALA, B805201, B805201-LALA, where the LALA mutation is the L234A / L235A mutation in Fc, and the same applies below). [Figure 9] The results show the results of detecting the inhibition of IFN-β activity by the fusion protein of anifrolumab and TACI-T4, of which Figure 9A shows the results of inhibition of IFN-β activity by B385801, B385802, B385803, and B385804, Figure 9B shows the results of inhibition of IFN-β activity by B385804, B498301, and B498302, and Figure 9C shows the results of inhibition of IFN-β activity by B606401. Anifrolumab and the IgG1 isotype were used as controls. [Figure 10]Figure 10 shows the ELISA results for the binding ability of anifrolumab and TACI-T4 fusion proteins to BAFF. Figure 10A shows the results for the binding ability of B385801, B385802, B385803, and B385804 to BAFF, with atacicept, telitacicept, and the IgG1 isotype used as controls. Figure 10B shows the results for the binding ability of B385804, B498301, and B498302 to BAFF. Figure 10C shows the results for the binding ability of B606401 to BAFF, with telitacicept and the IgG1 isotype used as controls. [Figure 11] Schematic structure of the anifrolumab and TACI-BCMA fusion protein, containing B613301, B613302, B613303, and B613304. [Figure 12] These results demonstrate the inhibition of IFN-β activity by anifrolumab and TACI-BCMA fusion proteins (B613301, B613302, B613303). Anifrolumab and the IgG1 isotype were used as controls. [Figure 13] ELISA results for the binding ability of anifrolumab and TACI-BCMA fusion proteins (B613301, B613302, B613303) to BAFF, with telitacicept and IgG1 isotype used as controls. [Figure 14] ELISA results for the binding ability of anifrolumab and TACI-BCMA fusion proteins (B613301, B613302, B613303) to APRIL, with telitacicept and IgG1 isotype used as controls. [Figure 15] Schematic diagrams of the structures of the fusion protein of anifrolumab with TACI-19-16 and BCMA-ECD-1 (B637301), and the fusion proteins of anifrolumab with TACI-19-16 and BCMA-ECD-2 (B637302, B637302-LALA, B746201, B746201-LALA). [Figure 16]These results demonstrate the inhibition of 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). Anifrolumab and the IgG1 isotype were used as controls. [Figure 17] ELISA measurement of the binding ability of B637302, B746201, B606401, and B805201 to BAFF. Telitaccept and the IgG1 isotype were used as controls. [Figure 18] ELISA measurement of the binding ability of B637302, B746201, B606401, and B805201 to APRIL. Telitaccept and the IgG1 isotype were used as controls. [Figure 19] Experimental results of pDC function in PBMC. Human PBMC cells 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. Secreted IFN-α levels were measured in the supernatant 24 hours later. [Figure 20] These are the results of an in vitro plasma cell differentiation experiment. B cells selected from human PBMCs were cultured in vitro, treated with CpG-B and IFN-α, and gradient concentrations of B637302, B746201, B606401, and B805201. Anifrolumab, IgG1 isotype, was used as a control. After 4 days, the differentiation rate of plasma cells (CD27+CD38+) was detected by flow cytometry. [Figure 21]These results show the effect of BAFF on in vitro B cell proliferation. Human PBMC-selected B cells were labeled with CTV and cultured in vitro. IL-4, anti-IgM, CD40L, and IL-17 were added as basal stimulation signals. BAFF was then added to induce B cell proliferation under basal conditions. The B cells were then treated with gradient concentrations of B637302, B606401, and B805201, with telitacicept, IgG1 isotype, used as a control. On day 5, B cell proliferation signals were detected by flow cytometry at drug concentrations of 100 nM, 300 nM, and 1000 nM. [Figure 22] This study investigated the induction of in vitro B cell proliferation by APRIL. B cells selected from human PBMCs were labeled with CTV and cultured in vitro. IL-4, anti-IgM, CD40L, and IL-17 were added as basal stimulation signals. Under these basal conditions, APRIL was added to induce B cell proliferation. The cells were then treated with gradient concentrations of B637302 and B606401, and telitacicept, IgG1 isotype, was used as a control. B cell proliferation signals were detected by flow cytometry on day 5 at drug concentrations of 100nM, 300nM, and 1000nM. [Figure 23] This study investigated the co-induction of in vitro plasma cell generation with BAFF and APRIL. B cells selected from human PBMCs were cultured in vitro and stimulated with CpG-B on days 1-4. CpG-B was then removed from the culture on days 4-10, and IL-6, IL-10, and IL-21 were added. Under basal conditions, 500 ng / mL BAFF and 50 ng / mL APRIL were added on days 4-7, and 50 ng / mL BAFF and 500 ng / mL APRIL were added on days 7-10 to induce plasma cell generation. The cells were treated with the drugs B637302, B606401, B606401, and B805201 at different concentrations (10, 100, and 1000 nM) on days 4-10. Telitacic receptor (IgG1 isotype) was used as a control. Plasma cell counts were detected by flow cytometry on day 10. [Figure 24]This is a co-induction experiment of in vitro plasma cell generation using three factors: IFN-α, BAFF, and APRIL. Figure 24A shows a schematic diagram of the experimental process. B cells selected from human PBMCs were cultured in vitro and stimulated with CpG-B and IFN-α on days 1-4. CpG-B was removed on days 4-10, and IL-6, IL-10, and IL-21 were added for continued culture. Under these basal conditions, 500 ng / mL BAFF and 50 ng / mL APRIL were added on days 4-7, and then 50 ng / mL BAFF and 500 ng / mL APRIL were added on days 7-10 to induce in vitro plasma cell generation. On days 0 to 10, the cells were treated with the drugs B637302 and B606401 at different concentrations of 10, 100, and 1000 nM. Telitaccept and IgG1 isotype were used as controls. The number of plasma cells was detected by flow cytometry on day 10. Figure 24B, C, and D show the detection results for the drugs 10 nM, 100 nM, and 1000 nM, respectively. [Figure 25] 25 shows the results of an experiment to induce PD in PBMC-humanized mice using PEG-IFN-α. Figure 25A shows a schematic diagram of the experimental process. B-NDG immunodeficient mice were used, and PBMCs were humanized via tail vein injection and PEG-IFN-α was induced via intraperitoneal injection. In this model, the mice were treated with different concentrations of the drugs B637302 and B606401 via intraperitoneal injection. Anifrolumab and PBS were used as controls. Figure 25B shows a schematic diagram of the induction of downstream signaling pathways by IFN-α. Figure 25C shows the results of PBMCs taken at 2 hours and the pSTAT1 level was measured. Figure 25D, E, and F show PBMCs taken on days 1, 3, and 7, respectively, and the expression levels of IFN-α downstream gene mRNA were measured by QPCR. [Figure 26]These are the results of a mouse PD co-induction experiment using BAFF and APRIL. Figure 26A shows a schematic diagram of the experimental process. C57 / B6 mice were induced by intraperitoneal injection of BAFF and APRIL. The mice were then treated with intraperitoneal injections of different concentrations of the drugs B637302 and B606401. Telitaccept 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 26C show the results of blood collection on days 4 and 7, respectively, for plasma IgA levels. For Figures 21 to 26, the statistical significance of the Student's t-test was *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. DETAILED DESCRIPTION OF THE INVENTION

[0127] The present disclosure will be further described below in conjunction with examples, but these examples do not limit the scope of the present disclosure.

[0128] Experimental methods for which specific conditions are not specified in the examples or experimental examples of this disclosure generally follow conventional conditions or conditions recommended by the manufacturers of materials or products. See Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, and Modern Methods in Molecular Biology, Ausubel et al., Greene Publishing Company, Wiley Interscience, NY. Reagents for which specific sources are not specified are conventional, commercially available reagents.

[0129] Example 1: Analysis and modification of TACI sequences Because the TACI sequence contains multiple protease cleavage sites, full-length TACI is susceptible to cleavage after expression. In this experiment, we designed TACI sequence fragments of different lengths and directly fused the C-terminus of TACI-ECD or its fragments to the N-terminus of human IgG1 Fc (SEQ ID NO: 3). TACI-Fc fusion proteins (including full-length TACI-ECD-Fc and TACI-9-Fc) were constructed and expressed in 293E (human embryonic kidney cells modified with the EBNA1 gene) cells. Purification yielded TACI-Fc fusion proteins containing two identical polypeptide chains (see Figure 1 for their structures). RCT-18 (telitacicept) was used as a positive control.

[0130] The sequence is as follows:

[0131] >TACI-ECD (amino acid residues 1-165 of wild-type human TACI) [ka] >TACI-9 (68th to 108th positions in the natural count of human TACI-ECD) [ka] >Human IgG1 Fc sequence [ka] >Telitacicept (RCT-18) [ka] >Atacicept [ka]

[0132] Note: In the Telitacicept (RCT-18) sequence, the ununderlined portion is the TACI sequence (human TACI extracellular domain positions 13-118, natural counting), and the underlined portion is the Fc sequence. In the Atacicept sequence, the ununderlined portion is the TACI sequence (human TACI extracellular domain positions 30-110, natural counting), and the linker EPKSS (shown in italics), and the underlined portion is the Fc sequence.

[0133] The resulting TACI-Fc fusion proteins were subjected to mass spectrometry analysis. Experimental results showed that TACI-9-Fc had no cleavage fragments, whereas the positive control RCT-18 and full-length TACI-ECD-Fc both had TACI cleavage fragments (results not shown).

[0134] Furthermore, we examined the blocking function of the TACI-Fc fusion protein on the binding of BAFF and BAFF-R. The method was as follows: The receptor protein was diluted to 2 μg / mL in PBS (B320, Genbioso) buffer, pH 7.4, and added to a 96-well microplate (Corning, 3590) at a volume of 100 μL per well. The plate was then 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. The plate was then 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) before use. A fixed concentration of biotin-labeled ligand protein was mixed with gradient-diluted antibody or fusion protein, preincubated at 37°C for 30 minutes, and then added to a blocked microplate and incubated at 37°C for 1.5 hours. After incubation, the plate was washed three times with PBST. 100 μL of streptavidin-HRP (Invitrogen, 434323, diluted 1:4000) was added to each well and incubated at 37°C for 1 hour. The supernatant was discarded, and the plate was washed three times with PBST. 100 μL of TMB chromogenic substrate (KPL, 5120-0077) was added to each well and incubated at room temperature for 10–15 minutes. The reaction was stopped by adding 50 μL of 1 M H2SO4 to each well. The absorbance at 450 nm was measured using a plate reader, and the IC was calculated by fitting a curve for ligand-receptor binding inhibition using software. 50 The origins of the ligand and receptor proteins used were BAFF (Sino biological, 10056-HNCH) and BAFF-R (Sino biological, 16079-H02H).

[0135] As a result, TACI-9-Fc and RCT-18 act as ICs that block the binding of BAFF 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 to be a promising candidate molecule.

[0136] Example 2. Analysis of TACI cleavage fragments Based on TACI-9, the TACI sequence was further truncated and the functional activity of the truncated TACI fragment was detected. The sequence of the truncated TACI fragment is as follows:

[0137] >TACI-10 (TACI-9 truncated by one amino acid "L" at the C-terminus) (natural counting of human TACI-ECD positions 68-107) [ka] TACI-11 (TACI-9 truncated at the C-terminus by two amino acids "KL") (natural counting of human TACI-ECD from positions 68 to 106) [ka] >TACI-12 (TACI-9 truncated at the C-terminus by three amino acids "NKL") (natural counting of human TACI-ECD from positions 68 to 105) [ka] The C-terminus of the above-mentioned cleaved 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 then 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).

[0138] The TACI-Fc fusion proteins constructed as above were subjected to mass spectrometry, and the experimental results are shown in Table 2. As can be seen from the experimental results, none of the TACI-Fc fusion proteins constructed with TACI-9 cleavage fragments resulted in cleavage of the TACI polypeptide.

[0139] In addition, the BAFF-binding activity of the constructed TACI-Fc fusion protein was detected, and the blocking activity of the TACI-Fc fusion protein against the binding of BAFF to BAFF-R was detected (see Example 1 for experimental methods).

[0140] The BAFF-binding activity of the TACI-Fc fusion protein was detected as follows: BAFF (Sino biological, 10056-HNCH) protein was diluted to 1 μg / mL in pH 7.4 PBS (Genbyou Biological, B320) buffer and added to a 96-well microplate (Corning, 3590) at a volume of 100 μL per well and incubated at 4°C overnight. After discarding the liquid, 300 μL of 5% nonfat milk (BD, 232100) diluted in PBS was added to each well for blocking and incubation at 37°C for 2 hours. After discarding the blocking solution and washing the plate three times with PBST buffer (PBS, pH 7.4, containing 0.1% Tween-20), 100 μL of a gradient-diluted antibody or fusion protein solution to be assayed was added to each well and incubated at 37°C for 1 hour. After incubation, the plate was washed three times with PBST, and 100 μL of mouse anti-human IgG (H+L) (Jackson ImmunoResearch, 209-035-088, diluted 1:8000) was added to each well and incubated at 37°C for 1 hour. After washing the plate three times with PBST, 100 μL of TMB chromogenic substrate (KPL, 5120-0077) was added to each well and incubated at room temperature for 10-15 minutes. The reaction was stopped by adding 50 μL of 1M H2SO4 to each well, and the absorbance value at 450 nm was read using a plate reader. The antibody-antigen binding curve was fitted using software to calculate the EC 50 The value was calculated.

[0141] As is clear from the results in Table 2, the TACI-10-Fc, TACI-11-Fc, and TACI-12-Fc fusion proteins constructed from the TACI-9 cleavage fragments TACI-10, TACI-11, and TACI-12 have binding activity to BAFF and blocking activity against the binding of BAFF to BAFF-R at the same levels as the TACI-9-Fc fusion protein.

[0142] [Table 2]

[0143] Example 3. Modification of the sequence of TACI cleavage fragments TACI protein is prone to aggregation in neutral solution. To further improve TACI stability, we used Molecular Operating Environment (MOE) software to analyze the hydrophobic and ionic groups in the TACI fragment based on the TACI crystal structure (PDB ID: 1XU1). We then performed amino acid substitutions on several amino acid residues in TACI-9, reducing the exposed hydrophobic and ionic groups in the TACI protein. This reduced the aggregation tendency of TACI and improved its stability while still maintaining its functional activity. The amino acid sequence of the modified TACI fragment is as follows:

[0144] >TACI-9-1 (TACI-9 containing the L69T substitution) [ka] >TACI-9-2 (TACI-9 contains the R72S mutation) [ka] >TACI-9-3 (TACI-9 contains the K73E mutation) [ka] >TACI-9-4 (TACI-9 contains the K73Q mutation) [ka] >TACI-9-5 (TACI-9 contains the K77E mutation) [ka] >TACI-9-6 (TACI-9 contains L69R and D85T mutations) [ka] >TACI-9-7 (TACI-9 contains L69R and D85A mutations) [ka] >TACI-9-8 (TACI-9 contains the Y102A mutation) [ka] >TACI-9-9 (TACI-9 contains L69R, D85T, and Y102R mutations) [ka] >TACI-9-10 (TACI-9 contains K73E and K77E mutations) [ka] >TACI-9-11 (TACI-9 contains R72S, K73E, and K77E mutations) [ka] >TACI-9-12 (TACI-9 contains L69T and Y102A mutations) [ka] >TACI-9-13 (TACI-9 contains L69T and F103Y mutations) [ka] >TACI-9-14 (TACI-9 contains L69T, Y102A, and F103Y mutations) [ka] >TACI-9-15 (TACI-9 contains L69T, K73E, K77E, and Y102A mutations) [ka]

[0145] Note: In the above sequence, the underlined amino acid residues are those after mutation.

[0146] The C-terminus of the TACI fragment modified with TACI-9 was fused to the N-terminus of human IgG Fc (SEQ ID NO: 3) to construct a TACI-Fc fusion protein (structure see Figure 1). After transfection and expression in 293E, a TACI-Fc fusion protein containing two identical polypeptide chains was obtained by Protein A affinity purification.

[0147] The BAFF-binding activity of the constructed TACI-Fc fusion protein was detected (see Example 2 for the experimental method), and the experimental results are shown in Table 3.

[0148] [Table 3-1] [Table 3-2]

[0149] Note: In the table, the mutation site is the amino acid residue site (natural order numbering) relative to TACI-ECD (SEQ ID NO: 1). For example, "L69T" indicates that the 69th amino acid residue (natural order numbering) in the sequence of SEQ ID NO: 1 has been mutated from L to T.

[0150] In addition, a portion of the purified TACI-Fc fusion protein was transferred to PBS solution using an ultrafiltration tube, and the appearance of the solution was examined to determine whether or not a precipitate was present in the TACI-Fc fusion protein solution. The experimental results clearly showed that the solution of the TACI-Fc fusion protein constructed with the modified TACI fragment did not contain a precipitate, whereas the PBS solution of RCT-18 did. (Results not shown.)

[0151] Additionally 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: >TACI-9-15a [ka] >TACI-9-15b [ka] >TACI-9-15c (i.e. TACI-19-16) [ka]

[0152] Example 4. Construction, expression and purification of TACI-BCMA fusion protein As mentioned above, atacicept and telitacicept are both BAFF / APRIL antagonists in the form of a TACI-Fc molecule. As shown in Figure 2, both TACI and BCMA can bind to BAFF and APRIL. TACI has relatively strong binding ability to BAFF but relatively weak binding ability to APRIL, while BCMA has relatively strong binding ability to APRIL. To enhance the neutralizing activity against APRIL, BCMA and TACI were designed as a fusion protein.

[0153] As shown in Figure 3, TACI-ECD is the TACI extracellular domain containing amino acid residues 1 to 165, TACI-d2 is the CRD2 ligand-binding domain containing amino acid residues 69 to 111, TACI-T2 is the CRD1 and CRD2 ligand-binding domain containing amino acid residues 13 to 118, TACI-T4 is the CRD1 and CRD2 ligand-binding domain containing amino acid residues 30 to 110, and BCMA-ECD is the BCMA extracellular domain containing amino acid residues 1 to 54.

[0154] As shown in Figure 4, four types of fusion proteins containing TACI polypeptide and BCMA polypeptide were designed, of which fusion proteins B575701 and B575702 are composed of a combination of TACI-d2 and BCMA-ECD sequences fused with human wild-type IgG1-Fc, and fusion proteins B575703 and B575704 are composed of a combination of TACI-T2 and BCMA-ECD sequences fused with human wild-type IgG1-Fc.

[0155] The sequence of TACI-ECD is shown in SEQ ID NO: 1. The remaining sequences are as follows, where the underlined part is human IgG1 Fc, and where the G at the last position of SEQ ID NO: 27 is optional.

[0156] >TACI-d2 (SEQ ID NO: 27) [ka] >TACI-T2 (SEQ ID NO: 28) [ka] >TACI-T4 (SEQ ID NO: 29) [ka] >BCMA-ECD (SEQ ID NO: 30) [ka] >TACI-d2-BCMA-ECD (SEQ ID NO: 31) [ka] >BCMA-ECD-TACI-d2 (SEQ ID NO: 32) [ka] >TACI-T2-BCMA-ECD (SEQ ID NO: 33) [ka] >BCMA-ECD-TACI-T2 (SEQ ID NO: 34) [ka] >B575701 (SEQ ID NO: 35) [ka] [ka] >B575702 (SEQ ID NO: 36) [ka] >B575703 (SEQ ID NO: 37) [ka] >B575704 (SEQ ID NO: 38) [ka]

[0157] The coding gene sequences for the above proteins were synthesized and subcloned into pcDNA3.4. 50 μg of expression plasmid was diluted with culture medium and mixed to homogeneity. 200 μL of transfection reagent was diluted with culture medium and mixed to homogeneity. The mixture was then mixed and incubated at 37°C for 15 minutes. The mixture was added dropwise to HEK293 cell suspension. The cell suspension was cultured at 37°C on a shaker for 1 week, then centrifuged at 8000 rpm for 5 minutes to collect the supernatant. A Protein A affinity chromatography column was equilibrated with 20 mL of 1x 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 off with 20 mL of 1x 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 1x PBS, replacing the PBS storage buffer. Upon detection, the target protein was obtained.

[0158] Example 5. In vitro binding experiments of TACI-BCMA fusion proteins to BAFF and APRIL The binding ability of TACI-BCMA fusion proteins (B575701, B575702, B575703, B575704) to BAFF and APRIL, respectively, was detected by ELISA.

[0159] The method is as follows: BAFF (PeproTech, Cat. 310-13) or ARPIL (Acro Biosystems, Cat. APL-H52D1) was diluted to 1 μg / mL in PBS and coated onto a 96-well plate (Costar, Cat. 3590) overnight at 4°C. The plate was washed with PBST and blocked with blocking buffer (PBST containing 1% BSA) at 37°C for 1 hour. The plate was then tapped, and antibodies to be measured were diluted in blocking buffer and incubated at 37°C for 1.5 hours. The plate was 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, and 100 μL of TMB color developing solution (Biopanda, Cat: TMB-S-003) was added to develop the color at 37°C for 3 minutes. 100 μL of ELISA stopping solution (Solarbio, Cat: C1058) was then added to detect OD450.

[0160] As a result, as shown in Table 4 and Figure 5, all four fusion proteins were able to clearly bind to BAFF, and the binding ability was stronger than that of telitacicept. Among them, B575702 and B575704 had the same binding ability to BAFF, and the EC 50 The EC2 activity of B575701 and B575703 was comparable to that of telitacicept, and the EC2 activity was reduced to one-quarter of that of telitacicept. 50 was reduced to half that level.

[0161] [Table 4]

[0162] As shown in Table 5 and Figure 6, the four fusion proteins were clearly able to bind to APRIL, and the binding abilities were almost identical. The binding ability of all four fusion proteins to APRIL was stronger than that of telitacicept, and the EC 50was reduced to 1 / 4 to 1 / 3 of the original level.

[0163] [Table 5]

[0164] Example 6. Construction, expression, and purification of an anifrolumab and TACI fusion protein Anifrolumab is an IFNAR1 antagonist that blocks type I interferon-mediated cell activation by IFNAR1 and is used to treat systemic lupus erythematosus. As shown in Figure 7, TACI-CRD2 is the CRD2 ligand-binding domain containing amino acid residues 68 to 105, and TACI-19-16 is a polypeptide with minor amino acid mutations (L69T, K73E, K77E, Y102A) in TACI-CRD2.

[0165] As shown in Figure 8, TACI-T4 or TACI-19-16 was linked to the N- or C-terminus of the heavy or light chain of anifrolumab, respectively, by linkers of different lengths. Among them, B385801 linked TACI-T4 to the N-terminus of the anifrolumab heavy chain via a (G4S)2 linker 1; B385802 linked TACI-T4 to the C-terminus of the anifrolumab heavy chain via a linker 1; B385803 linked TACI-T4 to the N-terminus of the anifrolumab light chain via a linker 1; B385804 linked TACI-T4 to the C-terminus of the anifrolumab light chain via a linker 1; B498301 linked TACI-T4 to the C-terminus of the anifrolumab light chain via a (G4S)3 linker 2; and B498302 linked TACI-T4 to the C-terminus of the anifrolumab light chain via a (G4S)4 linker 3. In B606401, TACI-19-16 was linked to the C-terminus of the anifrolumab light chain via linker 1, and in B805201, TACI-19-16 was linked to the C-terminus of the anifrolumab heavy chain via linker 2. In B606401-LALA, TACI-19-16 was linked to the C-terminus of the anifrolumab light chain via linker 1, and the Fc portion was a human IgG1 Fc with L234A and L235A mutations. In B805201-LALA, TACI-19-16 was linked to the C-terminus of the anifrolumab heavy chain via linker 2, and the Fc portion was a human IgG1 Fc with L234A and L235A mutations.

[0166] TACI-19-16 is represented by SEQ ID NO: 26, and the other sequences are as follows:

[0167] >Linker 1 (SEQ ID NO: 39) [ka] >Linker 2 (SEQ ID NO: 40) [ka] >Linker 3 (SEQ ID NO: 41) [ka] >TACI-CRD2 (SEQ ID NO: 42, same as SEQ ID NO: 8) [ka] >VH of anifrolumab (SEQ ID NO: 43) [ka] >VL of anifrolumab (SEQ ID NO: 44) [ka] Anifrolumab HCDR1 (SEQ ID NO: 45) [ka] > HCDR2 of anifrolumab (SEQ ID NO: 46) [ka] Anifrolumab HCDR3 (SEQ ID NO: 47) [ka] Anifrolumab LCDR1 (SEQ ID NO: 48) [ka] Anifrolumab LCDR2 (SEQ ID NO: 49) [ka] Anifrolumab LCDR3 (SEQ ID NO: 50) [ka] >Heavy chain of B385801 (SEQ ID NO: 51) [ka] >Heavy chain of B385802 (SEQ ID NO: 52) [ka] >Heavy chain of B805201 (SEQ ID NO: 53) [ka] Light chain of B385801, B385802, B805201, B805201-LALA (SEQ ID NO: 54) [ka] Heavy chains of B385803, B385804, B498301, B498302, and B606401 (SEQ ID NO: 55) [ka] >Light chain of B385803 (SEQ ID NO: 56) [ka] >Light chain of B385804 (SEQ ID NO: 57) [ka] >Light chain of B498301 (SEQ ID NO: 58) [ka] >Light chain of B498302 (SEQ ID NO: 59) [ka] >B606401, light chain of B606401-LALA (SEQ ID NO: 60) [ka] B606401 - Heavy chain of LALA (SEQ ID NO: 61) [ka] B805201-LALA heavy chain (SEQ ID NO: 62) [ka]

[0168] The underlined part of the heavy chain is the Fc region of IgG, the underlined part of the light chain is CH1, and the part in italics is the linker.

[0169] Using the protein preparation method provided in Example 1, transient transfection and protein expression were performed, and the protein was eluted through a Protein A affinity chromatography column. Upon detection, the target protein was obtained.

[0170] Example 7: Experiment on the inhibitory activity of the fusion protein of anifrolumab and TACI against type I interferon The HEK-Blue IFN-α / β cell (InvivoGen, Cat: hkb-ifnab) reagent kit was used to detect the inhibitory effects of the above-mentioned anifrolumab and TACI fusion proteins B385801, B385802, B385803, B385804, B498301, B498302, and B606401 on the activity of IFN-α / β.

[0171] As a method, HEK-Blue TM IFN-α / β cells were digested and resuspended at 2.8E5 / mL in DMEM medium (Gibco, Cat: 11995065) containing 10% FBS and 1% P / S, and seeded into a 96-well plate. Protein samples were 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 the 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.

[0172] As a result, all seven of the above fusion proteins had inhibitory effects on the activity of IFN-α / β, of which B385801 and B385803 had relatively weak activities, B385802 had the next weakest activity, and B385804, B498301, B498302, and B606401 had activities almost equivalent to that of anifrolumab (Figures 9A, 9B, and 9C).

[0173] Example 8. In vitro binding experiment of fusion protein of anifrolumab and TACI to BAFF The binding ability of the anifrolumab and TACI fusion proteins B385801, B385802, B385803, B385804, B498301, B498302, and B606401 to BAFF was detected by ELISA.

[0174] BAFF (PeproTech, Cat: 310-13) was diluted to 1 μg / mL in PBS and coated onto a 96-well plate overnight at 4°C. The plate was washed with PBST and blocked with Blocking Buffer (PBST containing 1% BSA) at 37°C for 1 hour. The plate was then tapped, and antibodies to be measured were diluted in Blocking Buffer and incubated at 37°C for 1.5 hours. The plate was 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, and 100 μL of TMB color developing solution (Biopanda, Cat: TMB-S-003) was added to develop the color at 37°C for 3 minutes. 100 μL of ELISA stopping solution (Solarbio, Cat: C1058) was then added to detect OD450.

[0175] As a result, referring to Figures 10A to 10C and Table 6, it was shown that the seven fusion proteins could clearly bind to BAFF. B385801, B385803, and B606401 had the strongest binding ability to BAFF, stronger than Telitacicept and Atacicept. B385802 had a relatively weak binding ability to BAFF. B385804, B498301, and B498302 had similar binding ability to BAFF, slightly weaker than Telitacicept. The length of the linker did not affect the binding ability of TACI to BAFF.

[0176] [Table 6]

[0177] Example 9. Construction, expression, and purification of anifrolumab and TACI-BCMA fusion proteins 11 , the TACI-BCMA fusion proteins described in Example 4 were each linked to the C-terminus of the anifrolumab antibody light chain via linker 1. Of these, 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.

[0178] The heavy chains of B613301, B613302, B613303, and B613304 are all the full-length heavy chain of anifrolumab (SEQ ID NO: 55), and the specific sequences of the light chains are as follows, with the underlined parts being the light chain constant regions.

[0179] >Light chain of B613301 (SEQ ID NO: 63) [ka] >Light chain of B613302 (SEQ ID NO: 64) [ka] >Light chain of B613303 (SEQ ID NO: 65) [ka] >Light chain of B613304 (SEQ ID NO: 66) [ka] Using the protein preparation method provided in Example 4, transient transfection and protein expression were performed, and the protein was eluted through a Protein A affinity chromatography column. Upon detection, the target protein was obtained.

[0180] Example 10: Inhibitory activity of fusion protein of anifrolumab and TACI-BCMA against type I interferon The inhibitory effect of anifrolumab and TACI-BCMA fusion proteins B613301, B613302, and B613303 on the activity of IFN-α / β cells (Example 9) was detected using a HEK-Blue IFN-α / β cell (InvivoGen, Cat: hkb-ifnab) reagent kit.

[0181] As a method, HEK-Blue TM IFN-α / β cells were digested and resuspended at 2.8E5 / mL in DMEM medium (Gibco, Cat. No. 11995065) containing 10% FBS and 1% P / S, and seeded into a 96-well plate (Costar, Cat. No. 3599). The antibody to be assayed was added and incubated at 37°C for 30 minutes. IFN-β (Sino biological, Cat. No. 10704-HNAS) was added to a final concentration of 0.01 ng / mL and incubated at 37°C for 24 hours. 20 μL of the culture supernatant was mixed with 180 μL of Quanti-Blue (InvivoGen, Cat. No. rep-qbs) in a new 96-well plate and incubated at 37°C for 1 hour. OD655 was measured.

[0182] As a result, as shown in Figure 12, all three fusion proteins were able to inhibit the activity of IFN-α / β, and the activities of the three fusion proteins were comparable and consistent with those of anifrolumab, suggesting that the TACI-BCMA fusion did not affect the activity of anifrolumab, particularly at the C-terminus of the light chain.

[0183] Example 11. In vitro binding experiments of anifrolumab and TACI-BCMA fusion proteins to BAFF and APRIL ELISA was used to detect the binding ability of anifrolumab and TACI-BCMA fusion proteins B613301, B613302, and B613303 to BAFF and APRIL.

[0184] BAFF (PeproTech, Cat: 310-13) or ARPIL (Acro Biosystems, Cat: APL-H52D1) was diluted to 1 μg / mL in PBS and coated onto a 96-well plate (Costar, Cat: 3590) overnight at 4°C. The plate was washed with PBST and blocked with blocking buffer (PBST containing 1% BSA) at 37°C for 1 hour. The plate was then tapped, and the antibody to be assayed was diluted in blocking buffer and incubated at 37°C for 1.5 hours. The plate was 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, and 100 μL of TMB color developing solution (Biopanda, Cat: TMB-S-003) was added to develop the color at 37°C for 3 minutes. 100 μL of ELISA stopping solution (Solarbio, Cat: C1058) was then added to detect OD450.

[0185] Referring to Figure 13 and Table 7, the results showed that all three fusion proteins clearly bound to BAFF. B613302 had significantly stronger BAFF-binding ability than Telitacic. 50was reduced to 1 / 8 to 1 / 7 of the original level. Placing TACI at the C-terminus of BCMA (e.g., in the case of B613302) may be more advantageous for maintaining the ability to bind to BAFF.

[0186] [Table 7]

[0187] Referring to Figure 14 and Table 8, the results showed that all three fusion proteins clearly bound to APRIL. B613301, B613302, and B613303 had almost identical binding to APRIL, and showed a significantly higher EC than telitacicept. 50 The results showed that the BCMA-fused fusion protein indeed improved binding to APRIL.

[0188] [Table 8]

[0189] Example 12. Construction, expression, and purification of an anifrolumab and TACI-19-16-BCMA fusion protein BCMA-ECD-1 (amino acid residues 1-43) and BCMA-ECD-2 (amino acid residues 1-41) lack risk sites that may be present in the non-CRD domains of BCMA, such as glycosylation sites (amino acid residues 42-44, NAS) and deamidation sites (amino acid residues 47-48, NS). Removal of these risk sites can make the fusion proteins more homogeneous.

[0190] As shown in Figure 15, in B637301, TACI-19-16 was fused to the N-terminus of BCMA-ECD-1, and then TACI-19-16-BCMA-ECD-1 was linked to the C-terminus of the anifrolumab antibody light chain via linker 1; in B637302, TACI-19-16 was fused to the C-terminus of BCMA-ECD-2, and then BCMA-ECD-2-TACI-19-16 was linked to the C-terminus of the anifrolumab antibody light chain via linker 1; and in B746201, TACI-19-16 was fused to the C-terminus of BCMA-ECD-2, and then BCMA-ECD-2-TACI-19-16 was linked to the C-terminus of the anifrolumab antibody heavy chain via linker 2. In B637302-LALA, TACI-19-16 is fused to the C-terminus of BCMA-ECD-2, and then BCMA-ECD-2-TACI-19-16 is linked to the C-terminus of the antibody anifrolumab light chain via linker 1, and the Fc portion of the anifrolumab antibody is a human IgG1 Fc with L234A and L235A mutations. In B746201-LALA, TACI-19-16 is fused to the C-terminus of BCMA-ECD-2, and then BCMA-ECD-2-TACI-19-16 is linked to the C-terminus of the antibody anifrolumab heavy chain via linker 2, and the Fc portion of the anifrolumab antibody is a human IgG1 Fc with L234A and L235A mutations; see Figure 15.

[0191] >BCMA-ECD-1 (SEQ ID NO: 67) [ka] >BCMA-ECD-2 (SEQ ID NO: 68) [ka] >TACI-19-16-BCMA-ECD-1 (SEQ ID NO: 69) [ka] >BCMA-ECD-2-TACI-19-16 (SEQ ID NO: 70) [ka] The heavy chains of both B637301 and B637302 are the same as those of anifrolumab, SEQ ID NO: 55, and the light chain sequences are as follows: >Light chain of B637301 (SEQ ID NO: 71) [ka] >B637302, light chain of B637302-LALA (SEQ ID NO: 72) [ka] B637302-LALA has a heavy chain of SEQ ID NO: 61. >Heavy chain of B746201 (SEQ ID NO: 73) [ka] B746201 and B746201-LALA have a light chain that corresponds to the light chain of anifrolumab, sequence number 54. B746201 - Heavy chain of LALA (SEQ ID NO: 74) [ka] Fusion proteins of B637301, B637302, and B746201 were expressed and purified, and analyzed by SDS-PAGE. The heavy and light chains of the antibodies linked to TACI-BCMA each formed a single band (results not shown). This indicates that removing the glycosylation risk points in the BCMA protein sequence can make the fusion proteins more homogeneous.

[0192] Example 13: Inhibitory activity of fusion protein of anifrolumab and TACI-19-16-BCMA against type I interferon The inhibitory effect of anifrolumab and TACI-19-16-BCMA fusion proteins (B606401, B637302, B746201, and B805201) on IFN-α / β activity was detected using the HEK-Blue IFN-α / β cell (InvivoGen, Cat: hkb-ifnab) reagent kit.

[0193] As a method, HEK-Blue TM IFN-α / β cells were digested and resuspended at 2.8E5 / mL in DMEM medium (Gibco, Cat. No. 11995065) containing 10% FBS and 1% P / S, and seeded into a 96-well plate (Costar, Cat. No. 3599). The antibody to be assayed was added and incubated at 37°C for 30 minutes. IFN-β (Sino biological, Cat. No. 10704-HNAS) was added to a final concentration of 0.01 ng / mL and incubated at 37°C for 24 hours. 20 μL of the culture supernatant was mixed with 180 μL of Quanti-Blue (InvivoGen, Cat. No. rep-qbs) in a new 96-well plate and incubated at 37°C for 1 hour. OD655 was measured.

[0194] Referring to Figure 16 and Table 9, the results showed that all four fusion proteins could inhibit the activity of IFN-α / β, and the activities of the four fusion proteins were comparable and consistent with those of anifrolumab.

[0195] [Table 9]

[0196] Example 14. In vitro binding studies of fusion proteins of anifrolumab and TACI-19-16-BCMA to BAFF and APRIL The binding ability of the fusion proteins of B606401, B637302, B746201 and B805201 to BAFF and APRIL was detected by ELISA.

[0197] BAFF (PeproTech, Cat: 310-13) or ARPIL (Acro Biosystems, Cat: APL-H52D1) was diluted to 1 μg / mL in PBS and coated onto a 96-well plate overnight at 4°C. The plate was washed with PBST and blocked with blocking buffer (PBST containing 1% BSA) at 37°C for 1 hour. The plate was then tapped, and antibodies to be measured were diluted in blocking buffer and incubated at 37°C for 1.5 hours. The plate was 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, and 100 μL of TMB color developing solution (Biopanda, Cat: TMB-S-003) was added to develop the color at 37°C for 3 minutes. 100 μL of ELISA stopping solution (Solarbio, Cat: C1058) was then added to detect OD450.

[0198] Referring to Figure 17 and Table 10, all four fusion proteins clearly bound to BAFF, and all four fusion proteins had stronger BAFF-binding abilities than Telitaciccept. B606401 and B637302 had comparable BAFF-binding abilities and showed a higher EC 50 Both were reduced to 1 / 4 to 1 / 3 of the original level, and B746201 and B805201 had equivalent BAFF binding ability and showed a higher EC 50 Both were reduced to half the original level.

[0199] [Table 10]

[0200] Referring to Figure 18 and Table 11, all four fusion proteins clearly bound to APRIL, and B637302 had similar ability to bind to APRIL and a lower EC 50The EC20 binding capacity of B746201 and B805201 was comparable to that of Telitacic receptor β-blocker (Telitacicept). 50 was reduced to one-third of that level, and B606401 has the same ability to bind to APRIL as Telitaccept.

[0201] [Table 11]

[0202] Example 15. Functional experiments of plasmacytoid dendritic cells (pDC) To evaluate the in vitro efficacy of anifrolumab and the TACI-19-16-BCMA fusion protein, we established a method for testing the biological activity of IFNAR1 antagonists in vitro. The experiment to detect pDC function was as follows: Freshly isolated healthy human PBMCs were cultured in vitro in 1640 GlutaMAX (11 mM glucose concentration) basal medium. PBMCs were cultured at 3 × 10 6 The cells were seeded into a 96-well plate at a density of 1 / mL and stimulated with 0.5 μM CpG-A ODN 2216 (InvivoGen cat: tlrl-2216), and then treated with gradient concentrations of anifrolumab, B637302, B606401, and IgG1 isotype. 24 h later, the secretion level of IFN-α cytokine in the cell culture supernatant was detected using a human IFN-α reagent kit (Cisbio cat: 62HIFNAPEG).

[0203] As a result, B637302, B606401, and the control antibody anifrolumab were all able to inhibit IFN-α production in a dose-dependent manner, and the inhibitory activities of the three were comparable (FIG. 19).

[0204] Example 16. IFN-α-induced plasma cell differentiation in vitro functional experiments To evaluate the antagonistic biological activity of the fusion protein of anifrolumab and TACI-19-16-BCMA against IFNAR1, a functional experimental method for in vitro plasma cell differentiation was established. Specifically, human B cells were isolated from fresh healthy human PBMCs using a B cell sorting reagent kit (Stemcell cat: 17954) and cultured in vitro in 1640 GlutaMAX (Gibco cat: 72400-47) basal medium. B cells were cultured at 1 × 10 5 The 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). They were then further treated with gradient concentrations of anifrolumab, B637302, B746201, B606401, B805201, and an IgG1 isotype control. Four days later, plasma cells (CD27 + CD38 + ) differentiation rate was detected.

[0205] Referring to Figure 20, the results show that B637302, B746201, B606401, B805201, and the control antibody anifrolumab can effectively inhibit the in vitro differentiation of B cells into plasma cells induced by IFN-α in a dose-dependent manner, and IC 50 The inhibitory activities of the fusion protein and the control antibody anifrolumab were 10.75 nM, 1.895 nM, 17.45 nM, 6.208 nM, and 12.66 nM, respectively, suggesting that the inhibitory activities of the fusion protein and the control antibody anifrolumab were equivalent.

[0206] Example 17. Functional experiment of BAFF-induced B cell proliferation To evaluate the in vitro efficacy of the anifrolumab and TACI-19-16-BCMA fusion protein, we established a method for testing the TACI-BCMA bioactive molecule in vitro. The steps of the BAFF-induced B cell proliferation experiment were as follows: Human B cells were isolated from fresh healthy human PBMCs using a B cell sorting reagent kit (Stemcell cat: 17954) and labeled with CTV (Invitrogen cat: C34557). They were cultured in vitro in 1640 GlutaMAX (Gibco cat: 72400-47) basal medium, and 1 x 10 B cells were cultured. 5 The cells were seeded into a 96-well plate at a density of 1 / well 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, the cells were stimulated with gradient concentrations of telitacicept, B637302, B606401, B80521, and IgG1. The isotype was added and treated, and B cell proliferation signals were detected by flow cytometry 5 days later.

[0207] As a result, referring to Table 12, B637302, B606401, B805201, and the control, telitacicept, all effectively inhibited BAFF-induced in vitro B cell proliferation in a dose-dependent manner. Referring to Figure 21, the inhibitory effects of B637302 and B805201 were significantly stronger than those of telitacicept. Under treatment conditions with a low drug concentration of 10 nM, telitacicept showed no inhibitory activity, while B637302, B606401, and B805201 all showed significant inhibitory activity against plasma cell differentiation.

[0208] [Table 12]

[0209] Example 18. Functional experiment of APRIL inducing B cell proliferation To evaluate the in vitro efficacy of the anifrolumab and TACI-19-16-BCMA fusion protein, we established a method for testing the TACI-BCMA bioactive molecule in vitro. The steps for the APRIL-induced B cell proliferation experiment were as follows: Human B cells were isolated from fresh healthy human PBMCs using a B cell sorting reagent kit (Stemcell cat: 17954), labeled with CTV (Invitrogen cat: C34557), and cultured in vitro in 1640 GlutaMAX (Gibco cat: 72400-47) basal medium. At 1 × 10 B cells, 5 The cells were seeded into a 96-well plate at a density of 1 / well 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). The isotype was added and treated, and B cell proliferation signals were detected by flow cytometry 5 days later.

[0210] As a result, referring to 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 superior to that of telitacicept. Under the condition of treatment with 1000 nM of the drug, the efficacy of B606401 was also significantly superior to that of the control telitacicept.

[0211] [Table 13]

[0212] Example 19. Experimental study of induction of plasma cell production by BAFF + APRIL To evaluate the biological activity of TACI-BCMA in the fusion protein of anifrolumab and TACI-19-16-BCMA, we established an experimental method for in vitro plasma cell generation induction using BAFF and APRIL. The specific procedure was as follows: Human B cells were isolated from fresh healthy human PBMCs using a B cell sorting reagent kit (Stemcell cat: 17954) and cultured in vitro in 1640 GlutaMAX (Gibco cat: 72400-47) basal medium. B cells were then cultured at a density of 1 × 10 5Cells were seeded at a density of 1000 / 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 and stimulated 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). After 7 days, half of the medium was replaced and the concentrations of BAFF and ARPIL were adjusted to 50 ng / mL and 500 ng / mL, respectively, while the concentrations of the remaining stimulatory signals remained unchanged. During this process, gradient concentrations of telitacicept, B637302, B746201, B606401, and B805201 were added to the IgG1 isotype on days 4 to 10, and plasma cells (CD27 + CD38 + ) number was detected.

[0213] Referring to Table 14, the experimental results of detecting plasma cell counts by flow cytometry on day 10 showed that BAFF and ARPIL could effectively induce in vitro plasma cell production. Referring to Figure 23, under 10 nM drug treatment conditions, telitacicept was unable to inhibit plasma cell production, while B637302, B746201, B606401, and B805201 significantly inhibited plasma cell production, suggesting that B637302, B746201, B606401, and B805201 have superior inhibitory activity against BAFF and ARPIL to telitacicept. Under 100 nM drug treatment conditions, all four test drugs and telitacicept significantly inhibited plasma cell production. Under 1000 nM drug treatment conditions, B637302 had the strongest inhibitory activity against plasma cell production, superior to telitacicept.

[0214] [Table 14]

[0215] Example 20: Experimental study of induction of plasma cell production by IFN-α + BAFF + APRIL To evaluate the synergistic biological activity of anifrolumab and TACI-BCMA in the fusion protein of anifrolumab and TACI-19-16-BCMA on the differentiation and generation of B cells into plasma cells, we established an experimental method for in vitro induction of plasma cell generation with IFN-α, BAFF, and APRIL. The specific procedure was as follows: Human B cells were isolated from fresh healthy human PBMCs using a B cell sorting reagent kit (Stemcell cat: 17954) and cultured in vitro in 1640 GlutaMAX (Gibco cat: 72400-47) basal medium. At 1 × 10 B cells, 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 and cells were stimulated 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). After 7 days, half of the medium was replaced, and the concentrations of BAFF and ARPIL were adjusted to 50 ng / mL and 500 ng / mL, respectively, while the concentrations of the remaining stimulatory signals remained unchanged. During this process, gradient concentrations of anifrolumab, telitacicept, B637302, B606401, and IgG1 isotype were added from days 0 to 10, and plasma cells (CD27) were counted by flow cytometry on day 10. + CD38 + ) was detected (Fig. 24A).

[0216] In a synergistic experiment on in vitro plasma cell production, experimental conditions were optimized and it was found that, under the condition of 15 ng / mL IL-21, IFN-α and BAFF+APRIL synergistically promoted plasma cell differentiation and production, and the promoting effects of both drugs were comparable. Therefore, the inhibitory activity of each protein drug at different concentrations was compared under these conditions. B637302, B606401, telitacicept, and anifrolumab all inhibited plasma cell production in a dose-dependent manner (see Table 15 and Figure 24, B–D). Under treatment conditions with low drug concentrations of 10 nM, telitacicept or anifrolumab alone had no significant inhibitory activity on plasma cell production, while B637302 and B606401 exhibited significant inhibitory activity (see Figure 24, B).

[0217] Under treatment conditions with a drug concentration of 100 nM, anifrolumab alone had no obvious inhibitory activity, and telitacicept alone had some activity, but the activity of B637302 was clearly superior to that of telitacicept (see Figure 24C). Under treatment conditions with a drug concentration of 1000 nM, anifrolumab alone had some activity, but the activity of the fusion protein of B637302 and B606401 was clearly superior to that of anifrolumab (see Figure 24D).

[0218] [Table 15]

[0219] Example 21: Experiment on induction of PD in PBMC-humanized mice using PEG-IFN-α To detect the in vivo efficacy of anifrolumab and the anifrolumab terminus in the TACI-19-16-BCMA fusion protein, B-NDG immunodeficient mice were used. 7PBMC cells were humanized via tail vein injection and 0.6 μg of PEG-IFN-α (PEGASYS®, Roche) was injected intraperitoneally to induce IFN-α expression. Mice were treated with different concentrations of the drugs anifrolumab, B637302, and B606401, or PBS intraperitoneally. PBMCs were harvested at 2 hours and pSTAT1 (#9167, Cell Signaling Technology) levels were assayed by flow cytometry. Peripheral blood was collected on days 1 and 3, and PBMCs were isolated and subjected to QPCR analysis to detect the mRNA expression levels of IFN-α downstream genes (e.g., ISG-15, IFI13, MX-1, HERC5, etc.). The results are shown in Figures 25A and 25B.

[0220] Referring to Figure 25C, PBMCs were collected 2 hours after induction and pSTAT1 was detected by flow cytometry. The results showed that B637302, B606401, and anifrolumab had comparable hIFN-α inhibitory activity. Referring to Figure 25D and E, the ISG mRNA expression levels detected by QPCR on days 1, 3, and 7 also showed similar results, consistent with the results of previous in vitro reporter molecule assays and biological activity experiments.

[0221] Example 22. Experiment on induction of PD in mice using BAFF+APRIL To assess the in vivo efficacy of the TACI-19-16-BCMA terminus in the anifrolumab and TACI-19-16-BCMA fusion protein, C57 / B6 mice were induced by intraperitoneal injection of 3 μg of BAFF (BAF-H52D4-1 mg, AcroBio) and 0.5 μg of APRIL (APL-H52D1-1 mg, AcroBio). In this model, mice were treated intraperitoneally with different concentrations of the drugs telitacicept, B637302, B606401, or PBS. Peripheral blood samples were collected on days 4 and 7, and plasma IgA levels were measured by ELISA (Figure 26A).

[0222] As a result, although there was no clear difference in IgA between the PBS and treated groups on days 0 and 2 (results not shown), on days 4 and 7, B637302, B606401, and telitacicept were able to significantly inhibit IgA production compared to the PBS control group (see Figure 26B and Figure 26C). Furthermore, the results on day 7 showed that under equimolar administration conditions, the efficacy of B637302 > B606401 > telitacicept (see Figure 26C), which is consistent with the results of the previous in vitro BAFF and APRIL ELISA binding experiment and in vitro biological activity experiment.

Claims

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

2. The TACI polypeptide (1) amino acid residues 68 to 105 of SEQ ID NO: 1 or a mutant thereof; (2) SEQ ID NO: 1 or a polypeptide having at least 95% sequence identity thereto; (3) amino acid residues 33 to 67 of SEQ ID NO: 1; (4) amino acid residues 70 to 104 of SEQ ID NO: 1 or a mutant thereof; (5) amino acid residues 30 to 110, 69 to 111, 69 to 112, 13 to 118, or 33 to 104 of SEQ ID NO: 1, or a mutant thereof; (6) Amino acid residues 68 to 106, 68 to 107, or 68 to 108 of SEQ ID NO: 1, or a mutant thereof The polypeptide comprises any one of (1) to (6) above, 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 replacements selected from 69T or 69R, 72S, 73E or 73Q, 77E, 85T or 85A, 102A or 102R, 103Y; Most preferably, the variant is 69T, 72S, 73E, 73Q, 77E, a combination of 69R and 85T, a combination of 69R and 85A, 102A, a combination of 69R, 85T and 102R, a combination of 73E and 77E, a combination of 72S, 73E and 77E, a combination of 69T and 102A, a combination of 69T and 103Y, a combination of 69T, 102A and 103Y, a combination of 69T, 73E, 77E and 102A, and having any one or combination of amino acid substitutions selected from: The amino acid residues are numbered according to the natural order of the sequence shown in SEQ ID NO:

1. The fusion protein of claim 1.

3. The TACI polypeptide comprises an amino acid sequence represented by any one of SEQ ID NOs: 1, 2, 6 to 29; A fusion protein according to any one of claims 1 to 2.

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

5. the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:43 or having at least 90% sequence identity thereto; The light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 44 or having at least 90% sequence identity thereto. The fusion protein of claim 4.

6. comprising an immunoglobulin Fc region, preferably an IgG1 Fc region, more preferably an Fc region set forth in SEQ ID NO: 3; A fusion protein according to any one of the preceding claims.

7. the Fc region comprises one or more amino acid mutations, which are capable of reducing Fc-FcR binding, preferably mutations capable of reducing Fc-FcγR binding, and more preferably any one of the following amino acid mutations or combinations 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. The fusion protein of claim 6.

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

9. (I) a first polypeptide chain which is, from the N-terminus to the C-terminus, [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 is, from the N-terminus to the 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 that is an antibody heavy chain, and a second polypeptide chain that is, in order from the N-terminus to the C-terminus, [TACI polypeptide]-[linker 3]c-[antibody light chain]; (IV) a first polypeptide chain that is an antibody heavy chain, and a second polypeptide chain that is, in order from the N-terminus to the C-terminus, [antibody light chain]-[linker 4]d-[TACI polypeptide]; (V) a first polypeptide chain consisting of, from the N-terminus to the C-terminus, [TACI polypeptide 1]-[linker 1]a-[antibody heavy chain], and a second polypeptide chain consisting of, from the N-terminus to the C-terminus, [antibody light chain]-[linker 4]d-[TACI polypeptide 2]; (VI) a first polypeptide chain consisting of, from the N-terminus to the C-terminus, [antibody heavy chain]-[linker 2]b-[TACI polypeptide 1]; and a second polypeptide chain consisting of, from the N-terminus to the C-terminus, [TACI polypeptide 2]-[linker 3]c-[antibody light chain]. (VII) a first polypeptide chain which is, from the N-terminus to the 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 that is an antibody heavy chain, and a second polypeptide chain that is, from the N-terminus to the C-terminus, [TACI polypeptide 1]-[linker 3]c-[antibody light chain]-[linker 4]d-[TACI polypeptide 2]; The polypeptide chain is represented by any one of (I) to (VIII): Among them, - indicates 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 polypeptides defined in any one of claims 1 to 3, and TACI polypeptide 1 and TACI polypeptide 2 may be the same or different; a, b, c, and d each independently represent 0 or 1; The antibody is an anti-IFNAR1 antibody defined in any one of claims 4 to 5, Preferably, the linker 1, linker 2, linker 3, and linker 4 are each independently (GxS) ｙ wherein 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 represented by any one of SEQ ID NOs: 39 to 41. A fusion protein according to any one of the preceding claims.

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

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

12. (i) a first polypeptide chain which is, from the N-terminus to the 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 is, 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 that is an antibody heavy chain, and a second polypeptide chain that is, in order from the N-terminus to the C-terminus, [TACI polypeptide and BCMA polypeptide domain]-[linker 3]c-[antibody light chain]; (iv) a first polypeptide chain that is an antibody heavy chain, and a second polypeptide chain that is, in N-terminus to C-terminus order, [antibody light chain]-[linker 4]d-[TACI polypeptide and BCMA polypeptide domain]; (v) a first polypeptide chain which is, from the N-terminus to the C-terminus, [TACI polypeptide and BCMA polypeptide domain 1]-[linker 1]a-[antibody heavy chain]; and a second polypeptide chain which is, from the N-terminus to the C-terminus, [antibody light chain]-[linker 4]d-[TACI polypeptide and BCMA polypeptide domain 2]; (vi) a first polypeptide chain consisting of, from N-terminus to C-terminus, [antibody heavy chain]-[linker 2]b-[TACI polypeptide and BCMA polypeptide domain 1]; and a second polypeptide chain consisting of, from N-terminus to C-terminus, [TACI polypeptide and BCMA polypeptide domain 2]-[linker 3]c-[antibody light chain]; (vii) a first polypeptide chain which is, from N-terminus to C-terminus, [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 that is an antibody heavy chain, and a second polypeptide chain that is, from N-terminus to C-terminus, [TACI polypeptide and BCMA polypeptide domain 1]-[linker 3]c-[antibody light chain]-[linker 4]d-[TACI polypeptide and BCMA polypeptide domain 2]; The polypeptide chain is represented by any one of (i) to (viii): Among them, 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, from the N-terminus to the C-terminus, [TACI polypeptide]-[linker 5]e-[BCMA polypeptide], 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 polypeptides defined in any one of claims 1 to 3, the BCMA polypeptide is selected from the BCMA polypeptides 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, Among them, - indicates a peptide bond, The linkers 1, 2, 3, 4, and 5 may be the same or different, and a, b, c, d, and e each independently represent 0 or 1; Preferably, the linker 1, linker 2, linker 3, linker 4, and linker 5 are each independently (GxS) ｙ wherein 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 represented by any one of SEQ ID NOs: 39 to 41. A fusion protein according to any one of claims 1 to 11.

13. Two identical first polypeptide chains represented by SEQ ID NO: 55 and two identical second polypeptide chains represented by any one of SEQ ID NOs: 63 to 66, 71, and 72; two identical first polypeptide chains set forth in SEQ ID NO: 61 and two identical second polypeptide chains set forth in any one of SEQ ID NOs: 63-66, 71, 72; or Two identical first polypeptide chains set forth in SEQ ID NO: 73 or 74 and two identical second polypeptide chains set forth in any one of SEQ ID NO: 54, A fusion protein according to any one of claims 1 to 12.

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

15. The TACI polypeptide (1) amino acid residues 68 to 105 of SEQ ID NO: 1 or a mutant thereof; (2) SEQ ID NO: 1 or a polypeptide having at least 95% sequence identity thereto; (3) amino acid residues 33 to 67 of SEQ ID NO: 1; (4) amino acid residues 70 to 104 of SEQ ID NO: 1 or a mutant thereof; (5) amino acid residues 30 to 110, 69 to 111, 69 to 112, 13 to 118, or 33 to 104 of SEQ ID NO: 1, or a mutant thereof; (6) Amino acid residues 68 to 106, 68 to 107, or 68 to 108 of SEQ ID NO: 1, or a mutant thereof The polypeptide comprises any one of (1) to (6) above, 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 replacements selected from 69T or 69R, 72S, 73E or 73Q, 77E, 85T or 85A, 102A or 102R, 103Y; Most preferably, the variant has any one amino acid substitution or combination of amino acid substitutions selected from 69T, 72S, 73E, 73Q, 77E, a combination of 69R and 85T, a combination of 69R and 85A, 102A, a combination of 69R, 85T and 102R, a combination of 73E and 77E, a combination of 72S, 73E and 77E, a combination of 69T and 102A, a combination of 69T and 103Y, a combination of 69T, 102A and 103Y, a combination of 69T, 73E, 77E and 102A; The amino acid residues are numbered according to the natural order of the sequence shown in SEQ ID NO:

1. The fusion protein of claim 14.

16. The TACI polypeptide comprises an amino acid sequence represented by any one of SEQ ID NOs: 1, 2, 6 to 29; A fusion protein according to claim 14 or 15.

17. The BCMA polypeptide comprises amino acid residues 7 to 41 of SEQ ID NO:

30. A fusion protein according to any one of claims 14 to 16.

18. The amino acid sequence of the BCMA polypeptide comprises any one of the sequences shown in SEQ ID NOs: 30, 67, and 68; 18. The fusion protein of claim 17.

19. Further comprising an immunoglobulin Fc region, preferably an IgG1 Fc region, more preferably an Fc region set forth in SEQ ID NO: 3; A fusion protein according to any one of claims 14 to 18.

20. the Fc region comprises one or more amino acid mutations, which are capable of reducing Fc-FcR binding, preferably mutations capable of reducing Fc-FcγR binding, and more preferably any one of the following amino acid mutations or combinations 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.

20. The fusion protein of claim 19.

21. (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]; The polypeptide chain is represented by any one of (I) to (VIII): Among them, - 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 each independently represent 0 or 1; Preferably, the linker 1, linker 2, linker 3, and linker 4 are each independently (GxS) ｙ wherein 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 represented by any one of SEQ ID NOs: 39 to 41. A fusion protein according to any one of claims 14 to 20.

22. Contains an amino acid sequence represented by any one of SEQ ID NOs: 31 to 38, 69, and 70; A fusion protein according to any one of claims 14 to 21.

23. It is a dimer, A fusion protein according to any one of claims 14 to 22.

24. A composite, a TACI polypeptide as defined in any one of claims 1 to 3, an anti-IFNAR1 antibody as defined in any one of claims 4 to 8, and a BCMA polypeptide as defined in claim 1 or 11; or A TACI polypeptide as defined in any one of claims 14 to 23 and a BCMA polypeptide as defined in any one of claims 14 to 23. Composite.

25. 1. A pharmaceutical composition comprising: A fusion protein according to any one of claims 1 to 23 or a conjugate according to claim 24; one or more pharmaceutically acceptable carriers, diluents or excipients; A pharmaceutical composition comprising:

26. A polynucleotide comprising: A fusion protein according to any one of claims 1 to 23, or 25. Encoding the compound of claim 24 Polynucleotide.

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

28. 1. 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 conjugate 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, said 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. method.

29. A method for preparing a fusion protein according to any one of claims 1 to 23 or a conjugate according to claim 24, comprising culturing a host cell according to claim 27 and expressing said fusion protein or conjugate, and optionally isolating or purifying said fusion protein or conjugate. method.

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