Methods for screening and preparing immunomodulators

Abstract

The present disclosure provides methods for screening and preparing immunomodulatory agents, and the present disclosure further relates to the use of said immunosuppressive agents for treating immune-related diseases.

Description

[Technical Field]

[0001] This disclosure claims priority to a Chinese patent application filed on August 29, 2022, bearing application number CN202211043007.2 and entitled "Method for screening and preparing immunomodulatory agents," the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to the field of biomedicine, and in particular, the disclosure relates to methods for screening and preparing immunomodulatory agents and the use of said immunomodulatory agents to treat diseases, particularly autoimmune diseases. [Background technology]

[0003] Many autoimmune diseases, such as systemic lupus erythematosus (SLE), lupus nephropathy, and multiple sclerosis, are associated with the production of autoantibodies against self-tissue antigens, triggering a systemic immune response that ultimately affects different organs and causes the corresponding disease symptoms. From a biological perspective, a series of key biological processes—B cell differentiation into plasma cells, plasma cell production and maintenance, and plasma cell antibody secretion—play a central role in the onset and progression of autoimmune diseases. Therefore, many drugs targeting B cells and plasma cells have entered clinical trials, including the approved belimumab (trade name Benlysta), a B-lymphocyte-stimulating factor (BLyS, also known as BAFF)-specific inhibitor for the treatment of patients with active, autoantibody-positive systemic lupus erythematosus who are receiving standard therapy. In addition, telitacicept (RC18, trade name: Tai'ai) is a TACI-Ig fusion protein that inhibits B cells and plasma cells by simultaneously targeting BLyS / APRIL, and was approved by the China National Medical Products Administration in 2021 as a treatment for systemic lupus erythematosus.

[0004] B cell differentiation is divided into several stages, including five stages: precursor B cells, immature B cells, mature B cells, activated B cells, and plasma cells. The first three stages are antigen-independent and mainly occur in the bone marrow. After maturation, B cells migrate to peripheral immune organs and become activated after antigen stimulation, thereby proliferating and further differentiating into memory B cells or plasma cells.

[0005] In the development of new drugs for autoimmune diseases (especially SLE), B cell-targeted therapy is one of the main directions for new drug development. Establishing experimental methods to evaluate B cell-related biological functions is a crucial step in the development of drugs for autoimmune diseases. Therefore, establishing a convenient, efficient, and stable experimental system to evaluate B cell-related functions will help advance the process of new drug development for autoimmune diseases.

[0006] However, conventional B cell function evaluation experiments are mainly performed by labeling thymidine (TdR) with 3H. 3H-TdR, which is a TdR labeled with the isotope 3H, can be incorporated into the DNA synthesis metabolic process as a precursor for DNA synthesis. Each newly synthesized DNA double strand then emits a radioactive signal, and measuring the cellular radioactivity intensity can reflect the cellular DNA metabolism and cell proliferation status. However, this method mainly evaluates B cell proliferation, and it must be performed in a licensed and secure laboratory. Experimenters also require rigorous specialized training. Radioactivity detection equipment is relatively expensive, and there is a risk of radionuclide contamination. Therefore, B cell proliferation experiments cannot be performed in ordinary laboratories, and are therefore not widely used.

[0007] In the present disclosure, after exploring a series of experimental conditions, a simple and efficient experimental system for B cell differentiation into plasma cells was developed. Using related commercially available or late-stage clinical drugs, it was confirmed that the method is convenient and feasible and can be used to screen immunomodulators that can be used for drug preparation. Summary of the Invention

[0008] The present disclosure provides methods for screening and preparing immunomodulatory agents, and the use of such immunomodulatory agents to prepare medicaments for treating or ameliorating disease.

[0009] Methods for screening and preparing immunomodulators The present disclosure provides methods for promoting B cell differentiation. The present disclosure also provides methods for screening and preparing immunomodulatory agents. The methods can be in vitro or in vivo.

[0010] In some embodiments, methods for promoting B cell differentiation and screening and preparation of immunomodulatory agents are provided, comprising administering to B cells a TLR agonist, and / or an interleukin, and / or retinoic acid (RA), and / or a tumor necrosis factor family member. For example, the TLR agonist is selected from a TLR1 agonist, a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR6 agonist, a TLR7 agonist, a TLR8 agonist (e.g., R848), and a TLR9 agonist (e.g., CpG-ODN, e.g., CpG-A, CpG-B). For example, the interleukin is selected from IL-3, IL-4, IL-6, IL-10, and IL-21. For example, the tumor necrosis factor family member is BAFF or APRIL.

[0011] In some embodiments, the TLR agonist is Heplisav, SD-101, resiquimod, Dynavax, DV-281, imiquimod, cobitolimod, entolimod, lefitolimod, Poly-ICLC, Grass MATA MPL, G-100, AST-008, GSK-1795091, tilsotolimod, KMRC-011, CMB-305, rintatolimod, AZD-1419, influenza-PAL, SAR-439794, MIS-416, MGN-1601, GS K-2245035, VTX-1463, motolimod, GS-9688, LHC-165, BDB-001, PGV-001, AV-7909, DSP-0509, DPX-E7, RG-7854, telratolimod, vesatolimod, poly-ICLC adjuvanted vaccines, MVAME-03, Riboxxim, G-305, PUL-042, litenimod, DRibbles The compound may be selected from the group consisting of cyclohexyl methyl methacrylate (CPM), ...

[0012] In a first aspect, some embodiments of the present disclosure provide a method for promoting B cell differentiation, comprising: a) IL-3 and CpG-A, b) IL-3, CpG-A, and IL-21; c) IL-3, CpG-A and IFNα, or d) IL-3, CpG-A, IFNα, and IL-21; administering The above method can improve the rate of differentiation of B cells into plasma cells and / or improve the survival rate of B cells.

[0013] In some embodiments, any of a) to d) can improve the differentiation rate of B cells into plasma cells, and d) can improve the survival rate of B cells.

[0014] In the above-mentioned method, the concentration of IL-3 is 0.1 to 50 ng / mL, 1 to 30 ng / mL, or 5 to 20 ng / mL, for example, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 ng / mL; The concentration of CpG-A is 0.01 to 10 μM, 0.05 to 5 μM, 0.1 to 2 μM, or 0.2 to 1 μM, for example, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1 μM; the concentration of IFNα is 1 to 10,000 U / mL, 100 to 5,000 U / mL, or 500 to 2,000 U / mL, for example, about 500, about 600, about 700, about 800, about 900, about 1,000, about 1,100, about 1,200, about 1,300, about 1,400, or about 1,500 U / mL; The concentration of IL-21 is 0.1 to 1000 ng / mL, 1 to 200 ng / mL, 10 to 100 ng / mL, 20 to 50 ng / mL, for example, about 6.6, about 16.5, about 20, about 25, about 30, about 33, about 35, about 40, about 45, about 50, or about 66 ng / mL.

[0015] In some embodiments, the B cells are a-1) 5 to 20 ng / mL IL-3 and 0.2 to 1 μM CpG-A; b-1) 5 to 20 ng / mL IL-3, 0.2 to 1 μM CpG-A, and 16.5 to 66 ng / mL IL-21; c-1) 5 to 20 ng / mL IL-3, 0.2 to 1 μM CpG-A, and 500 to 2000 U / mL IFNα, or d-1) 5 to 20 ng / mL IL-3, 0.2 to 1 μM CpG-A, 500 to 2000 U / mL IFNα, and 16.5 to 66 ng / mL IL-21; The method includes administering

[0016] In some embodiments, the B cells are a-2) about 10 ng / mL IL-3 and about 0.5 μM CpG-A; b-2) about 10 ng / mL IL-3, about 0.5 μM CpG-A, and about 33 ng / mL IL-21; c-2) about 10 ng / mL IL-3, about 0.5 μM CpG-A, and about 1000 U / mL IFNα, or d-2) about 10 ng / mL IL-3, about 0.5 μM CpG-A, about 1000 U / mL IFNα, and about 33 ng / mL IL-21; The method includes administering

[0017] In some embodiments, a method for screening an immunomodulatory agent is provided, comprising: Step 1) administering a TLR agonist to B cells and culturing them for M days, where M is an integer selected from 1 to 28; Step 2) Removing the TLR agonist; Step 3) adding one or more interleukins and one or more TNF family members and culturing for N days, where N is an integer selected from 1 to 28; Step 4) Adjust the concentration of TNF family members and continue culturing. Step 5) Adding the sample to be measured; Step 6) determining the differentiation degree of B cells; Among them, the step 5) is carried out simultaneously with, during, or after any one of the steps 1) to 4).

[0018] In some embodiments, a method for screening an immunomodulatory agent is provided, comprising: Step 1) administering a TLR agonist and IFN to B cells and culturing them for M days, where M is an integer selected from 1 to 28; Step 2) Removing TLR agonists and IFN; Step 3) adding one or more interleukins and one or more TNF family members and culturing for N days, where N is an integer selected from 1 to 28; Step 4) Adjust the concentration of TNF family members and continue culturing. Step 5) Adding the sample to be measured; Step 6) determining the differentiation degree of B cells; Among them, the step 5) is carried out simultaneously with, during, or after any one of the steps 1) to 4).

[0019] In some embodiments, the method further comprises: Step 7) further comprises selecting the sample to be measured as an immunomodulatory agent capable of activating or inhibiting B cell differentiation, and optionally, step 6) further comprises selecting the sample to be measured as an immunomodulatory agent capable of activating or inhibiting B cell differentiation, and + CD38 + The present invention includes determining the degree of B cell differentiation by detecting the percentage of B cells in the blood.

[0020] In some embodiments, the TLR agonist is any one or more selected from TLR7, TLR8, and TLR9 agonists, illustratively any one or more selected from R848, CpG, and LPS, illustratively CpG-A or CpG-B, more illustratively CpG-B; If present, the IFN is IFNα or IFNβ, illustratively IFNα; The interleukin is one or more selected from IL-3, IL-6, IL-10, and IL-21, and illustratively, the interleukin is a combination of IL-6, IL-10, and IL-21; The TNF family member is any one or more selected from BAFF and APRIL, and illustratively, the TNF family member is a combination of BAFF and APRIL.

[0021] In some embodiments, when the TNF family members are BAFF and APRIL, modulating the concentration of the TNF family members comprises decreasing the concentration of BAFF and / or increasing the concentration of APRIL; Exemplarily, the concentration of BAFF in step 4) is reduced to 0.01 to 0.9 times, more exemplarily 0.02 to 0.5 times, and further exemplarily about 0.02, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, or about 0.9 times the concentration of BAFF added in step 3); Illustratively, the concentration of APRIL in step 4) is increased by 1.1 to 100 times, more illustratively 5 to 50 times, and further illustratively about 1.5, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 15, about 20, about 25, about 30, about 35, about 40, or about 50 times the concentration of APRIL added in step 3).

[0022] In some embodiments, a method for screening an immunomodulatory agent is provided, comprising: 1) Administering TLR agonists and interferon to B cells, 2) Add the sample to be measured on any of days 0 to 21 and continue culturing. 3) determining the degree of differentiation of B cells; Illustratively, the method further comprises: 4) Selecting the sample to be measured as an immunomodulatory agent capable of activating or inhibiting B cell differentiation.

[0023] In some embodiments, the TLR agonist is one or more selected from TLR7, TLR8, and TLR9 agonists, illustratively, the TLR agonist is one or more selected from R848 and CpG, illustratively, the TLR agonist is CpG-A or CpG-B, more illustratively, CpG-B; The IFN is IFNα or IFNβ, illustratively IFNα.

[0024] In some embodiments, a method for screening an immunomodulatory agent is provided, comprising: 1) administering 0.1-20 μg / mL of CpG-B and 50-1000 U / mL of IFNα to B cells; 2) Add the sample to be measured on any of days 0 to 21 and continue culturing. 3) determining the degree of differentiation of B cells; Illustratively, the method further comprises: 4) selecting the sample to be measured as an immunomodulatory agent capable of activating or inhibiting B cell differentiation; Illustratively, step 1) administering 0.5 to 5 μg / mL of CpG-B and 100 to 800 U / mL of IFNα to B cells; More illustratively, step 1) includes administering approximately 1 to 2 μg / mL of CpG-B and 250 to 500 U / mL of IFNα to B cells.

[0025] In some embodiments, the immunomodulatory agent is a type I interferon pathway modulator and / or a TNF pathway modulator; Illustratively, the type I interferon pathway modulator is an IFNAR1 signaling pathway modulator; Illustratively, the TNF pathway modulator is a BAFF and / or ARRIL pathway modulator.

[0026] In some embodiments, activating or inhibiting the differentiation of the B cells is achieved by detecting the number or proportion of B cells differentiated into plasma cells, and the plasma cells are illustratively CD27 + CD38 + These are B cells.

[0027] In some embodiments, the concentration of the B cells is 1 x 10 in a 96-well plate. 5 / Well.

[0028] In some embodiments, the B cells are 1) isolating B cells from PBMCs; 2) culturing in 1640 medium containing 50 μM β-mercaptoethanol, 100 μM MEM non-essential amino acids, and sodium pyruvate, wherein the 1640 medium contains GlutaMAX; is obtained by

[0029] In some embodiments, the immunomodulatory agent is selected from a protein or polypeptide, a nucleic acid, an aptamer, a small molecule compound, and a molecule comprising the protein or polypeptide, nucleic acid, aptamer, or small molecule compound, and the protein or polypeptide is, for example, an antibody or antigen-binding fragment thereof, a cytokine, a receptor, or a ligand; Illustratively, the immunomodulator is for treating a B cell disorder or an autoimmune disease, Further illustratively, the B cell disorder or autoimmune disease is a disease or condition associated with TACI and / or BCMA expression; Further illustratively, 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, and the B-cell disorder is selected from tumors, chronic leukemia, multiple myeloma, non-Hodgkin's lymphoma, post-transplant lymphoproliferation, and light chain gammopathy.

[0030] In a second aspect, some embodiments of the present disclosure provide a method for promoting B cell differentiation, comprising: e) CpG-B and IFNα, f) R484 and IFNα; g) CpG-B, BAFF, and APRIL; h) R484, BAFF and APRIL, i) CpG-B, IFNα, RA, BAFF, and APRIL, or j) R484, IFNα, RA, BAFF, and APRIL; administering The above method can improve the rate of differentiation of B cells into plasma cells and / or improve the survival rate of B cells.

[0031] In some embodiments, e), f), i), and j) can promote the differentiation rate of B cells into plasma cells, and e) to j) can all increase the survival rate of B cells, with i) and j) improving the survival rate of B cells to a greater extent than g) and h).

[0032] In the above method, the concentration of CpG-B is 0.1 to 100 μg / mL, 0.2 to 20 μg / mL, 0.5 to 10 μg / mL, or 0.5 to 5 μg / mL, for example, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, or about 2 μg / mL; the concentration of IFNα is 1 to 10,000 U / mL, 100 to 2,000 U / mL, 200 to 800 U / mL, or 100 to 500 U / mL, for example, about 50, about 100, about 150, about 200, about 250, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1,000 U / mL; The concentration of R484 is 0.1 to 100 μg / mL, 0.2 to 20 μg / mL, 0.5 to 10 μg / mL, or 0.5 to 5 μg / mL, for example, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, or about 2 μg / mL; The concentration of RA is 0.1 to 50 μg / mL, 0.2 to 20 μg / mL, 0.2 to 10 μg / mL, 1 to 5 μg / mL, for example, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 μg / mL; the concentration of BAFF or APRIL is 1 to 10,000 ng / mL, 100 to 5,000 ng / mL, or 200 to 1,000 ng / mL, for example, about 200, about 300, about 400, about 500, about 600, about 7,000, about 800, about 900, or about 1,000 ng / mL; or the concentration of BAFF or APRIL is 0.1 to 100 nM, 0.5 to 20 nM, or 1 to 10 nM, for example, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nM; In some embodiments, the B cells are e-1) 0.5 to 2 μg / mL of CpG-B and 200 to 800 U / mL of IFNα, or about 1 to 3 μg / mL of CpG-B and 100 to 500 U / mL of IFNα; f-1) 0.5 to 2 μg / mL of R484 and about 200 to 800 U / mL of IFNα, or 1 to 3 μg / mL of R484 and about 100 to 500 U / mL of IFNα; g-1) 0.5-2 μg / mL CpG-B, 200-1000 ng / mL BAFF, and 200-1000 ng / mL APRIL, h-1) 0.5-2 μg / mL of R484, 200-1000 ng / mL of BAFF, and 200-1000 ng / mL of APRIL, i-1) 0.5 to 2 μg / mL CpG-B, 200 to 1000 U / mL IFNα, 2 to 5 μg / mL RA, 200 to 1000 ng / mL BAFF, and 200 to 1000 ng / mL APRIL, or about 1 to 3 μg / mL CpG-B, 100 to 500 U / mL IFNα, 2 to 5 μg / mL RA, 200 to 1000 ng / mL BAFF, and 200 to 1000 ng / mL APRIL; j-1) 0.5 to 2 μg / mL of R484, 200 to 1000 U / mL of IFNα, 2 to 5 μg / mL of RA, 200 to 1000 ng / mL of BAFF, and 200 to 1000 ng / mL of APRIL, or about 1 to 3 μg / mL of R484, 100 to 500 U / mL of IFNα, 2 to 5 μg / mL of RA, 200 to 1000 ng / mL of BAFF, and 200 to 1000 ng / mL of APRIL; is administered.

[0033] In some embodiments, the B cells are e-2) about 1 μg / mL CpG-B and about 500 U / mL IFNα, or about 2 μg / mL CpG-B and 250 U / mL IFNα; f-2) about 1 μg / mL R484 and about 500 U / mL IFNα, or about 1 μg / mL R484 and about 250 U / mL IFNα; g-2) about 1 μg / mL or about 2 μg / mL CpG-B, 500 ng / mL BAFF, and 500 ng / mL APRIL; h-2) about 1 μg / mL or about 2 μg / mL R484, 500 ng / mL BAFF, and 500 ng / mL APRIL; i-2) about 1 μg / mL or about 2 μg / mL CpG-B, about 500 U / mL IFNα, about 3 μg / mL RA, about 500 ng / mL BAFF, and about 500 ng / mL APRIL, or about 1 μg / mL or about 2 μg / mL CpG-B, 250 U / mL IFNα, about 3 μg / mL RA, about 500 ng / mL BAFF, and about 500 ng / mL APRIL; j-2) about 1 μg / mL or about 2 μg / mL R484, about 500 U / mL IFNα, about 3 μg / mL RA, about 500 ng / mL BAFF, and about 500 ng / mL APRIL, or about 1 μg / mL or about 2 μg / mL R484, 250 U / mL IFNα, about 3 μg / mL RA, about 500 ng / mL BAFF, and about 500 ng / mL APRIL; is administered.

[0034] In the above-described configuration, after stimulation with a factor, B cell differentiation is detected (e.g., plasma cells (CD27 + CD38 + ) The rate of differentiation) The time can be any day, for example, day 1, day 2, day 3, day 4, day 5, or day 6, for example, day 4.

[0035] In some embodiments, a method of inducing B cell differentiation is provided, comprising administering to the B cells a TLR agonist and an IFN, Wherein, the TLR agonist is one or more of TLR7, TLR8 and TLR9 agonists, illustratively, the TLR agonist is one or more of R848 or CpG, illustratively, the TLR agonist is CpG-A or CpG-B, further illustratively CpG-B; The IFN is IFNα or IFNβ, illustratively IFNα.

[0036] In some embodiments, a method of inducing B cell differentiation is provided, comprising: administering CpG-A and IFNα to B cells; or administering CpG-B or R484 and IFNα to B cells; Optionally, the method further comprises: administering CpG-A and IFNα to B cells, or administering CpG-B or R484 and IFNα to B cells; and simultaneously administering IL-3 and IL-21 to the B cells.

[0037] In some embodiments, a method of inducing B cell differentiation is provided, comprising: administering to the B cells 5 to 20 ng / mL of IL-3, 0.2 to 1 μM of CpG-A, 500 to 2000 U / mL of IFNα, and 16.5 to 66 ng / mL of IL-21; or administering 0.5 to 5 μg / mL of CpG-B or R484 and 100 to 800 U / mL of IFNα to B cells; For example, administering to the B cells about 10 ng / mL IL-3, about 0.5 μM CpG-A, about 1000 U / mL IFNα, and about 33 ng / mL IL-21; or The method includes administering 1 to 2 μg / mL of CpG-B or R484 and 250 to 500 U / mL of IFNα to B cells.

[0038] In an embodiment of the method for inducing B cell differentiation, the method further comprises simultaneously adding retinoic acid to the B cells, and the concentration of the retinoic acid is illustratively 2 to 5 μg / mL, more illustratively about 3 μg / mL.

[0039] In some embodiments, a cellular model for screening immunomodulatory agents is provided, which comprises B cells induced to differentiate by the methods described herein.

[0040] In a third aspect, some embodiments of the present disclosure provide methods for screening and preparing immunomodulatory agents, which target B cells by: k) CpG-B, IFNα, and immunosuppressants; The method includes administering plasma cells (CD27 + CD38 + ) can promote differentiation into

[0041] In the above embodiment, the concentration of CpG-B is 0.1 to 100 μg / mL, 0.2 to 20 μg / mL, 0.5 to 10 μg / mL, 0.5 to 5 μg / mL, for example, about 1, about 1.2, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, or about 2 μg / mL; The concentration of IFNα is 1 to 10,000 U / mL, 50 to 1,000 U / mL, 100 to 500 U / mL, for example, about 150, about 200, about 250, about 300, about 350, about 400, or about 500 U / mL.

[0042] In some embodiments, methods for screening and preparing immunomodulatory agents are provided, which target B cells: k-1) an immunomodulator having a concentration gradient of 1 to 5 μg / mL CpG-B and 100 to 500 U / mL IFNα; k-2) an immunomodulator having a concentration gradient of about 2 μg / mL CpG-B and about 250 U / mL IFNα; k-3) an immunomodulator having a concentration gradient of about 1 μg / mL CpG-B and about 500 U / mL IFNα; The method includes administering

[0043] In a fourth aspect, in some embodiments of the present disclosure, there is provided a method for screening, preparing or manufacturing an immunomodulatory agent, comprising: Induction step: administering a TLR agonist to B cells, removing the TLR agonist on day M, adding an interleukin, adding a TNF family member, and adjusting the concentration of the TNF family member on day N after addition of the TNF family member; A detection step: adding a sample to be measured during or after the induction step, detecting B cell differentiation, and selecting the sample to be measured that can activate or inhibit B cell differentiation as an immunomodulator; In this case, M and N are integers of 1 to 30, for example, 2, 3, 4, 5, 6, 7, 8, 9, and 10, respectively.

[0044] In some embodiments of the present disclosure, there is provided a method for screening, preparing, or producing an immunomodulatory agent, which includes steps 1), 6), and 7), and may optionally further include steps 2) to 3), and may optionally further include steps 4) to 5), Step 1) administering a TLR agonist to B cells; Step 2) Removing the TLR agonist on day M; Step 3) Adding interleukins Step 4) Adding TNF family members, Step 5) adjusting the concentration of TNF family members; Step 6) Adding samples to be measured and detecting B cell differentiation; Step 7) selecting the sample to be measured as an immunomodulatory agent capable of activating or inhibiting B cell differentiation; In this case, M and N are integers of 1 to 30, for example, 2, 3, 4, 5, 6, 7, 8, 9, and 10, respectively.

[0045] In some specific embodiments, the TLR agonist includes, but is not limited to, a TLR7, TLR8, or TLR9 agonist, for example, R848, LPS, or CpG, for example, CpG-A, CpG-B, for example, CpG-B; the interleukin is selected from, for example, IL-4, IL-3, IL-6, IL-10 and / or IL-21, for example, IL-6, IL-10 and / or IL-21, for example, IL-6, IL-10 and IL-21; The TNF family member is a molecule capable of regulating the TNF signaling pathway, and is selected from, for example, BAFF and APRIL, for example, BAFF and APRIL; the concentration of the TLR agonist is selected from 0.1 to 100 μg / mL, 0.2 to 20 μg / mL, 0.5 to 10 μg / mL, 0.5 to 5 μg / mL, for example, about 1, about 2, about 3, about 4, or about 5 μg / mL; Adjusting the concentration of the TNF family member in step 5 above can be done by, for example, decreasing the concentration of BAFF (e.g., 0.01 to 0.99 times, 0.05 to 0.9 times, 0.05 to 0.5 times, 0.05 to 0.2 times the original concentration, e.g., about 0.02, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, or about 0.9 times the original concentration), and increasing the concentration of APRIL (for example, adjusting to 1.1 to 1000 times, 2 to 100 times, 5 to 50 times, or 10 to 20 times the original concentration, for example, about 1.5, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 15, about 20, about 25, about 30, about 35, about 40, or about 50 times the original concentration), Detecting the differentiation of the B cells can be, for example, detecting plasma cells (CD27 + CD38 + ) detecting the number of The sample to be measured is an immunomodulator (eg, an immunoagonist, an immunosuppressant) or a B cell differentiation modulator, such as a TNF pathway modulator (eg, a BAFF, APRIL pathway agonist or inhibitor).

[0046] In some embodiments, a method for screening, preparing, or manufacturing an immunomodulatory agent is provided, comprising: Step 1) administering CpG (e.g., CpG-A or CpG-B), R848, or LPS to B cells; Step 2) Removing CpG (e.g., CpG-A or CpG-B) or R848 or LPS on day M; step 3) adding interleukins IL-6, IL-10 and / or IL-21; Optionally, step 4) adding BAFF, APRIL; Optionally, step 5) adjusting the concentration of BAFF and APRIL; Optionally, step 6) adding a sample to be measured and detecting B cell differentiation; Optionally, step 7) selecting the sample to be measured as an immunomodulatory agent capable of activating or inhibiting B cell differentiation; In this case, M and N are integers of 1 to 30, respectively.

[0047] In some embodiments, a method for screening, preparing, or manufacturing an immunomodulatory agent is provided, comprising: Step 1) administering CpG-B to B cells; Step 2) Removing CpG-B on day M; Step 3) adding interleukins IL-6, IL-10, and IL-21; Optionally, step 4) adding BAFF and APRIL; Optionally, step 5) decreasing the concentration of BAFF and increasing the concentration of APRIL; Optionally, step 6) adding a sample to be measured and detecting B cell differentiation; Optionally, step 7) selecting the sample to be measured as an immunomodulatory agent capable of activating or inhibiting B cell differentiation; In the formula, M and N are each an integer of 1 to 20, where M is 3, 4, 5, or 6, and N is 2, 3, or 4.

[0048] In the above embodiment, the concentration of CpG-B is selected from 0.1 to 100 μg / mL, 0.2 to 20 μg / mL, 0.5 to 10 μg / mL, and 0.5 to 5 μg / mL, and is, for example, about 0.5, about 0.8, about 1, about 1.2, about 1.4, about 1.5, about 1.6, about 1.8, about 2, about 2.2, about 2.5, about 2.8, about 3, about 3.5, about 4, about 4.5, or about 5 μg / mL; The concentration of CpG-A or LPS is 0.01 to 10 μM, 0.05 to 5 μM, 0.1 to 2 μM, or 0.2 to 1 μM, for example, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.5, about 2, about 2.5, or about 3 μM; The concentration of R484 is 0.1 to 100 μg / mL, 0.2 to 20 μg / mL, 0.5 to 10 μg / mL, or 0.5 to 5 μg / mL, for example, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 2, about 3, or about 4 μg / mL; The concentrations of interleukins (e.g., IL-3, IL-4, IL-6, IL-10, IL-21) are 0.1 to 1000 ng / mL, 1 to 500 ng / mL, 5 to 200 ng / mL, for example, about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, and about 200 ng / mL, respectively; The concentration of the TFN pathway regulator (e.g., BAFF, APRIL) is 1 to 10,000 ng / mL, 10 to 5,000 ng / mL, 50 to 2,000 ng / mL, 20 to 200 ng / mL, for example, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 250, about 300, about 400, about 500, about 600, about 7,000, about 800, about 900, or about 1,000 ng / mL.

[0049] In some embodiments, a method for screening, preparing, or manufacturing an immunomodulatory agent is provided, comprising: Step 1) Administer 0.5-10 μg / mL of CpG-B to B cells; Step 2) Removal of CpG-B on days 2 to 8 Step 3) Add 2 to 100 ng / mL of IL-6, 10 to 1000 ng / mL of IL-10, and 10 to 1000 ng / mL of IL-21. Optionally, step 4) adding 10-2000 ng / mL BAFF and 10-2000 ng / mL APRIL and stimulating for an additional 2-5 days; Optionally, step 5) decreasing the concentration of BAFF and increasing the concentration of APRIL; Optionally, step 6) adding samples to be measured on days 0 to 21 to detect B cell differentiation; Optionally, step 7) selecting the sample to be measured as an immunomodulatory agent capable of activating or inhibiting B cell differentiation; Includes:

[0050] In some embodiments, a method for screening, preparing, or manufacturing an immunomodulatory agent is provided, comprising: Step 1) Administer 1-5 μg / mL of CpG-B to B cells; Step 2) Remove CpG-B on days 3 to 5. Step 3) Add 5-50 ng / mL of IL-6, 100-500 ng / mL of IL-10, and 100-500 ng / mL of IL-21. Step 4) Add 200-1000 ng / mL of BAFF and 20-200 ng / mL of APRIL and stimulate for another 2-4 days. Step 5) Decrease the concentration of BAFF to 20-200 ng / mL and increase the concentration of APRIL to 200-1000 ng / mL; Step 6) Add samples to be measured in a concentration gradient on days 0 to 14 to detect B cell differentiation. Step 7) selecting the sample to be measured as an immunomodulatory agent capable of activating or inhibiting B cell differentiation; Includes:

[0051] In some embodiments, a method for screening, preparing, or manufacturing an immunomodulatory agent is provided, comprising: Step 1) administering approximately 2 μg / mL of CpG-B to B cells; Step 2) Removing CpG-B on day 4 or 5; Step 3) adding about 10 ng / mL IL-6, about 50 ng / mL IL-10, and about 50 ng / mL IL-21; Step 4) adding about 500 ng / mL of BAFF and about 50 ng / mL of APRIL; Step 5) After 3 days, adjust the concentration of BAFF to about 50 ng / mL and the concentration of APRIL to about 500 ng / mL; Step 6) Add samples to be measured in a concentration gradient on days 0 to 14 to detect B cell differentiation. Step 7) selecting the sample to be measured as an immunomodulatory agent capable of activating or inhibiting B cell differentiation; Includes.

[0052] In the above embodiments, the immunomodulator includes, but is not limited to, a type I interferon pathway modulator, such as an IFNAR1 signaling pathway modulator, and the IFNAR1 modulator includes, but is not limited to, an antibody or an antigen-binding fragment thereof, a small molecule compound, a nucleic acid, an aptamer, a receptor, a ligand, and any molecule including the antibody or an antigen-binding fragment thereof, a small molecule compound, a nucleic acid, an aptamer, a receptor, or a ligand. Exemplary immunomodulators include, but are not limited to, anifrolumab and other anti-IFNAR1 antibodies of the present disclosure, and fusion proteins comprising the anti-IFNAR1 antibodies (e.g., fusion proteins comprising a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody, fusion proteins comprising a TACI polypeptide and an anti-IFNAR1 antibody, fusion proteins comprising a BCMA polypeptide and an anti-IFNAR1 antibody), and fusion proteins comprising a TACI polypeptide and a BCMA polypeptide of the present disclosure.

[0053] In a fifth aspect, in some embodiments of the present disclosure, there is provided a method for screening, preparing or manufacturing an immunomodulatory agent, comprising: Induction step: administering a TLR agonist and IFN to B cells, removing the TLR agonist and IFN on day M, adding an interleukin, adding a TNF family member, and adjusting the concentration of the TNF family member on day N after the addition of the TNF family member; A detection step: adding a sample to be measured during or after the induction step, detecting B cell differentiation, and selecting the sample to be measured that can activate or inhibit B cell differentiation as an immunomodulator; In this case, M and N are integers of 1 to 30, for example, 2, 3, 4, 5, 6, 7, 8, 9, and 10, respectively.

[0054] In some embodiments of the present disclosure, there is provided a method for screening, preparing, or producing an immunomodulatory agent, which includes steps 1), 6), and 7), and may optionally further include steps 2) to 3), and may optionally further include steps 4) to 5), Step 1) administering TLR agonists and IFN to B cells; Step 2) Removal of TLR agonist and IFN on day M; Step 3) Adding interleukins Step 4) Adding TNF family members, Step 5) adjusting the concentration of TNF family members; Step 6) Adding samples to be measured and detecting B cell differentiation; Step 7) selecting the sample to be measured as an immunomodulatory agent capable of activating or inhibiting B cell differentiation; In this case, M and N are integers of 1 to 30, for example, 2, 3, 4, 5, 6, 7, 8, 9, and 10, respectively.

[0055] In some specific embodiments, the TLR agonist includes, but is not limited to, a TLR7, TLR8, or TLR9 agonist, for example, R848, LPS, or CpG, for example, CpG-A, CpG-B, for example, CpG-B; The IFN is selected from IFNβ and IFNα, the interleukin is selected from, for example, IL-4, IL-3, IL-6, IL-10 and / or IL-21, for example, IL-6, IL-10 and / or IL-21, for example, IL-6, IL-10 and IL-21; The TNF family member is a molecule capable of regulating the TNF signaling pathway, and is selected from, for example, BAFF and APRIL, for example, BAFF and APRIL; the concentration of the TLR agonist is selected from 0.1 to 100 μg / mL, 0.2 to 20 μg / mL, 0.5 to 10 μg / mL, 0.5 to 5 μg / mL, for example, about 1, about 2, about 3, about 4, or about 5 μg / mL; Adjusting the concentration of the TNF family member may involve, for example, reducing the concentration of BAFF (e.g., 0.01 to 0.99 times, 0.05 to 0.9 times, 0.05 to 0.5 times, 0.05 to 0.2 times the original concentration, e.g., about 0.02, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.2, about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 times the original concentration), and increasing the concentration of APRIL (for example, adjusting to 1.1 to 1000 times, 2 to 100 times, 5 to 50 times, 10 to 20 times the original concentration, for example, about 1.5, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 15, about 20, about 25, about 30, about 35, about 40, or about 50 times the original concentration), Detecting the differentiation of the B cells can be, for example, detecting plasma cells (CD27 + CD38 + ) detecting the number of The sample to be measured is an immunomodulator (eg, an immunoagonist, an immunosuppressant) or a B cell differentiation modulator, such as a TNF pathway modulator (eg, a BAFF, APRIL pathway agonist or inhibitor).

[0056] In some embodiments, a method for screening, preparing, or manufacturing an immunomodulatory agent is provided, comprising: Step 1) administering CpG (e.g., CpG-A or CpG-B) or R848 or LPS and IFNα to B cells; Step 2) On day M, removing CpG (e.g., CpG-A or CpG-B) or R848 or LPS, and IFNα; step 3) adding interleukins IL-6, IL-10 and / or IL-21; Optionally, step 4) adding BAFF, APRIL; Optionally, step 5) adjusting the concentration of BAFF and APRIL; Optionally, step 6) adding a sample to be measured and detecting B cell differentiation; Optionally, step 7) selecting the sample to be measured as an immunomodulatory agent capable of activating or inhibiting B cell differentiation; In this case, M and N are integers of 1 to 30, respectively.

[0057] In some embodiments, a method for screening, preparing, or manufacturing an immunomodulatory agent is provided, comprising: Step 1) administering CpG-B and IFNα to B cells; Step 2) Removing CpG-B and IFNα on day M; Step 3) adding interleukins IL-6, IL-10, and IL-21; Optionally, step 4) adding BAFF and APRIL; Optionally, step 5) decreasing the concentration of BAFF and increasing the concentration of APRIL; Optionally, step 6) adding a sample to be measured and detecting B cell differentiation; Optionally, step 7) selecting the sample to be measured as an immunomodulatory agent capable of activating or inhibiting B cell differentiation; In the formula, M and N are each an integer of 1 to 20, where M is 3, 4, 5, or 6, and N is 2, 3, or 4.

[0058] In the above embodiment, the concentration of CpG-B is selected from 0.1 to 100 μg / mL, 0.2 to 20 μg / mL, 0.5 to 10 μg / mL, and 0.5 to 5 μg / mL, and is, for example, about 0.5, about 0.8, about 1, about 1.2, about 1.4, about 1.5, about 1.6, about 1.8, about 2, about 2.2, about 2.5, about 2.8, about 3, about 3.5, about 4, about 4.5, or about 5 μg / mL; The concentration of CpG-A or LPS is 0.01 to 10 μM, 0.05 to 5 μM, 0.1 to 2 μM, or 0.2 to 1 μM, for example, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.5, about 2, about 2.5, or about 3 μM; The concentration of R484 is 0.1 to 100 μg / mL, 0.2 to 20 μg / mL, 0.5 to 10 μg / mL, or 0.5 to 5 μg / mL, for example, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 2, about 3, or about 4 μg / mL; The concentration of IFN (e.g., IFNα) is 1 to 10,000 U / mL, 50 to 1,000 U / mL, 100 to 500 U / mL, for example, about 150, about 200, about 250, about 300, about 350, about 400, about 500, about 600, about 700, about 800, about 900, or about 1,000 U / mL; The concentrations of interleukins (e.g., IL-3, IL-4, IL-6, IL-10, IL-21) are 0.1 to 1000 ng / mL, 1 to 500 ng / mL, 5 to 200 ng / mL, for example, about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, and about 200 ng / mL, respectively; The concentration of the TFN pathway regulator (e.g., BAFF, APRIL) is 1 to 10,000 ng / mL, 10 to 5,000 ng / mL, 50 to 2,000 ng / mL, 20 to 200 ng / mL, for example, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 250, about 300, about 400, about 500, about 600, about 7,000, about 800, about 900, or about 1,000 ng / mL.

[0059] In some embodiments, a method for screening, preparing, or manufacturing an immunomodulatory agent is provided, comprising: Step 1) On day 0, administer 0.5 to 10 μg / mL of CpG-B and 50 to 1000 U / mL of IFNα to B cells; Step 2) Remove CpG-B and IFNα on days 2 to 8. Step 3) Add 2 to 100 ng / mL of IL-6, 10 to 1000 ng / mL of IL-10, and 2 to 30 ng / mL of IL-21. Optionally, step 4) adding 10-2000 ng / mL BAFF and 10-2000 ng / mL APRIL and stimulating for an additional 2-5 days; Optionally, step 5) decreasing the concentration of BAFF and increasing the concentration of APRIL; Optionally, step 6) adding samples to be measured on days 0 to 21 to detect B cell differentiation; Optionally, step 7) selecting the sample to be measured as an immunomodulatory agent capable of activating or inhibiting B cell differentiation; Includes:

[0060] In some embodiments, a method for screening, preparing, or manufacturing an immunomodulatory agent is provided, comprising: Step 1) On day 0, administer 1 to 5 μg / mL of CpG-B and 100 to 500 U / mL of IFNα to B cells; Step 2) Remove CpG-B and IFNα on days 3 to 5. Step 3) Add 5-50 ng / mL of IL-6, 100-500 ng / mL of IL-10, and 5-20 ng / mL (or 5-21, 5-22, 5-23, or 5-24 ng / mL) of IL-21. Step 4) Add 200-1000 ng / mL of BAFF and 20-200 ng / mL of APRIL and stimulate for another 2-4 days. Step 5) Decrease the concentration of BAFF to 20-200 ng / mL and increase the concentration of APRIL to 200-1000 ng / mL; Step 6) Add samples to be measured in a concentration gradient on days 0 to 14 to detect B cell differentiation. Step 7) selecting the sample to be measured as an immunomodulatory agent capable of activating or inhibiting B cell differentiation; Includes:

[0061] In some embodiments, a method for screening, preparing, or manufacturing an immunomodulatory agent is provided, comprising: Step 1) administering approximately 2 μg / mL of CpG-B and 250 U / mL of IFNα to B cells; Step 2) Removing CpG-B and IFNα on day 4 or 5; step 3) adding about 10 ng / mL IL-6, about 50 ng / mL IL-10, and about 15 ng / mL IL-21; Step 4) adding about 500 ng / mL of BAFF and about 50 ng / mL of APRIL; Step 5) After 3 days, adjust the concentration of BAFF to about 50 ng / mL and the concentration of APRIL to about 500 ng / mL; Step 6) Add samples to be measured in a concentration gradient on days 0 to 14 to detect B cell differentiation. Step 7) selecting the sample to be measured as an immunomodulatory agent capable of activating or inhibiting B cell differentiation; Includes:

[0062] In some embodiments, the present disclosure provides a cell model for screening immunomodulatory agents, which comprises B cells induced to differentiate by any one of the methods described above.

[0063] In the fourth and fifth aspects, the immunomodulator includes, but is not limited to, a type I interferon pathway modulator (e.g., an IFNAR1 signaling pathway modulator) and a TNF pathway modulator (e.g., a BAFF or ARRIL pathway modulator). The IFNAR1 modulator, BAFF, and / or ARRIL pathway modulator include, but are not limited to, an antibody or antigen-binding fragment thereof, a small molecule compound, a nucleic acid, an aptamer, a receptor, a ligand, and any molecule including the antibody or antigen-binding fragment thereof, a small molecule compound, a nucleic acid, an aptamer, a receptor, or a ligand. Exemplary IFNAR1 modulators include, but are not limited to, anifrolumab and other anti-IFNAR1 antibodies of the present disclosure, and fusion proteins comprising the anti-IFNAR1 antibodies (e.g., a fusion protein comprising a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody, a fusion protein comprising a TACI polypeptide and an anti-IFNAR1 antibody, or a fusion protein comprising a BCMA polypeptide and an anti-IFNAR1 antibody), as well as a fusion protein comprising a TACI polypeptide and a BCMA polypeptide of the present disclosure.

[0064] Immunomodulators (e.g., BAFF and / or ARRIL pathway modulators) screened, prepared, or manufactured using the methods provided in the present disclosure include, but are not limited to, TACI, Telitacicept, and the TACI polypeptides, BCMA polypeptides, and fusion proteins comprising the TACI polypeptides and / or BCMA polypeptides provided in the present disclosure below (e.g., fusion proteins comprising a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody, a fusion protein comprising a TACI polypeptide and an anti-IFNAR1 antibody, a fusion protein comprising a BCMA polypeptide and an anti-IFNAR1 antibody, and a fusion protein comprising a TACI polypeptide and a BCMA polypeptide).

[0065] In the above, B cells are, for example, 1 × 10 5B cells were isolated from PBMCs and cultured in 1640 basal medium supplemented with 50 μM β-mercaptoethanol, MEM non-essential amino acids (glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine), sodium pyruvate, and GlutaMAX.

[0066] In a sixth aspect, the present disclosure further provides a pharmaceutical composition comprising an immunomodulator obtained by screening using the above method and one or more pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the amount of the immunomodulator in the pharmaceutical composition is 0.1 to 2000 mg, and in some specific embodiments, 1 to 1000 mg.

[0067] The present disclosure also provides use of an immunomodulator obtained by screening using the above method for preparing a pharmaceutical composition. The present disclosure also provides a method for producing or preparing a pharmaceutical composition comprising an immunomodulator obtained by screening using the above method. The method for producing or preparing the pharmaceutical composition comprises mixing any one of the above immunomodulators with one or more pharmaceutically acceptable carriers, diluents, or excipients, or comprises preparing or producing any one of the above immunomodulators and mixing the obtained immunomodulator with one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0068] In a seventh aspect, the present disclosure provides a method for treating or ameliorating a disease or condition of the immunomodulatory agent or pharmaceutical composition, the method comprising administering the immunomodulatory agent or pharmaceutical composition to a subject in need thereof; In some embodiments, the disease or condition is a B cell disorder or an autoimmune disease; In some specific embodiments, the B cell disorder or autoimmune disease is a disease or condition associated with TACI and / or BCMA expression; In some specific 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, and the B-cell disorder is selected from tumors, chronic leukemia, multiple myeloma, non-Hodgkin's lymphoma, post-transplant lymphoproliferation, and light chain gammopathy.

[0069] In some embodiments, a method for preparing the immunomodulatory agent is provided. These immunomodulatory agents can be prepared by conventional methods known in the art, including, but not limited to, expressing the antibody or antigen-binding fragment in a host cell and isolating the fusion protein from the host cell. Optionally, a purification step may be included. Optionally, a filtration or concentration step may be included. Soluble compounds and multimers 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.

[0070] The present disclosure also 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.

[0071] fusion proteins The present disclosure provides a fusion protein, the fusion protein comprising: (1) comprising a TACI polypeptide; (2) comprising a BCMA polypeptide; (3) comprising a TACI polypeptide and a BCMA polypeptide; (4) A TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), (5) a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody); or (6) Includes a TACI polypeptide, a BCMA polypeptide, and an antibody (e.g., an anti-IFNAR1 antibody).

[0072] Regarding the TACI polypeptide in the fusion protein: In some embodiments, the TACI polypeptide comprises CRD1 and / or CRD2 of the TACI extracellular domain, eg, CRD2 of the TACI extracellular domain.

[0073] In some embodiments, the TACI polypeptide comprises the following amino acid sequence: (1) SEQ ID NO: 1 or an amino acid sequence having at least 90% sequence identity thereto; (2) amino acid residues 33 to 67 of SEQ ID NO: 1; (3) amino acid residues 70 to 104 of SEQ ID NO: 1 or a mutant thereof; (4) 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; or (5) Amino acid residues at positions 68 to 105, 68 to 106, 68 to 107, or 68 to 108 of SEQ ID NO: 1, or a mutant thereof.

[0074] In some specific embodiments, the mutant has one or more amino acid mutations selected from positions 69, 72, 73, 77, 85, 102, and 103.

[0075] In some specific embodiments, the variant has one or more amino acid substitutions selected from 69T or 69R, 72S, 73E or 73Q, 77E, 85T or 85A, 102A or 102R, and 103Y.

[0076] 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.

[0077] In some specific embodiments, a combination mutation at multiple given positions is indicated by connecting each single amino acid substitution with a " / ". For example, a combination mutation at positions 69T and 102A may be indicated as 69T / 102A.

[0078] In some specific embodiments, the mutant has the following amino acid substitution combination: 69T / 73E / 77E / 102A, for example, the following amino acid substitution combination: L69T / K73E / K77E / Y102A.

[0079] The site of the above amino acid mutation is an amino acid residue site numbered in the natural order of the TACI extracellular domain (sequence number 1), for example, "the above mutant has an amino acid mutation selected from position 69" means that there is a mutation in the amino acid residue corresponding to position 69 of sequence number 1.

[0080] 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.

[0081] 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.

[0082] Regarding the BCMA polypeptide in the fusion protein: In some embodiments, the BCMA polypeptide comprises CRD1 of the BCMA extracellular domain.

[0083] In some embodiments, the BCMA polypeptide comprises amino acid residues 7-41 of SEQ ID NO:30.

[0084] 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.

[0085] Regarding antibodies in fusion proteins: In some embodiments, the antibody in the fusion protein is an anti-IFNAR1 antibody.

[0086] In some embodiments, the anti-IFNAR1 antibody 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 respectively set forth in SEQ ID NOs: 45 to 47, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, whose amino acid sequences are respectively set forth in SEQ ID NOs: 48 to 50.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] In some embodiments, the present disclosure relates to anifrolumab, sifalimumab, and compounds generally disclosed in WO20062002177A, WO2009100309A (e.g., 3F11, 4G5, 11E2, 9D4), WO2020156474A (e.g., 7G4, 10C5, etc.), and WO2020057541A (e.g., 8G11H and 485G10H). , CN201610634601.7 (e.g., H19B7+L16C11, H19B7+L8C3, H15D10+L16C11, H15D10+L8C3 or anti-IFNAR1-C1), CN201510685200.X, and WO2012162367A are incorporated as anti-IFNAR1 antibodies of the present disclosure.

[0091] About fusion proteins: As described above, the present disclosure provides a fusion protein comprising a TACI polypeptide and a BCMA polypeptide, a fusion protein comprising a TACI polypeptide and an anti-IFNAR1 antibody, or a fusion protein comprising a TACI polypeptide, a BCMA polypeptide and an anti-IFNAR1 antibody.

[0092] 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.

[0093] In some embodiments, the Fc region in the fusion protein comprises one or more amino acid substitutions relative 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 Fcγ 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.

[0094] 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 comprise one or more amino acid substitutions selected from positions 354, 356, 358, and 366, and the amino acid residues of the second subunit comprise 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.

[0095] Regarding fusion proteins 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.

[0096] 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 comprising two or more of the TACI polypeptides, the TACI polypeptide may be a TACI polypeptide with the same or different sequence as provided in the present disclosure, and when comprising two or more of the BCMA polypeptides, the BCMA polypeptide may be a BCMA polypeptide with the same or different sequence as provided in the present disclosure.

[0097] In some embodiments, in the fusion protein, the TACI polypeptide and one subunit of the Fc region are linked in any order, and optionally, the TACI polypeptide and the first or second subunit of the Fc region are linked directly or via a linker. In some embodiments, in any one of the fusion proteins, the BCMA polypeptide and one subunit of the Fc region are linked in any order, and optionally, the BCMA polypeptide and the first or second subunit of the Fc region are linked directly or via a linker. In some embodiments, the C-terminus of the TACI polypeptide and the N-terminus of the first or second Fc region subunit are linked directly or via a linker, or the N-terminus of the TACI polypeptide and the C-terminus of the first or second Fc region subunit are linked directly or via a linker. In some embodiments, the C-terminus of the BCMA polypeptide and the N-terminus of the first or second subunit of the Fc domain are linked via a linker or directly, or the N-terminus of the BCMA polypeptide and the C-terminus of the first or second subunit of the Fc domain are linked via a linker or directly.

[0098] In some embodiments, the fusion protein comprising the TACI polypeptide and the BCMA polypeptide 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, in scheme (II), In I) or (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 0 and c and d are 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 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.

[0099] 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 (G4S) y(SEQ ID NO: 78), and y is independently selected from an integer of 1 to 6 (e.g., y is 1, 2, 3, 4, 5, or 6). For example, the linker may be x S) 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 (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).

[0100] In some embodiments, a fusion protein is provided comprising a TACI polypeptide and a BCMA polypeptide, which comprises an amino acid sequence set forth in any one of SEQ ID NOs: 31-38, 69, and 70, or having at least 90% sequence identity thereto.

[0101] In some embodiments, the fusion protein is a multimer, eg, a dimer (eg, a homodimer or heterodimer).

[0102] 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.

[0103] 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.

[0104] Regarding the 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.

[0105] 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.

[0106] 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; 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.

[0107] In some embodiments, the linker is m S n ) 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 (G4S) y and y is independently selected from an integer of 1 to 6 (e.g., y is 1, 2, 3, 4, 5, or 6). For example, the linker has an amino acid sequence represented by the formula (G x S) yIt is a linker, in which x is selected from integers of 1 to 5 and y is selected from integers of 1 to 6, for example, a linker represented by any one of SEQ ID NOs: 39 to 41.

[0108] 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.

[0109] In some embodiments, the fusion protein comprises two identical first polypeptide chains and two identical second polypeptide chains.

[0110] 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.

[0111] 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.

[0112] Regarding fusion proteins 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.

[0113] 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.

[0114] In some embodiments, the fusion protein is any one of the TACI polypeptide, BCMA polypeptide, and anti-IFNAR1 antibody described above, 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 that 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 that 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 that 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 that 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, and 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.

[0115] 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 (G4S) y and y is independently selected from an integer of 1 to 6 (e.g., y is 1, 2, 3, 4, 5, or 6). For example, the linker has an amino acid sequence represented by the formula (G x S) 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).

[0116] 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.

[0117] In some embodiments, the fusion protein comprises two identical first polypeptide chains and two identical second polypeptide chains.

[0118] 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.

[0119] 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.

[0120] Regarding the above fusion proteins: In some embodiments, the fusion protein comprising a TACI polypeptide and a BCMA polypeptide, the fusion protein comprising a TACI polypeptide and an IFNAR1 antibody, or the fusion protein comprising a TACI polypeptide, a BCMA polypeptide, and an IFNAR1 antibody has one or more of the following properties or functions: (a) BAFF-binding activity; (b) TALL-2 / 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.

[0121] 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 (coated with antigen protein), 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 3, e. Inhibiting the induction of B cell proliferation by APRIL, and in some embodiments, IC 50values ​​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. binding 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, less than 9E-11 M, less than 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, less than 2.0E-11 M, less than 1.9E-11 M, less than 1.3E-11 M or less; g. good pharmacokinetics in vivo, 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.

[0122] 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. 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, d. Good pharmacokinetics in the body; e.Good stability.

[0123] 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 an IFNα / β reporter gene method, for example, the experiment in Example 10; or the inhibitory rate is detected in vivo by detecting mRNA expression of IFNα downstream gene ISG in PBMC, for example, 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 values ​​are detected by an 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. 50The 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 an ELISA method, for example, the ELISA experiment of Example 11, e. inhibiting IFNα secretion; in some embodiments, the fusion protein has an inhibitory ability on IFNα 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 in vitro differentiation of B cells into plasma cells induced by IFNα 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 plasma cell production induced by BAFF and APRIL; in some embodiments, the fusion protein inhibits plasma cell production induced by BAFF and APRIL 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%, 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 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 pharmacokinetics in the body; k.Good stability.

[0124] Furthermore, in some embodiments, there is provided a product or combination comprising any one of the fusion proteins comprising a TACI polypeptide and a BCMA polypeptide of the present disclosure, further comprising an antibody. In some embodiments, there is provided a product or combination comprising any one of the fusion proteins comprising a TACI polypeptide of the present disclosure, further comprising an antibody. In some embodiments, the antibody is an anti-IFNAR1 antibody, such as anifrolumab.

[0125] 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 to the TACI polypeptide and the BCMA polypeptide directly or via any one of the linkers disclosed herein, or unlinked.

[0126] 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.

[0127] 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).

[0128] 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.

[0129] 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.

[0130] 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.

[0131] In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0132] 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).

[0133] 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.

[0134] Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0135] 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.

[0136] The cells of the present disclosure are incapable of developing into complete plants or animals.

[0137] 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 included. Optionally, a filtration or concentration step may be included. Soluble compounds and multimers 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.

[0138] 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. A mammalian expression system will 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 antibody. 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.

[0139] 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.

[0140] 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 amount of any one of the above polypeptides, fusion proteins, products, combinations, composites, or polynucleotides contained in a unit dose of the pharmaceutical composition is 0.1 to 2000 mg, and in some specific embodiments, 1 to 1000 mg.

[0141] 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.

[0142] 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 any one 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, complex, polynucleotide, or pharmaceutical composition of the present disclosure. In some embodiments, the disease or condition is a disease or condition associated with TACI, BCMA, and / or IFN expression.

[0143] In some embodiments, a method for treating or pharmaceutical use of any one of the TACI polypeptides, BCMA polypeptides, and anti-IFNAR1 antibodies disclosed herein in combination for a disease or condition is provided. In some embodiments, a method for treating or pharmaceutical use of any one of the TACI polypeptides and anti-IFNAR1 antibodies disclosed herein in combination for a disease or condition is provided, which in some specific embodiments further comprises combination with a BCMA polypeptide. In some embodiments, a method for treating or pharmaceutical use of any one of the fusion proteins comprising the BCMA polypeptide and anti-IFNAR1 antibody disclosed herein in combination for a disease or condition is provided, which in some specific embodiments further comprises combination with a TACI polypeptide. The anti-IFNAR1 antibody is, for example, anifrolumab.

[0144] 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.

[0145] 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.

[0146] In some embodiments, the disease or condition is a B cell disorder or an autoimmune disease.

[0147] 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.

[0148] 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, and in some embodiments, the B-cell disorder is selected from tumors, chronic leukemia, multiple myeloma, non-Hodgkin's lymphoma, post-transplant lymphoproliferation, and light chain gammopathy. In some embodiments, the autoimmune disease is systemic lupus erythematosus.

[0149] Definition of Terms In order that the present disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless expressly defined otherwise herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0150] 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).

[0151] As used in this disclosure, the singular forms "a," "an," and "the above" include plural referents unless the context clearly dictates otherwise.

[0152] Unless the context clearly indicates otherwise, in the patent specification and claims, the words "comprises," "has," "includes," and the like are to be understood in the sense of "including, but not limited to," rather than in the exclusive or exhaustive sense.

[0153] The term "cytokine" or "factor" is a general term for proteins released by one cell population that act on other cells as intercellular mediators. 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.

[0154] 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 N-terminal region (amino acid residues 1-165, see SEQ ID NO: 1 in this disclosure), the transmembrane region (amino acid residues 166-186), and the 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 CRD1 of human TACI (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.

[0155] The BCMA (B cell maturation antigen) described in the present disclosure is a membrane-bound receptor. Human BCMA protein is a 184-amino acid polypeptide, including 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.

[0156] 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.

[0157] The terms "interferon α," "IFNα," "IFNa," "IFNA," and "IFN alpha" can be used interchangeably and should 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," should 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.

[0158] The terms "interferon alpha receptor-1," "IFNAR1," "IFNAR-1," and "IFNAR-1 antigen" can be 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 can 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 and do not exhibit species or other types of cross-reactivity. The complete cDNA sequence of human IFNAR-1 has GenBank accession number NM_000629.

[0159] 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).

[0160] 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 identical heavy chains and two identical 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, and the corresponding heavy chains are 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," which include single-chain antibodies (i.e., full-length heavy and light chains), Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single-domain antibodies (e.g., VH or VL or VHH), scFv, bivalent, 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).

[0161] The 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.

[0162] 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 some 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; "AbM™, 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.

[0163] [Table 1]

[0164] 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 position 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.

[0165] 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. Typically, two sequences are compared when aligned to obtain the maximum percentage of homology.

[0166] 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.

[0167] 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.

[0168] 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 apply to naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise specified, a particular polypeptide sequence also implicitly covers conservatively modified variants thereof.

[0169] The term "nucleic acid" may be used interchangeably with the term "polynucleotide" herein and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term covers nucleic acids containing known nucleotide 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 nucleotide. 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 the polypeptide or 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 its conservatively modified variants (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 generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues.

[0170] 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 that 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] "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 treating, for example, cells with a reagent, diagnostic, binding composition, or via another cell, in vitro and ex vivo. "Treatment," when applied to a human, veterinary, or research subject, refers to therapeutic treatment, preventative or prophylactic measures, and research and diagnostic uses.

[0175] "Treatment" refers to administering to a subject an internal or external therapeutic agent, e.g., a composition comprising any one of the antibodies or antigen-binding fragments thereof, or fusion proteins thereof, wherein the subject is suffering from, at risk of suffering from, or prone to suffering from one or more diseases or symptoms thereof, and the therapeutic agent is known to have a therapeutic effect on these 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 agent 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 should alleviate the target disease symptom in a statistically significant number of subjects, as determined by 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.

[0176] An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also refers to an amount sufficient to allow 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.

[0177] 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 completely identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are progeny 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.

[0178] The terms "optionally," "optionally," and "selectively" mean that the subsequently described event or circumstance may occur, but does not necessarily 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.

[0179] The term "fusion protein comprising a TACI polypeptide and a BCMA polypeptide" according to the present disclosure covers all molecules of fusion proteins comprising a TACI polypeptide and a BCMA polypeptide according to the present disclosure; the term "fusion protein comprising a TACI polypeptide and an anti-IFNAR1 antibody" covers all molecules of fusion proteins comprising a TACI polypeptide and an anti-IFNAR1 antibody (or antigen-binding fragment thereof) according to the present disclosure; and the term "fusion protein comprising a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody (or antigen-binding fragment thereof)" covers all molecules of fusion proteins 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 proteins may comprise one or more effector molecules, e.g., multiplexed. 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]

[0180] [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; the LALA mutations above are L234A / L235A mutations in Fc, and the same applies below). [Figure 9] These are the results of detecting the inhibitory activity of the fusion protein of anifrolumab and TACI-T4 against IFN-β, of which Figure 9A shows the results of the inhibitory activity of B385801, B385802, B385803, and B385804 against IFN-β, Figure 9B shows the results of the inhibitory activity of B385804, B498301, and B498302 against IFN-β, and Figure 9C shows the results of the inhibitory activity of B606401 against IFN-β. Anifrolumab and the IgG1 isotype were used as controls in all of Figure 9A to C. [Figure 10] These are the ELISA measurement results of the binding ability of the fusion protein of anifrolumab and TACI-T4 to BAFF, of which, A in Figure 10 shows the results of the binding ability of B385801, B385802, B385803, and B385804 to BAFF, with Atacicept, Telitacicept, and the IgG1 isotype used as controls, B in Figure 10 shows the results of the binding ability of B385804, B498301, and B498302 to BAFF, and C in Figure 10 shows the results of 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 inhibitory activity of anifrolumab and TACI-BCMA fusion proteins (B613301, B613302, B613303) on IFN-β. 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 inhibitory effect of anifrolumab on the IFN-β activity of fusion proteins of anifrolumab with TACI-19-16 and BCMA (B637302, B746201), and 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 IFNα 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. Telitacic receptor (IgG1 isotype) was 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 B cell proliferation in vitro with 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. On day 5, B cell proliferation signals were detected by flow cytometry 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 (10, 100, and 1000 nM). Telitaccept and IgG1 isotype were used as controls. On day 10, the number of plasma cells was detected by flow cytometry. 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. [Figure 27] 27A and 27B show the results of an in vitro B cell differentiation experiment. B cells selected from human PBMCs were cultured in vitro, and IL-3, CpG-A, IFNα, and IL-21 were added to stimulate B cell differentiation. After 6 days, cell viability and the percentage of differentiated plasma cells (CD27+CD38+) were measured. Figure 27A shows the FACS results for IL-3+CpG-A, IL-3+CpG-A+IL-21, and IL-3+CpG-A+IFNα, using 1640 medium as the control. Figure 27B shows the corresponding results for the percentage of live cells, and Figure 27C shows the corresponding results for the percentage of plasma cells. [Figure 28]These are the results of an in vitro B cell differentiation experiment. B cells selected from human PBMCs were cultured in vitro and stimulated with R848, CpG-B, IFNα, BAFF, APRIL, and RA. After 6 days, cell viability and the percentage of differentiated plasma cells (CD27+CD38+) were measured. Figure 28A shows the FACS analysis results for CpG-B + IFNα, R484 + IFNα, CpG-B + BAFF + APRIL, R484 + BAFF + APRIL, CpG-B + IFNα + RA + BAFF + APRIL, and R484 + IFNα + RA + BAFF + APRIL. 1640 medium was used as a control. Figure 28B shows the corresponding percentage of viable cells, and Figure 28C shows the corresponding percentage of plasma cells (CD27+CD38+). [Figure 29] These are the results of an in vitro B cell differentiation experiment. B cells selected from human PBMCs were cultured in vitro, and B cell differentiation was stimulated with CpG-B and IFNα. Anifrolumab control was also added, and the percentage of differentiated plasma cells (CD27+CD38+) was measured after 4 days. [Figure 30] These are the results of an in vitro B cell differentiation experiment. B cells selected from human PBMCs were cultured in vitro, and B cell differentiation was stimulated with CpG-B and IFNα. A concentration gradient of anifrolumab control was added, and the differentiation rate of plasma cells (CD27+CD38+) was measured after 4 days. Under these conditions, anifrolumab was able to stimulate plasma cell differentiation in a concentration-dependent manner. IgG1 isotype was used as a control. [Figure 31]These are the results of a plasma cell generation experiment. B cells selected from human PBMCs were cultured in vitro and plasma cell generation was induced with IL-3, CpG-A, IFNα, BAFF, and APRIL. After 6 days, the percentage of differentiated plasma cells (CD27+CD38+) was measured. Figure 31A shows the FACS detection results for IL-3 + CpG-A, IL-3 + CpG-A + BAFF + APRIL, IL-3 + CpG-A + IFNα, and IL-3 + CpG-A + IFNα + BAFF + APRIL. 1640 medium was used as a control. Figure 31B shows the corresponding plasma cell (CD27+CD38+) percentage detection results. [Figure 32] These are the results of a plasma cell generation experiment. B cells selected from human PBMCs were cultured in vitro, and plasma cell generation was induced with CpG-B, IFNα, BAFF, and APRIL. The number of differentiated plasma cells (CD27+CD38+) was measured after 7, 10, 14, and 21 days. [Figure 33] These are the results of a plasma cell generation experiment. B cells selected from human PBMCs were used and cultured in vitro. From day 0 to day 4, they were stimulated with CpG-B and IFNα. After day 4, CpG-B and IFNα were removed, and IL-6, BAFF, and APRIL were added to induce plasma cell generation. The number of differentiated plasma cells (CD27+CD38+) was measured after days 7, 11, and 12. [Figure 34] These are the results of a plasma cell generation experiment. B cells selected from human PBMCs were cultured in vitro and stimulated with CpG-B from days 0 to 4. CpG-B was removed on day 4, and plasma cell generation was induced by adding IL-6, IL-10, IL-21 (5 ng / mL), BAFF, and APRIL. From days 4 to 7, 500 ng / mL BAFF and 50 ng / mL APRIL were added, and from days 7 to 10, 50 ng / mL BAFF and 500 ng / mL APRIL were added. The number of differentiated plasma cells (CD27+CD38+) was measured after 10 days. [Figure 35]These are the results of a plasma cell generation experiment. B cells selected from human PBMCs were cultured in vitro and stimulated with CpG-B from days 0 to 4. CpG-B was removed on day 4, and plasma cell generation was induced by adding IL-6, IL-10, IL-21 (50 ng / mL), BAFF, and APRIL. From days 4 to 7, 500 ng / mL BAFF and 50 ng / mL APRIL were added, and from days 7 to 10, 50 ng / mL BAFF and 500 ng / mL APRIL were added. The number of differentiated plasma cells (CD27+CD38+) was measured on days 9, 10, and 11. [Figure 36] These are the results of a plasma cell generation experiment. B cells selected from human PBMCs were cultured in vitro and stimulated with CpG-B on days 0–4. CpG-B was removed on day 4, and plasma cell generation was induced by adding IL-6, IL-10, IL-21 (15, 25, or 40 ng / mL), BAFF, and APRIL. On days 5–7, 500 ng / mL BAFF and 50 ng / mL APRIL were added, and on days 7–10, 50 ng / mL BAFF and 500 ng / mL APRIL were added. A concentration gradient of Telitacicep control was added on day 4, and the number of differentiated plasma cells (CD27+CD38+) was measured 10 days later. Figure 36, A, B, and C, show the results of plasma cell (CD27+CD38+) counts at 15 ng / mL, 25 ng / mL, and 40 ng / mL IL-21, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0181] The present disclosure will be further described below in conjunction with examples, but these examples do not limit the scope of the present disclosure.

[0182] 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.

[0183] 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 (structures shown in Figure 1) containing two identical polypeptide chains. RCT-18 (telitacicept) was used as a positive control.

[0184] The sequence is as follows: >TACI-ECD (amino acid residues 1-165 of wild-type human TACI) [ka] >TACI-9 (human TACI-ECD, natural counting, 68th to 108th) [ka] >Human IgG1 Fc sequence [ka] >Telitacicept (RCT-18) [ka] >Atacicept [ka] 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.

[0185] The resulting TACI-Fc fusion proteins were subjected to mass spectrometry analysis. The experimental results revealed 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).

[0186] Furthermore, the blocking function of the TACI-Fc fusion protein on the binding of BAFF and BAFF-R was examined as follows. The receptor protein was diluted to 2 μg / mL in pH 7.4 PBS (B320, Corning) buffer, added to a 96-well microplate (Corning, 3590) at a volume of 100 μL per well, and 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, followed by incubation at 37°C for 2 hours. After blocking, the blocking solution was discarded, and the plate was washed three times with PBST buffer (PBS, pH 7.4, containing 0.1% Tween-20) before use. A fixed concentration of biotin-labeled ligand protein was mixed with 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 IC2000 curve was fitted using software to calculate the inhibition of ligand-receptor binding. 50 The origins of the ligand and receptor proteins used were BAFF (Sino biological, 10056-HNCH) and BAFF-R (Sino biological, 16079-H02H).

[0187] As a result, TACI-9-Fc and RCT-18 act as ICs that block the binding of BAFF to BAFF-R. 50The values ​​are 5.02 nM and 27.48 nM, respectively. The functional activity of TACI-9-Fc is significantly stronger than that of the control RCT-18. Therefore, TACI-9 is considered to be a promising candidate molecule.

[0188] Example 2. Analysis of TACI cleavage fragments Based on TACI-9, the length of 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: >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 positions 68-106) [ka] >TACI-12 (3 amino acids "NKL" truncated at the C-terminus of TACI-9) (natural counting of human TACI-ECD from 68th to 105th amino acids) [ka] The C-terminus of the above-mentioned cleaved TACI fragment was fused to the N-terminus of human IgG Fc (sequence number 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).

[0189] Mass spectrometry was performed on the TACI-Fc fusion proteins constructed as described above, 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.

[0190] 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).

[0191] 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.

[0192] 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.

[0193] [Table 2]

[0194] Example 3. Sequence modification of TACI cleavage fragments TACI protein is prone to aggregation in neutral solution. To further improve the stability of TACI, the hydrophobic and ionic groups in the TACI fragment were analyzed using MOE (Molecular Operating Environment) software based on the TACI crystal structure (PDB ID: 1XU1). Amino acid substitutions were made on multiple amino acid residues in TACI-9, reducing the exposed hydrophobic and ionic groups on the TACI protein, reducing the aggregation tendency of TACI and improving TACI stability while still essentially maintaining the functional activity of TACI. The amino acid sequence of the modified TACI fragment is as follows: >TACI-9-1 (TACI-9 contains 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] Note: In the above sequence, the underlined amino acid residues are those after mutation.

[0195] 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.

[0196] 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.

[0197] [Table 3]

[0198] 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.

[0199] 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.)

[0200] 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]

[0201] 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. BCMA and TACI were designed as a fusion protein to enhance the neutralizing activity against APRIL.

[0202] 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.

[0203] 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.

[0204] 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.

[0205] >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]

[0206] B575701, B575702, B575703, and B575704 are TACI-d2-BCMA-ECD, BCMA-ECD-TACI-d2, TACI-T2-BCMA-ECD, and BCMA-ECD-TACI-T2, respectively, with human IgG1 linked to the C-terminus of the Fc (SEQ ID NO: 3). In the following sequences, the underlined portion represents the Fc.

[0207] >B575701 (SEQ ID NO: 35) [ka] >B575702 (SEQ ID NO: 36) [ka] >B575703 (SEQ ID NO: 37) [ka] >B575704 (SEQ ID NO: 38) [ka]

[0208] 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.

[0209] 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.

[0210] 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.

[0211] The results are shown in Table 4 and Figure 5. All four fusion proteins can clearly bind to BAFF, and their binding ability is stronger than that of telitacicept. Among them, B575702 and B575704 have the same binding ability to BAFF, and have a higher EC than telitacicept. 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.

[0212] [Table 4]

[0213] 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.

[0214] [Table 5]

[0215] 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 in which TACI-CRD2 has some amino acid mutations (L69T, K73E, K77E, Y102A).

[0216] 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.

[0217] TACI-19-16 is shown in SEQ ID NO: 26, and other sequences are as follows: >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] 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.

[0218] Using the protein preparation method provided in Example 1, transient transfection and protein expression were performed, and the protein was passed through a Protein A affinity chromatography column and eluted. Upon detection, the target protein was obtained.

[0219] Example 7: Experimental study 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.

[0220] The method is as follows: HEK-Blue™ 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 to be measured was added and incubated at 37°C for 30 minutes. IFN-β (Sino biological, Cat: 10704-HNAS) was added to a final concentration of 0.01 ng / mL and incubated at 37°C for 24 hours. 20 μL of the culture supernatant was removed and mixed with 180 μL of Quanti-Blue (InvivoGen, Cat: rep-qbs) in a new 96-well plate. The plate was incubated at 37°C for 1 hour, and OD655 was measured.

[0221] The results show that all seven of the above fusion proteins have inhibitory effects on the activity of IFN-α / β, with the activities of B385801 and B385803 being relatively weak, followed by B385802, and the activities of B385804, B498301, B498302, and B606401 being almost equivalent to that of anifrolumab (Figures 9A, 9B, and 9C).

[0222] 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.

[0223] The method is as follows: 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.

[0224] As a result, referring to Figures 10A to 10C and Table 6, it can be seen that the seven fusion proteins can clearly bind to BAFF. B385801, B385803, and B606401 have the strongest binding ability to BAFF, stronger than Telitacicept and Atacicept. B385802 has a relatively weak binding ability to BAFF. B385804, B498301, and B498302 have similar binding abilities to BAFF, slightly weaker than Telitacicept. The length of the linker does not affect the binding ability of TACI to BAFF.

[0225] [Table 6]

[0226] 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.

[0227] 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.

[0228] >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 passed through a Protein A affinity chromatography column and eluted. Upon detection, the target protein was obtained.

[0229] 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.

[0230] The method was as follows: HEK-Blue™ 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 removed and mixed with 180 μL of Quanti-Blue (InvivoGen, Cat. No. rep-qbs) in a new 96-well plate. The plate was incubated at 37°C for 1 hour, and OD655 was measured.

[0231] 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 does not essentially affect the activity of anifrolumab, particularly at the C-terminus of the light chain.

[0232] 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.

[0233] 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.

[0234] Referring to Figure 13 and Table 7, the results show that all three fusion proteins clearly bound to BAFF. B613302 had significantly stronger binding ability to BAFF than Telitaciccept, and the EC50 was reduced to 1 / 8 to 1 / 7 of the original level. It has been shown that placing TACI at the C-terminus of BCMA (e.g., in the case of B613302) may be even more advantageous for retaining the ability to bind to BAFF.

[0235] [Table 7]

[0236] Referring to Figure 14 and Table 8, the results show that all three fusion proteins clearly bound to APRIL. B613301, B613302, and B613303 showed almost identical binding to APRIL, and had a significantly higher EC than telitacicept. 50 The results showed that the BCMA-fused fusion protein indeed improved binding to APRIL.

[0237] [Table 8]

[0238] 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.

[0239] 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.

[0240] >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] In both B637301 and B637302, the heavy chain is the heavy chain of anifrolumab, SEQ ID NO: 55, and the light chain sequence is 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.

[0241] >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.

[0242] B746201 - Heavy chain of LALA (SEQ ID NO: 74) [ka] Fusion proteins of B637301, B637302, and B746201 were expressed and purified, and detected by SDS-PAGE. Both the heavy and light chains of the antibody linked to TACI-BCMA appeared as single bands (results not shown). This indicates that removing the glycosylation risk points in the BCMA protein sequence can make the fusion proteins more homogeneous.

[0243] 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.

[0244] The method was as follows: HEK-Blue™ 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 removed and mixed with 180 μL of Quanti-Blue (InvivoGen, Cat. No. rep-qbs) in a new 96-well plate. The plate was incubated at 37°C for 1 hour, and OD655 was measured.

[0245] Referring to Figure 16 and Table 9, the results show that all four fusion proteins can inhibit the activity of IFN-α / β, and the activities of the four fusion proteins are comparable and consistent with that of anifrolumab.

[0246] [Table 9]

[0247] Example 14. In vitro binding experiments 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.

[0248] 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 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.

[0249] 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.

[0250] [Table 10]

[0251] Referring to Figure 18 and Table 11, all four fusion proteins clearly bound to APRIL, and B637302 had the ability to bind to APRIL at the EC2 of Telitaccept. 50B746201 and B805201 have comparable binding ability to APRIL and have a lower EC2 than telitacicept. 50 was reduced to one-third of that level, and B606401 has the same ability to bind to APRIL as Telitaccept.

[0252] [Table 11]

[0253] 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. Functional experiments to detect pDCs were performed as follows: Freshly isolated healthy human PBMCs were cultured in vitro in 1640 GlutaMAX (11 mM glucose) basal medium. 3 × 10 PBMCs were cultured at 1000 x g for 1 h. 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 IFN-α cytokine secretion levels in the cell culture supernatant were detected using a human IFN-α reagent kit (Cisbio cat: 62HIFNAPEG).

[0254] The results show that B637302, B606401, and the control antibody anifrolumab are all capable of inhibiting IFN-α production in a dose-dependent manner, and the inhibitory activities of the three are comparable (FIG. 19).

[0255] Example 16. IFN-α-induced plasma cell differentiation and function experiments in vitro 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. After 4 days, plasma cells (CD27 + CD38 + The differentiation rate of the cells was detected by flow cytometry.

[0256] 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 are equivalent.

[0257] Example 17. Functional studies of the induction of B cell proliferation by BAFF To evaluate the in vitro efficacy of the fusion protein of anifrolumab and TACI-19-16-BCMA, we established a method for testing the TACI-BCMA bioactive molecule in vitro. The procedure for the BAFF-induced B cell proliferation experiment was 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. After treatment with isotype, B cell proliferation signals were detected by flow cytometry 5 days later.

[0258] As a result, Table 12 shows that B637302, B606401, B805201, and the control, telitacicept, can all effectively inhibit BAFF-induced in vitro B cell proliferation in a dose-dependent manner. Figure 21 shows that 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, whereas B637302, B606401, and B805201 all showed clear inhibitory activity on plasma cell differentiation.

[0259] [Table 12]

[0260] Example 18. Functional studies of induction of B cell proliferation by APRIL 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 procedure for the APRIL-induced B cell proliferation experiment was 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). After treatment with isotype, B cell proliferation signals were detected by flow cytometry 5 days later.

[0261] As a result, referring to Table 13 and Figure 22, it can be seen that B637302, B606401 and the control Telitaccept can all effectively inhibit the in vitro proliferation of B cells induced by APRIL in a dose-dependent manner, and the efficacy of B637302 is significantly superior to that of Telitaccept, and the efficacy of B606401 is also significantly superior to that of the control Telitaccept under treatment conditions with 1000 nM of the drug.

[0262] [Table 13-1] [Table 13-2]

[0263] Example 19. Functional 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, B805201, and IgG1 isotype were added on days 4 to 10, and plasma cells (CD27 + CD38 + ) were detected by flow cytometry.

[0264] Referring to Table 14, the experimental results of detecting plasma cell counts by flow cytometry on day 10 indicate that BAFF and ARPIL can effectively induce in vitro plasma cell production. Referring to Figure 23, under treatment conditions with 10 nM of the drug, 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 treatment conditions with 100 nM of the drug, all four test drugs and telitacicept significantly inhibited plasma cell production. Under treatment conditions with 1000 nM of the drug, B637302 had the strongest inhibitory activity against plasma cell production, superior to telitacicept.

[0265] [Table 14]

[0266] Example 20. Functional 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 generated on day 10. + CD38 + The number of nuclei was detected by flow cytometry (Fig. 24A).

[0267] 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 under these conditions was compared. It was found that 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 had significant inhibitory activity (see Figure 24, B).

[0268] 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).

[0269] [Table 15]

[0270] 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 mRNA expression of IFN-α downstream genes (e.g., ISG-15, IFI13, MX-1, HERC5, etc.). The results are shown in Figures 25A and 25B.

[0271] 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.

[0272] 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).

[0273] As a result, while 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 shown to be able to significantly inhibit IgA production compared to the PBS control group (see Figure 26B and Figure 26C). The results on day 7 also showed that under equimolar administration conditions, the efficacy of B637302 was greater than that of B606401 and telitacicept (see Figure 26C), which is consistent with the results of the previous in vitro ELISA binding experiments of BAFF and APRIL and the in vitro biological activity experiments.

[0274] Example 23. Establishment of B cell in vitro differentiation conditions Conventional experiments evaluating B cell-related functions are mainly performed by labeling thymidine (TdR) with 3H. This disclosure provides a novel in vitro experimental system for B cell differentiation into plasma cells, which can be used to evaluate immunosuppressants such as IFNAR1 inhibitors and BAFF / ARPIL antagonists. To establish an in vitro B cell differentiation experimental system, it is necessary to explore basal stimulation conditions that effectively induce in vitro B cell differentiation.

[0275] B cell selection and in vitro culture conditions included isolating human B cells from freshly isolated healthy human PBMCs using a B cell sorting kit (Stemcell cat: 17954) and culturing them in vitro in 1640 GlutaMAX (Gibco cat: 72400-47) basal medium supplemented with 50 μM β-mercaptoethanol (Sigma-Aldrich cat: M3148), MEM non-essential amino acids (Gibco cat: 11140050), sodium pyruvate (Gibco cat: 11360070), and GlutaMAX™ (Gibco cat: 35050061).

[0276] 1) B cell in vitro differentiation conditions 1 × 10 B cells 5The cells were seeded into a 96-well plate at a density of 1 / well. 10 ng / mL recombinant human IL-3, 0.5 μM CpG-A, and 1000 U / mL IFNα or 33 ng / mL recombinant human IL-21 (control) were added for stimulation. After 6 days, plasma cells (CD27 + CD38 + The differentiation rate and cell viability of the cells were detected by flow cytometry.

[0277] The results showed that when IL-3 and CpG-A were used as basal stimuli, they were able to induce the differentiation of some B cells, and when IL-21 or IFNα was added based on this, both could further stimulate the differentiation of B cells, respectively (Figure 27A and B). However, IL-3, CpG-A, and IL-21 were unable to improve overall cell viability, and the addition of IFNα increased cell viability (Figure 27C).

[0278] 2) In vitro basal stimulation conditions for B cells 1 × 10 B cells 5 The cells were seeded into a 96-well plate at a density of 1 / well and stimulated with 1 μg / mL CpG-B, 1 μg / mL R848, 500 U / mL IFNα, 500 ng / mL recombinant human BAFF, 500 ng / mL recombinant human APRIL, and 3 μg / mL retinoic acid (RA). After 6 days, plasma cells (CD27 + CD38 + The differentiation rate of the cells was detected by flow cytometry.

[0279] The results show that CpG-B / R848 + IFNα can effectively stimulate B cell differentiation (Figure 28A and C). On the other hand, CpG-B / R848 + BAFF + APRIL cannot effectively stimulate B cell differentiation under such conditions and can only increase cell viability (Figure 28B). CpG-B / R848 + IFNα + BAFF + APRIL + RA tends to further promote B cell differentiation in the CpG-B / R848 + IFNα group (Figures 28A and 28C).

[0280] 3) In vitro B cell differentiation detection time and detection window As an example, let us consider the induction of differentiation by CpG-B + IFNα. 5 The cells were seeded at a density of 1 / well in a 96-well plate and stimulated with 1 μg / mL CpG-B and 500 U / mL IFNα for 4 days. During this period, 100 nM anifrolumab or control IgG was added on days 0 to 4, and plasma cells (CD27 + CD38 + The differentiation rate of the cells was detected by flow cytometry.

[0281] The results showed that when the induction time was 4 days, IFNα alone could not induce B cell differentiation, but it could promote B cell differentiation under the induction of CpG-B, and the control antibody anifrolumab could inhibit the action of IFNα, with a detection window and an inhibition rate of 75% (Figure 29).

[0282] Example 24. Screening of evaluation methods for IFNAR1 antagonists in B cell in vitro differentiation experiments As an example, let us consider the induction of differentiation by CpG-B + IFNα. 5 The cells were seeded into a 96-well plate at a density of 1 / well and stimulated with 2 μg / mL CpG-B and 250 U / mL IFNα for 4 days. During this period, gradient concentrations of anifrolumab were added on days 0 to 4, and plasma cells (CD27 + CD38 + The differentiation rate and number of erythrocytes were detected by flow cytometry. Repeated detections demonstrated good reproducibility and stability of the method. Table 16 shows the results of two of these experiments, Experiment 1 and Experiment 2.

[0283] The results showed that anifrolumab dose-dependently inhibited B cell differentiation into plasmablasts (Figure 30), suggesting that this screening method can be applied to the screening of molecules such as IFNAR1 antagonists. It also suggests that the concentrations of CpG-B and IFNα can be adjusted within a certain range.

[0284] [Table 16]

[0285] Example 25. Screening of evaluation methods for BAFF / ARPIL antagonists in B cell in vitro differentiation experiments To clarify the conditions for induction of plasma cell production by BAFF / ARPIL, a functional experimental method for in vitro plasma cell production was established.

[0286] 1) IL-3 + CpG-A + IFNα 1 × 10 B cells 5 The cells were seeded at a density of 1 / well into a 96-well plate, and stimulated with 10 ng / mL recombinant human IL-3, 0.5 μM CpG-A, and 1000 U / mL IFNα or 4 nM recombinant human BAFF and 3 nM recombinant human APRIL control. After 6 days, plasma cells (CD27 + CD38 + The differentiation rate of the cells was detected by flow cytometry.

[0287] The results show that when IL-3 and CpG-A are used as basal stimuli, some B cells can be induced to differentiate into plasma cells, but the percentage is relatively low, and when IFNα is added based on this, B cell differentiation can be further stimulated (Figure 31A and B). However, the improvement in the ability to induce B cell differentiation by adding BAFF + APRIL to the IL-3, CpG-A, or IL-3, CpG-A, and IFNα groups is relatively weak (Figure 31B).

[0288] 2) CpG-B + IFNα 1 × 10 B cells 5 The cells were seeded into a 96-well plate at a density of 1 / well and stimulated with 2 μg / mL of CpG-B for 4 days. On day 4, 250 U / mL of IFNα or 500 ng / mL of recombinant human BAFF and 50 ng / mL of recombinant human APRIL were added for 3 days. On days 7, 10, 14, and 18, half of the medium was replaced and the concentrations of recombinant human BAFF and recombinant human APRIL were adjusted to 50 ng / mL and 500 ng / mL, respectively. On days 7, 10, 14, and 21, plasma cells (CD27 + CD38 + ) were detected by flow cytometry.

[0289] The results show that under these conditions, CpG-B + BAFF + APRIL cannot induce in vitro generation of plasma cells. While CpG-B + IFNα can induce in vitro generation of plasma cells, BAFF + APRIL cannot induce further in vitro generation of plasma cells under the same conditions (Figure 32). Furthermore, the detection window for plasma cell generation was relatively large up to day 10 but narrowed from day 14 onwards.

[0290] 3) CpG-B + IFNα was used as the early stimulation condition, and IL-6 was used as the late stimulation condition. BAFF / APRIL acted at the late stage of plasma cell production. 5 The cells were seeded into a 96-well plate at a density of 1000 / well, and B cells were stimulated according to any one of the conditions a) to d) below. On days 7, 11, and 12, plasmablasts (CD27 + CD38 + ) were detected by flow cytometry.

[0291] a) Cells were stimulated with 2 μg / mL CpG-B and 250 U / mL IFN-α for 4 days. On day 4, CpG-B and IFN-α were removed, and 10 ng / mL recombinant human IL-6, 500 ng / mL recombinant human BAFF, and 50 ng / mL recombinant human APRIL were added and stimulated for 3 days. On days 7 and 11, half of the medium was replaced, and the concentrations of recombinant human BAFF and recombinant human APRIL were adjusted to 50 ng / mL and 500 ng / mL, respectively.

[0292] b) 2 μg / mL CpG-B was added and stimulation was continued for 4 days, and on the fourth day, CpG-B was removed and 10 ng / mL recombinant human IL-6 was added to continue stimulation.

[0293] c) 2 μg / mL CpG-B and 250 U / mL IFNα were added and stimulated for 4 days, and on the 4th day, CpG-B and IFNα were removed, and 10 ng / mL recombinant human IL-6 was added and stimulation continued.

[0294] d) 2 μg / mL CpG-B was added and cells were stimulated for 4 days. On day 4, CpG-B was removed, and 10 ng / mL recombinant human IL-6, 500 ng / mL recombinant human BAFF, and 50 ng / mL recombinant human APRIL were added and cells were stimulated for 3 days. On days 7 and 11, half of the medium was replaced, and the concentrations of recombinant human BAFF and recombinant human APRIL were adjusted to 50 ng / mL and 500 ng / mL, respectively.

[0295] As a result, when CpG-B is used as the early basal stimulus, under these conditions, IL-6+BAFF+APRIL cannot induce plasma cell production alone in the late phase, nor can it further induce plasma cell production in the presence of early induction by IFNα (Figure 33).

[0296] 4) CpG-B is used as the early stimulation condition, and IL-6 + IL-10 + IL-21 is used as the late stimulation condition. 1 × 10 B cells 5The cells were seeded into a 96-well plate at a density of 1 / well, and stimulated with 2 μg / mL of CpG-B (Invivogen cat: tlrl-2006) for 4 days. On day 4, CpG-B was removed, and 10 ng / mL of recombinant human IL-6, 50 ng / mL of recombinant human IL-10, 5 ng / mL of recombinant human IL-21, 500 ng / mL of recombinant human BAFF, and 50 ng / mL of recombinant human APRIL were added and stimulated for 3 days. On day 7, half of the medium was replaced, and the concentrations of recombinant human BAFF and recombinant human APRIL were adjusted to 50 ng / mL and 500 ng / mL, respectively. On day 10, plasmablasts (CD27 + CD38 + ) were detected by flow cytometry.

[0297] The results show that when CpG-B is used as the early basal stimulus, IL-6+IL-10+BAFF+APRI and 5 ng / mL of IL-21 cannot induce plasma cell generation in the late phase (Figure 34).

[0298] 5) CpG-B is used as the early stimulation condition, and IL-6 + IL-10 + IL-21 is used as the late stimulation condition. 1 × 10 B cells 5 The cells were seeded at a density of 1 / well into a 96-well plate, and stimulated with 2 μg / mL of CpG-B for 5 days. On day 5, CpG-B was removed, and 10 ng / mL of recombinant human IL-6, 50 ng / mL of recombinant human IL-10, 50 ng / mL of recombinant human IL-21, 500 ng / mL of recombinant human BAFF, and 50 ng / mL of recombinant human APRIL were added and stimulated for 2 days. On day 7, half of the medium was replaced, and the concentrations of recombinant human BAFF and recombinant human APRIL were adjusted to 50 ng / mL and 500 ng / mL, respectively. On days 9, 10, and 11, plasmablasts (CD27 + CD38 + ) were detected by flow cytometry.

[0299] As a result, when CpG-B was used as the early basal stimulation, plasma cell production could be induced in the late stage by IL-6+IL-10+BAF+APRIL and 50 ng / mL of IL-21, and there was a detection window on both days 9 and 11, which tended to gradually increase (Figure 35).

[0300] 6) CpG-B is used as the early stimulation condition, and IL-6 + IL-10 + IL-21 is used as the late stimulation condition. 1 × 10 B cells 5 The cells were seeded at a density of 1 / well into a 96-well plate and stimulated with 2 μg / mL of CpG-B for 4 days. On day 4, CpG-B was removed, and 10 ng / mL of recombinant human IL-6, 50 ng / mL of recombinant human IL-10, 50 ng / mL of recombinant human IL-21, 500 ng / mL of recombinant human BAFF, and 50 ng / mL of recombinant human APRIL were added and stimulated for 3 days. On day 7, half of the medium was replaced, and the concentrations of recombinant human BAFF and recombinant human APRIL were adjusted to 50 ng / mL and 500 ng / mL, respectively. On day 10, plasmablasts (CD27 + CD38 + ) were detected by flow cytometry.

[0301] Using this in vitro cell model, we screened target fusion proteins. Specifically, gradient concentrations of target fusion proteins, such as telitacicept, were added on days 4 to 10. The results are shown in Figure 23. Using this screening method, telitacicept was shown to dose-dependently inhibit the in vitro generation of plasma cells (Figure 23), suggesting that this screening method can be applied to the screening of drugs targeting BAFF / APRIL, such as TACI-Fc molecules and other similar drugs (e.g., telitacicept, B637302, and B606401).

[0302] Repeated detections showed good reproducibility and stability of the method, and Table 17 shows the results of two of these detections, Experiment 1 and Experiment 2.

[0303] [Table 17]

[0304] 7) CpG-B + IFNα is used as the early stimulation condition, and IL-6 + IL-10 + IL-21 is used as the late stimulation condition. 1 × 10 B cells 5 The cells were seeded at a density of 1 / well into a 96-well plate and stimulated with 2 μg / mL CpG-B and 250 U / mL IFNα for 5 days. On day 5, CpG-B and IFNα were removed, and 10 ng / mL recombinant human IL-6, 50 ng / mL recombinant human IL-10, different concentrations of recombinant human IL-21 (15, 25, or 40 ng / mL), 500 ng / mL recombinant human BAFF, and 50 ng / mL recombinant human APRIL (R&D cat: 5860-AP-010 / CF) were added for 2 days. On day 7, half of the medium was replaced, and the concentrations of recombinant human BAFF and recombinant human APRIL were adjusted to 50 ng / mL and 500 ng / mL, respectively. On day 10, plasmablasts (CD27 + CD38 + ) were detected by flow cytometry.

[0305] The results show that when CpG-B+IFNα is used as early basal stimulation, IL-6+IL-10+BAF+APRIL can synergistically induce plasma cell production with 15 ng / mL of IL-21, but cannot synergistically induce plasma cell production with 25 or 40 ng / mL of IL-21 (Figure 36).

[0306] The reagents used were IL-3 (R&D cat: 203-IL-010 / CF), CpG-A (Invivogen cat: tlrl-2216), CpG-B (Invivogen cat: tlrl-2006), IFNα (Biolegen cat: 592704), human BAFF (R&D cat: 7537-BF-025 / CF), APRIL (R&D cat: 5860-AP-010 / CF), IL-10 (Peprotech cat: 200-10), IL-21 (Peprotech cat: AF-200-21-10), R848 (Invivogen cat: tlrl-r848), and retinoic acid (RA, Sigma-Aldrich cat: R2625).

[0307] Although specific embodiments of the present disclosure have been described above, those skilled in the art should understand that these are merely illustrative examples and that various changes and modifications can be made to these embodiments without departing from the principles and spirit of the present disclosure. Accordingly, the scope of the present disclosure is limited by the appended claims.

Claims

1. A method for screening an immunomodulatory agent, comprising: Step 1) administering a TLR agonist to B cells and culturing them for M days, where M is an integer selected from 1 to 28; Step 2) removing the TLR agonist; Step 3) adding one or more interleukins and one or more TNF family members and culturing for N days, where N is an integer selected from 1 to 28; Step 4) adjusting the concentration of TNF family members and continuing to culture; Step 5) Adding the sample to be measured; Step 6) determining the differentiation degree of B cells; wherein step 5) is performed simultaneously with, during, or after any one of steps 1) to 4); method.

2. A method for screening an immunomodulatory agent, comprising: Step 1) administering a TLR agonist and IFN to B cells and culturing them for M days, where M is an integer selected from 1 to 28; Step 2) removing the TLR agonist and IFN; Step 3) adding one or more interleukins and one or more TNF family members and culturing for N days, where N is an integer selected from 1 to 28; Step 4) adjusting the concentration of TNF family members and continuing to culture; Step 5) Adding the sample to be measured; Step 6) determining the differentiation degree of B cells; wherein step 5) is performed simultaneously with, during, or after any one of steps 1) to 4); method.

3. Step 7) further comprises selecting the sample to be measured as an immunomodulatory agent capable of activating or inhibiting B cell differentiation, and optionally, step 6) further comprises selecting the sample to be measured as an immunomodulatory agent capable of activating or inhibiting B cell differentiation, and ＋ CD38 ＋ determining the degree of B cell differentiation by detecting the percentage of B cells in the 3. The method according to claim 1 or 2.

4. the TLR agonist is any one or more selected from TLR7, TLR8 and TLR9 agonists, preferably any one or more selected from R848, CpG and LPS, preferably CpG-A or CpG-B, more preferably CpG-B; When present, the IFN is IFNα or IFNβ, preferably IFNα; the interleukin is one or more selected from IL-3, IL-6, IL-10, and IL-21, preferably the interleukin is a combination of IL-6, IL-10, and IL-21; The TNF family member is any one or more selected from BAFF and APRIL, and preferably, the TNF family member is a combination of BAFF and APRIL. The method according to any one of claims 1 to 3.

5. Where the TNF family members are BAFF and APRIL, modulating the concentration of the TNF family members includes decreasing the concentration of BAFF and / or increasing the concentration of APRIL; Preferably, the concentration of BAFF in step 4) is reduced to 0.01 to 0.9 times, more preferably 0.02 to 0.5 times, and even more preferably about 0.02, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, or about 0.9 times the concentration of BAFF added in step 3); Preferably, the concentration of APRIL in step 4) is increased by 1.1 to 100 times, more preferably 5 to 50 times, and even more preferably about 1.5, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 15, about 20, about 25, about 30, about 35, about 40, or about 50 times the concentration of APRIL added in step 3). The method according to any one of claims 1 to 3.

6. Step 1) administering 0.1 to 10 μg / mL of CpG-B, CpG-A, or R848 to B cells and culturing them for 2 to 8 days; Step 2) Remove CpG-B or CpG-A or R848; Step 3) adding 2 to 100 ng / mL of IL-6, 10 to 1000 ng / mL of IL-10, and 10 to 200 ng / mL of IL-21, as well as 10 to 2000 ng / mL of BAFF and 10 to 2000 ng / mL of APRIL, and culturing for 2 to 5 days; Step 4) Decreasing the concentration of BAFF and increasing the concentration of APRIL; Step 5) adding a sample to be measured on any one day during the culture of the B cells, and detecting B cell differentiation; Step 6) Selecting the sample to be measured as an immunomodulatory agent capable of activating or inhibiting B cell differentiation; Preferably, Step 1) On day 0, administer 0.5 to 5 μg / mL of CpG-B to B cells; Step 2) Remove CpG-B on days 3-5; Step 3) adding 10 to 50 ng / mL of IL-6, 20 to 200 ng / mL of IL-10, and 20 to 200 ng / mL of IL-21, and also adding 200 to 1000 ng / mL of BAFF and 20 to 200 ng / mL of APRIL, and stimulating for an additional 2 to 4 days; Step 4) reducing the concentration of BAFF to 20-200 ng / mL and increasing the concentration of APRIL to 200-1000 ng / mL; Step 5) Add samples to be measured on days 0-21 to detect B cell differentiation; Step 6) Selecting the sample to be measured as an immunomodulatory agent capable of activating or inhibiting B cell differentiation; More preferably, Step 1) On day 0, administer 1-2 μg / mL of CpG-B to B cells; Step 2) After 4 or 5 days, remove CpG-B; step 3) adding about 10 ng / mL IL-6, about 50 ng / mL IL-10, and about 50 ng / mL IL-21, and adding about 500 ng / mL BAFF and about 50 ng / mL APRIL; Step 4) After 2, 3, or 4 days, adjust the concentration of BAFF to about 50 ng / mL and the concentration of APRIL to about 500 ng / mL; Step 5) Add samples to be measured on days 0-21 to detect B cell differentiation; Step 6) Selecting the sample to be measured as an immunomodulatory agent capable of activating or inhibiting B cell differentiation; 10. A method according to any one of the preceding claims.

7. Step 1) administering 0.1 to 10 μg / mL of CpG-B, CpG-A, or R848 and 50 to 1000 U / mL of IFNα to B cells and culturing them for 2 to 8 days; Step 2) removing CpG-B or CpG-A or R848 and removing IFNα; Step 3) adding 2 to 100 ng / mL of IL-6, 10 to 1000 ng / mL of IL-10, 10 to 200 ng / mL of IL-21, and 10 to 2000 ng / mL of BAFF and 10 to 2000 ng / mL of APRIL, and culturing for 2 to 5 days; Step 4) Decreasing the concentration of BAFF and increasing the concentration of APRIL; Step 5) Add samples to be measured on days 0-21 to detect B cell differentiation; Step 6) Selecting the sample to be measured as an immunomodulatory agent capable of activating or inhibiting B cell differentiation; Preferably, Step 1) On day 0, B cells are stimulated by administering 0.5 to 5 μg / mL of CpG-B and 100 to 500 U / mL of IFNα to the B cells; Step 2) Remove CpG-B and IFNα on days 3-5; Step 3) adding 10 to 50 ng / mL of IL-6, 20 to 200 ng / mL of IL-10, and 5 to 22 ng / mL of IL-21, and also adding 200 to 1000 ng / mL of BAFF and 20 to 200 ng / mL of APRIL, and stimulating for an additional 2 to 4 days; Step 4) reducing the concentration of BAFF to 20-200 ng / mL and increasing the concentration of APRIL to 200-1000 ng / mL; Step 5) Add samples to be measured on days 0-21 to detect B cell differentiation; Step 6) Selecting the sample to be measured as an immunomodulatory agent capable of activating or inhibiting B cell differentiation; More preferably, Step 1) on day 0, administering about 1-2 μg / mL of CpG-B and about 250-500 U / mL of IFNα to B cells; Step 2) After 4 or 5 days, CpG-B and IFNα are removed; step 3) adding about 10 ng / mL IL-6, about 50 ng / mL IL-10, and about 15 ng / mL IL-21, and adding about 500 ng / mL BAFF and about 50 ng / mL APRIL; Step 4) After 2, 3, or 4 days, adjust the concentration of BAFF to about 50 ng / mL and the concentration of APRIL to about 500 ng / mL; Step 5) Add samples to be measured on days 0-21 to detect B cell differentiation; Step 6) Selecting the sample to be measured as an immunomodulatory agent capable of activating or inhibiting B cell differentiation; 10. A method according to any one of the preceding claims.

8. A method for screening an immunomodulatory agent, comprising: 1) administering a TLR agonist and interferon to B cells; 2) Adding a sample to be measured on any of days 0 to 21 and continuing to culture; 3) determining the degree of differentiation of B cells; Preferably, the method further comprises: 4) selecting the sample to be measured as an immunomodulatory agent capable of activating or inhibiting B cell differentiation; method.

9. the TLR agonist is one or more selected from TLR7, TLR8 and TLR9 agonists, preferably the TLR agonist is one or more selected from R848 and CpG, preferably the TLR agonist is CpG-A or CpG-B, more preferably CpG-B; The IFN is IFNα or IFNβ, preferably IFNα; The method of claim 8.

10. 1) administering 0.1 to 20 μg / mL of CpG-B and 50 to 1000 U / mL of IFNα to B cells; 2) Add the sample to be measured on any of days 0 to 21 and continue culturing. 3) determining the degree of differentiation of B cells; Preferably, the method further comprises: 4) selecting the sample to be measured as an immunomodulatory agent capable of activating or inhibiting B cell differentiation; Preferably, step 1) administering 0.5 to 5 μg / mL of CpG-B and 100 to 800 U / mL of IFNα to B cells; More preferably, the method comprises step 1) administering to B cells about 1-2 μg / mL of CpG-B and 250-500 U / mL of IFNα; 10. The method according to claim 8 or 9.

11. the immunomodulator is a type I interferon pathway modulator and / or a TNF pathway modulator; Preferably, the type I interferon pathway modulator is an IFNAR1 signaling pathway modulator; Preferably, the TNF pathway modulator is a BAFF and / or ARRIL pathway modulator.

10. A method according to any one of the preceding claims.

12. Activating or inhibiting the differentiation of the B cells is achieved by detecting the number or proportion of B cells differentiated into plasma cells, and the plasma cells are CD27 ＋ CD38 ＋ 2. The method according to claim 1, wherein B cells of the present invention are preferably B cells of the present invention.

13. The concentration of the B cells was 1 x 10 in a 96-well plate. ５ 10. The method of claim 9, wherein the number of cells in the well is 1 / well.

14. The B cells 1) isolating B cells from PBMCs; 2) culturing in 1640 medium containing 50 μM β-mercaptoethanol, 100 μM MEM non-essential amino acids, and sodium pyruvate, wherein the 1640 medium contains GlutaMAX; obtained by 10. A method according to any one of the preceding claims.

15. the immunomodulator is selected from a protein or polypeptide, a nucleic acid, an aptamer, a small molecule compound, and a molecule containing the protein or polypeptide, the nucleic acid, the aptamer, or the small molecule compound, and the protein or polypeptide is preferably an antibody or an antigen-binding fragment thereof, a cytokine, a receptor, or a ligand; Preferably, the immunomodulator is for treating a B cell disorder or an autoimmune disease; More preferably, the B cell disorder or autoimmune disease is a disease or condition associated with TACI and / or BCMA expression; More preferably, 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, and the B-cell disorder is selected from tumors, chronic leukemia, multiple myeloma, non-Hodgkin's lymphoma, post-transplant lymphoproliferation and light chain gammopathy.

10. A method according to any one of the preceding claims.

16. 16. A method for preparing or manufacturing an immunomodulatory agent, comprising screening said immunomodulatory agent according to the method of any one of claims 1 to 15.

17. A pharmaceutical composition comprising the immunomodulatory agent of any one of claims 1 to 16 and one or more pharmaceutically acceptable carriers, diluents or excipients.

18. 19. A method for preparing or manufacturing a pharmaceutical composition comprising the step of mixing an immunomodulatory agent according to any one of claims 1 to 16 with one or more pharmaceutically acceptable carriers, diluents or excipients, or the step of screening an immunomodulatory agent according to any one of claims 1 to 14, or the step of preparing or manufacturing an immunomodulatory agent according to claim 16, and mixing the obtained immunomodulatory agent with one or more pharmaceutically acceptable carriers, diluents or excipients.

19. 19. A method of treating or ameliorating a disease or condition using the immunomodulatory agent of the method of any one of claims 1 to 16 or the pharmaceutical composition of claim 17, said method comprising administering to a subject in need thereof the immunomodulatory agent of the method of any one of claims 1 to 16 or the pharmaceutical composition of claim 17; Preferably, the disease or condition is a B cell disorder or an autoimmune disease; More preferably, the B cell disorder or autoimmune disease is a disease or condition associated with TACI and / or BCMA expression; More preferably, 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, and the B-cell disorder is selected from tumors, chronic leukemia, multiple myeloma, non-Hodgkin's lymphoma, post-transplant lymphoproliferation and light chain gammopathy. method.

20. 1. A method of inducing B cell differentiation, comprising administering to B cells a TLR agonist and IFN; wherein the TLR agonist is one or more of TLR7, TLR8 and TLR9 agonists, preferably the TLR agonist is one or more of R848 or CpG, preferably the TLR agonist is CpG-A or CpG-B, more preferably CpG-B; The IFN is IFNα or IFNβ, preferably IFNα; method.

21. administering CpG-A and IFNα to the B cells; or administering CpG-B or R484 and IFNα to B cells; Optionally, the method further comprises: administering CpG-A and IFNα to B cells, or administering CpG-B or R484 and IFNα to B cells; and simultaneously administering IL-3 and IL-21 to the B cells.

20. The method of claim 19.

22. administering to the B cells 5-20 ng / mL IL-3, 0.2-1 μM CpG-A, 500-2000 U / mL IFNα, and 16.5-66 ng / mL IL-21; or administering to the B cells 0.5-5 μg / mL of CpG-B or R484 and 100-800 U / mL of IFNα; Preferably, administering to the B cells about 10 ng / mL IL-3, about 0.5 μM CpG-A, about 1000 U / mL IFNα, and about 33 ng / mL IL-21; or administering to B cells 1-2 μg / mL of CpG-B or R484 and 250-500 U / mL of IFNα; The method according to claims 20 to 21.

23. The method of any one of claims 20 to 22, further comprising simultaneously adding retinoic acid to the B cells, wherein the concentration of the retinoic acid is preferably 2 to 5 μg / mL, more preferably about 3 μg / mL.

24. A cell model for screening immunomodulatory agents, comprising B cells whose differentiation has been induced by the method of any one of claims 20 to 23.

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