Screening and preparation method of immunomodulator

Abstract

The present disclosure provides methods of screening and preparing immunomodulators. The disclosure also relates to the use of said immunomodulators for the treatment of immune-related diseases.

Description

Screening and preparation methods of immunomodulators

[0001] This disclosure claims priority to the following patent application: Chinese patent application filed on August 29, 2022, with application number CN202211043007.2 and invention name “Screening and Preparation Methods of Immunomodulators”; the entire contents of the above patent application are incorporated into this disclosure by reference. Technical Field

[0002] The present disclosure relates to the field of biomedicine, and in particular, to a method for screening and preparing an immunomodulator, and the use of the immunomodulator in treating diseases (especially autoimmune diseases). Background Art

[0003] Autoimmune diseases such as systemic lupus erythematosus (SLE), lupus nephropathy, and multiple sclerosis mostly involve the production of autoantibodies against self-tissue antigens, which triggers a systemic immune response that ultimately affects different organs and causes corresponding disease symptoms. From a biological mechanism perspective, a series of important biological processes, including the differentiation of B cells into plasma cells, the generation and maintenance of plasma cells, and the secretion of antibodies by plasma cells, play a central role in the onset and progression of autoimmune diseases. Therefore, a large number of drugs targeting B cells and plasma cells have entered the clinical stage. Among them, the approved belimumab (trade name Benlysta) is a specific inhibitor of B lymphocyte stimulator (BLyS, also known as BAFF) for the treatment of patients with active, autoantibody-positive systemic lupus erythematosus who are receiving standard treatment. In addition, tadalafil (RC18, trade name: Tai Ai) is a TACI-Ig fusion protein that simultaneously targets BLyS / APRIL to inhibit B cells and plasma cells. In 2021, it was approved by the China National Medical Products Administration for the treatment of systemic lupus erythematosus.

[0004] B cell differentiation is divided into five main stages: precursor B cells, immature B cells, mature B cells, activated B cells, and plasma cells. The first three stages are antigen-independent and primarily occur in the bone marrow. After maturation, B cells migrate to peripheral immune organs and become activated upon antigen stimulation, 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 therapies are one of the main directions of new drug development. Establishing experimental methods to evaluate B cell-related biological functions is a key step in the development of autoimmune disease-related drugs. Therefore, establishing a convenient, efficient and stable experimental system for evaluating B cell-related functions will help promote the development of new drugs for autoimmune diseases.

[0006] Existing functional tests for evaluating B cells primarily rely on 3H-labeled thymidine (TdR). This isotope, 3H-labeled TdR, acts as a precursor for DNA synthesis and is incorporated into the DNA synthesis and metabolism process. Each newly synthesized DNA double strand then carries a radioactive signal. Measuring the intensity of radioactivity in cells can reflect cellular DNA metabolism and cell proliferation. However, this method primarily evaluates B cell proliferation, and its implementation requires specialized and rigorous training for laboratory personnel. Radioactivity detection equipment is expensive, and there are concerns about radionuclide contamination. Therefore, this B cell proliferation assay is not readily available in conventional laboratories, making it difficult to perform.

[0007] After exploring a series of experimental conditions, the present invention has developed a simple and efficient experimental system for B cell differentiation into plasma cells. It has been confirmed by relevant drugs that are already on the market or in the late clinical stage that this method is convenient and feasible and can be used for screening immunomodulators, which can be used to prepare drugs.

[0008] Summary of the Invention

[0009] The present disclosure provides methods for screening and preparing immunomodulators, as well as uses of the immunomodulators for preparing drugs for treating or alleviating diseases.

[0010] Screening and preparation methods of immunomodulators

[0011] The present disclosure provides methods for promoting B cell differentiation. Furthermore, the present disclosure provides methods for screening and preparing immunomodulators. The methods can be performed in vitro or in vivo.

[0012] In some embodiments, a method for promoting B cell differentiation and a method for screening and preparing an immunomodulator are provided, comprising administering a TLR agonist, and / or an interleukin, and / or retinoic acid (RA), and / or a tumor necrosis factor family member to the B cell. 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), a TLR9 agonist (e.g., CpG-ODN, such as 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.

[0013] In some embodiments, the TLR agonist can be selected from 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 vaccine, TMX-202, ISA-201, PEPA-10, AL-034, CpG-ODN(K3), Vaxart, CBLB-612, Pseudomonas aeruginosa, IR-103, VAX-161, VAX-125, MEDI19197, R848, CpG-A, CpG-B or a complex thereof or a pharmaceutically acceptable salt thereof.

[0014] In a first aspect, in some embodiments of the present disclosure, there is provided a method for promoting B cell differentiation, comprising administering to B cells:

[0015] a) IL-3 and CpG-A;

[0016] b) IL-3, CpG-A, and IL-21;

[0017] c) IL-3 and CpG-A and IFNα; or

[0018] d) IL-3, CpG-A, IFNα, and IL-21;

[0019] The method can increase the rate of B cell differentiation into plasma cells and / or increase the viability of B cells.

[0020] In some embodiments, a)-d) can all increase the rate of B cell differentiation into plasma cells, and d) can increase the viability of B cells.

[0021] In the aforementioned method, the concentration of IL-3 is 0.1-50 ng / mL, 1-30 ng / mL, 5-20 ng / mL, for example, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15 ng / mL;

[0022] The concentration of CpG-A is 0.01-10 μM, 0.05-5 μM, 0.1-2 μM, 0.2-1 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 μM;

[0023] The concentration of IFNα is 1-10000 U / mL, 100-5000 U / mL, 500-2000 U / mL, for example, about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500 U / mL;

[0024] The concentration of IL-21 is 0.1-1000 ng / mL, 1-200 ng / mL, 10-100 ng / mL, 20-50 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, about 66 ng / mL.

[0025] In some embodiments, the method comprises administering to the B cells:

[0026] a-1) 5-20 ng / mL IL-3 and 0.2-1 μM CpG-A;

[0027] b-1) 5-20 ng / mL IL-3, 0.2-1 μM CpG-A, and 16.5-66 ng / mL IL-21;

[0028] c-1) 5-20 ng / mL IL-3 and 0.2-1 μM CpG-A and 500-2000 U / mL IFNα; or

[0029] d-1) 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.

[0030] In some embodiments, the method comprises administering to the B cells:

[0031] a-2) approximately 10 ng / mL IL-3 and approximately 0.5 μM CpG-A;

[0032] b-2) about 10 ng / mL IL-3, about 0.5 μM CpG-A, and about 33 ng / mL IL-21;

[0033] c-2) about 10 ng / mL IL-3 and about 0.5 μM CpG-A and about 1000 U / mL IFNα; or

[0034] 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.

[0035] In some embodiments, a method for screening an immunomodulator is provided, comprising the steps of:

[0036] Step 1) administering a TLR agonist to B cells and culturing for M days, wherein M is selected from an integer of 1-28;

[0037] Step 2) removing the TLR agonist;

[0038] Step 3) adding one or more interleukins and one or more TNF family members; culturing for N days, wherein N is an integer selected from 1-28;

[0039] Step 4) adjusting the concentration of TNF family members and continuing the culture;

[0040] Step 5) adding the sample to be tested; and

[0041] Step 6) determining the degree of B cell differentiation;

[0042] The step 5) is performed simultaneously with, during, or after any one of the steps 1) to 4).

[0043] In some embodiments, a method for screening an immunomodulator is provided, comprising the steps of:

[0044] Step 1) administering a TLR agonist and IFN to B cells and culturing for M days, wherein M is selected from an integer of 1-28;

[0045] Step 2) removing TLR agonists and IFN;

[0046] Step 3) adding one or more interleukins and one or more TNF family members; culturing for N days, wherein N is an integer selected from 1-28;

[0047] Step 4) adjusting the concentration of TNF family members and continuing the culture;

[0048] Step 5) adding the sample to be tested; and

[0049] Step 6) determining the degree of B cell differentiation;

[0050] The step 5) is performed simultaneously with, during, or after any one of the steps 1) to 4).

[0051] In some embodiments, the method further comprises:

[0052] Step 7) selecting a test sample that can activate or inhibit B cell differentiation as an immunomodulator; optionally, step 6) includes detecting CD27 + CD38 + The percentage of B cells was used to determine the degree of B cell differentiation.

[0053] In some embodiments, the TLR agonist is selected from any one or more of TLR7, TLR8, and TLR9 agonists; exemplarily, the TLR agonist is selected from any one or more of R848, CpG, and LPS; exemplarily, the TLR agonist is CpG-A or CpG-B, more exemplarily CpG-B;

[0054] When present, the IFN is IFNα or IFNβ, exemplified by IFNα;

[0055] The interleukin is selected from one or more of IL-3, IL-6, IL-10 and IL-21; illustratively, the interleukin is a combination of IL-6, IL-10 and IL-21;

[0056] The TNF family member is selected from any one or more of BAFF and APRIL; illustratively, the TNF family member is a combination of BAFF and APRIL.

[0057] In some embodiments, when the TNF family member is BAFF and APRIL, regulating the concentration of the TNF family member comprises decreasing the concentration of BAFF, and / or increasing the concentration of APRIL;

[0058] Illustratively, the BAFF concentration in step 4) is reduced to 0.01-0.9 times, more illustratively to 0.02-0.5 times, and more illustratively to 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 BAFF concentration added in step 3);

[0059] Illustratively, the concentration of APRIL in step 4) is increased to 1.1-100 times, more illustratively 5-50 times, and more 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).

[0060] In some embodiments, a method for screening an immunomodulator is provided, comprising:

[0061] 1) administering TLR agonists and interferon to B cells;

[0062] 2) Add the sample to be tested on any day from day 0 to day 21 and continue culturing;

[0063] 3) determine the degree of B cell differentiation;

[0064] Illustratively, the method further comprises:

[0065] 4) Selecting a test sample that can activate or inhibit B cell differentiation as an immunomodulator.

[0066] In some embodiments, the TLR agonist is selected from one or more of TLR7, TLR8, and TLR9 agonists; exemplarily, the TLR agonist is selected from one or more of R848 and CpG; exemplarily, the TLR agonist is CpG-A or CpG-B, more exemplarily CpG-B;

[0067] The IFN is IFNα or IFNβ, exemplified by IFNα.

[0068] In some embodiments, a method for screening an immunomodulator is provided, comprising:

[0069] 1) administering 0.1-20 μg / mL CpG-B and 50-1000 U / mL IFNα to B cells;

[0070] 2) Add the sample to be tested on any day from day 0 to day 21 and continue culturing;

[0071] 3) determine the degree of B cell differentiation;

[0072] Illustratively, the method further comprises:

[0073] 4) Selecting a test sample that can activate or inhibit B cell differentiation as an immunomodulator;

[0074] Exemplarily, step 1) administering 0.5-5 μg / mL CpG-B and 100-800 U / mL IFNα to B cells;

[0075] More exemplarily, step 1) administers about 1-2 μg / mL CpG-B and 250-500 U / mL IFNα to the B cells.

[0076] In some embodiments, the immunomodulator is a type I interferon pathway modulator and / or a TNF pathway modulator;

[0077] Exemplarily, the type I interferon pathway regulator is an IFNAR1 signaling pathway regulator;

[0078] Exemplarily, the TNF pathway regulator is a BAFF and / or ARRIL pathway regulator.

[0079] In some embodiments, the activation or inhibition of B cell differentiation is achieved by detecting the number or ratio of B cells differentiated into plasma cells, exemplified by CD27 + CD38 + of B cells.

[0080] In some embodiments, the concentration of the B cells is 1×10 5 / hole.

[0081] In some embodiments, the B cells are obtained by:

[0082] 1) Isolation of B cells from PBMCs, and

[0083] 2) Culture in 1640 medium supplemented with 50 μM β-mercaptoethanol, 100 μM MEM non-essential amino acids, sodium pyruvate, and GlutaMAX.

[0084] In some embodiments, the immunomodulator is selected from proteins or polypeptides, nucleic acids, aptamers, small molecule compounds, and molecules comprising the proteins or polypeptides, nucleic acids, aptamers, small molecule compounds; the proteins or polypeptides are exemplified by antibodies or antigen-binding fragments thereof, cytokines, receptors, and ligands;

[0085] Exemplarily, the immunomodulator is used to treat a B cell disorder or an autoimmune disease;

[0086] More exemplarily, the B cell disorder or autoimmune disease is a disease or condition associated with TACI and / or BCMA expression;

[0087] More exemplarily, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, insulin-dependent diabetes mellitus, Crohn's disease, rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis and psoriatic arthritis; and the B cell disorder is selected from the group consisting of tumors, chronic leukocytic leukemia, multiple myeloma, non-Hodgkin's lymphoma, post-transplant lymphoproliferative disease and light chain gammaglobulinopathy.

[0088] In a second aspect, in some embodiments of the present disclosure, there is provided a method for promoting B cell differentiation, comprising administering to B cells:

[0089] e) CpG-B and IFNα;

[0090] f) R484 and IFNα;

[0091] g) CpG-B, BAFF, and APRIL;

[0092] h) R484, BAFF, and APRIL;

[0093] i) CpG-B and IFNα and RA and BAFF and APRIL; or

[0094] j) R484, IFNα, RA, BAFF, and APRIL;

[0095] The method can increase the differentiation rate of B cells into plasma cells and / or enhance the viability of B cells.

[0096] In some embodiments, e), f), i), and j) can promote the rate of B cell differentiation into plasma cells, and e)-j) can all increase B cell viability, wherein i) and j) increase B cell viability to a greater extent than g) and h).

[0097] In the aforementioned method, the concentration of CpG-B is 0.1-100 μg / mL, 0.2-20 μg / mL, 0.5-10 μg / mL, 0.5-5 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 μg / mL;

[0098] IFNα concentration is 1-10000 U / mL, 100-2000 U / mL, 200-800 U / mL, 100-500 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, about 1000 U / mL;

[0099] R484 concentration is 0.1-100 μg / mL, 0.2-20 μg / mL, 0.5-10 μg / mL, 0.5-5 μg / mL, such as 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 μg / mL;

[0100] RA concentration is 0.1-50 μg / mL, 0.2-20 μg / mL, 0.2-10 μg / mL, 1-5 μg / mL, such as about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10 μg / mL;

[0101] The concentration of BAFF or APRIL is 1-10000 ng / mL, 100-5000 ng / mL, or 200-1000 ng / mL, such as about 200, about 300, about 400, about 500, about 600, about 7000, about 800, about 900, or about 1000 ng / mL; or the concentration of BAFF or APRIL is 0.1-100 nM, 0.5-20 nM, or 1-10 nM, such as about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nM;

[0102] In some embodiments, the B cells are administered:

[0103] e-1) 0.5-2 μg / mL CpG-B and 200-800 U / mL IFNα; or about 1-3 μg / mL CpG-B and 100-500 U / mL IFNα;

[0104] f-1) 0.5-2 μg / mL R484 and approximately 200-800 U / mL IFNα; or 1-3 μg / mL R484 and approximately 100-500 U / mL IFNα;

[0105] g-1) 0.5-2 μg / mL CpG-B, 200-1000 ng / mL BAFF, and 200-1000 ng / mL APRIL;

[0106] h-1) 0.5-2 μg / mL R484, 200-1000 ng / mL BAFF, and 200-1000 ng / mL APRIL;

[0107] i-1) 0.5-2 μg / mL CpG-B and 200-1000 U / mL IFNα and 2-5 μg / mL RA and 200-1000 ng / mL BAFF and 200-1000 ng / mL APRIL; or, about 1-3 μg / mL CpG-B and 100-500 U / mL IFNα and 2-5 μg / mL RA and 200-1000 ng / mL BAFF and 200-1000 ng / mL APRIL;

[0108] j-1) 0.5-2 μg / mL R484 and 200-1000 U / mL IFNα and 2-5 μg / mL RA and 200-1000 ng / mL BAFF and 200-1000 ng / mL APRIL; or, about 1-3 μg / mL R484 and 100-500 U / mL IFNα and 2-5 μg / mL RA and 200-1000 ng / mL BAFF and 200-1000 ng / mL APRIL.

[0109] In some embodiments, the B cells are administered:

[0110] 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;

[0111] 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;

[0112] g-2) about 1 μg / mL or about 2 μg / mL CpG-B, 500 ng / mL BAFF, and 500 ng / mL APRIL;

[0113] h-2) about 1 μg / mL or about 2 μg / mL R484 and 500 ng / mL BAFF and 500 ng / mL APRIL;

[0114] i-2) about 1 μg / mL or about 2 μg / mL CpG-B and about 500 U / mL IFNα and about 3 μg / mL RA and about 500 ng / mL BAFF and about 500 ng / mL APRIL; or, about 1 μg / mL or about 2 μg / mL CpG-B and 250 U / mL IFNα and about 3 μg / mL RA and about 500 ng / mL BAFF and about 500 ng / mL APRIL;

[0115] j-2) about 1 μg / mL or about 2 μg / mL R484 and about 500 U / mL IFNα and about 3 μg / mL RA and about 500 ng / mL BAFF and about 500 ng / mL APRIL; or, about 1 μg / mL or about 2 μg / mL R484 and 250 U / mL IFNα and about 3 μg / mL RA and about 500 ng / mL BAFF and about 500 ng / mL APRIL;

[0116] In the above scheme, after adding factor stimulation, the differentiation of B cells (e.g. differentiation into plasma cells (CD27 + CD38 + ) ratio) time is any day, such as the 1st day, the 2nd day, the 3rd day, the 4th day, the 5th day, the 6th day, for example, the 4th day.

[0117] In some embodiments, there is provided a method of inducing B cell differentiation, comprising the steps of administering a TLR agonist and IFN to the B cell;

[0118] wherein the TLR agonist is one or more of TLR7, TLR8 and TLR9 agonists; exemplarily, the TLR agonist is one or more of R848 or CpG; exemplarily, the TLR agonist is CpG-A or CpG-B, more exemplarily CpG-B;

[0119] The IFN is IFNα or IFNβ, exemplified by IFNα.

[0120] In some embodiments, a method of inducing B cell differentiation is provided, comprising:

[0121] administering CpG-A and IFNα to B cells; or

[0122] a step of administering CpG-B or R484, and IFNα to B cells;

[0123] Optionally, the method comprises:

[0124] administering CpG-A and IFNα to B cells; or administering CpG-B or R484, and IFNα to B cells; and,

[0125] A step of simultaneously administering IL-3 and IL-21 to B cells.

[0126] In some embodiments, a method of inducing B cell differentiation is provided, comprising:

[0127] administering 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 to B cells; or

[0128] B cells were administered 0.5-5 μg / mL CpG-B or R484 and 100-800 U / mL IFNα;

[0129] For example,

[0130] administering about 10 ng / mL IL-3, about 0.5 μM CpG-A, about 1000 U / mL IFNα, and about 33 ng / mL IL-21 to the B cells; or

[0131] B cells were administered 1-2 μg / mL CpG-B or R484, and 250-500 U / mL IFNα.

[0132] In an embodiment of the method of inducing B cell differentiation, the method further comprises simultaneously adding retinoic acid to the B cells, wherein the concentration of retinoic acid is exemplarily 2-5 μg / mL, more exemplarily about 3 μg / mL.

[0133] In some embodiments, a cell model for screening immunomodulators is provided, comprising: B cells induced to differentiate by the method described herein.

[0134] In a third aspect, in some embodiments of the present disclosure, a method for screening and preparing an immunomodulator is provided, comprising administering to B cells:

[0135] k) CpG-B and IFNα and immunosuppressants. The method can promote B cells to plasma cells (CD27 + CD38 + ) differentiation.

[0136] In the aforementioned scheme, the concentration of CpG-B is 0.1-100 μg / mL, 0.2-20 μg / mL, 0.5-10 μg / mL, 0.5-5 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, about 2 μg / mL;

[0137] The concentration of IFNα is 1-10000 U / mL, 50-1000 U / mL, 100-500 U / mL, for example, about 150, about 200, about 250, about 300, about 350, about 400, about 500 U / mL.

[0138] In some embodiments, a method for screening and preparing an immunomodulatory agent is provided, comprising administering to a B cell:

[0139] k-1) 1-5 μg / mL CpG-B and 100-500 U / mL IFNα and gradient concentrations of immunomodulators;

[0140] k-2) approximately 2 μg / mL CpG-B and approximately 250 U / mL IFNα and a gradient of immunomodulators;

[0141] k-3) about 1 μg / mL CpG-B and about 500 U / mL IFNα and a gradient of immunomodulatory agents.

[0142] In a fourth aspect, some embodiments of the present disclosure provide methods for screening, preparing or producing immunomodulators, comprising:

[0143] Induction steps: administering a TLR agonist to B cells; removing the TLR agonist on day M; adding interleukin; adding a TNF family member; adjusting the concentration of the TNF family member after the TNF family member has been added for N days;

[0144] Detection step: adding a test sample during or after the induction step to detect B cell differentiation; selecting a test sample that can activate or inhibit B cell differentiation as an immunomodulator;

[0145] Wherein, M and N are integers of 1-30, such as 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0146] In some embodiments of the present disclosure, a method for screening, preparing or producing an immunomodulator is provided, comprising steps 1), 6), and 7); optionally, further comprising steps 2)-3); optionally, further comprising steps 4)-5):

[0147] Step 1) administering a TLR agonist to B cells;

[0148] Step 2) removing the TLR agonist on day 24;

[0149] Step 3) adding interleukin;

[0150] Step 4) adding TNF family members;

[0151] Step 5) adjusting the concentration of TNF family members;

[0152] Step 6) adding the sample to be tested to detect B cell differentiation;

[0153] Step 7) selecting a test sample that can activate or inhibit B cell differentiation as an immunomodulator;

[0154] Wherein, M and N are integers of 1-30, such as 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0155] In some specific embodiments, the aforementioned TLR agonists include but are not limited to TLR7, TLR8, and TLR9 agonists, for example, selected from R848, LPS, or CpG; for example, selected from CpG-A, CpG-B; for example, CpG-B;

[0156] The aforementioned interleukin is, for example, selected from IL-4, IL-3, IL-6, IL-10 and / or IL-21; for example, selected from IL-6, IL-10 and / or IL-21; for example, IL-6, IL-10 and IL-21;

[0157] The aforementioned TNF family members are molecules capable of regulating the TNF signaling pathway, for example, selected from BAFF and APRIL; for example, BAFF and APRIL;

[0158] The concentration of the aforementioned TLR agonist is selected from 0.1-100 μg / mL, 0.2-20 μg / mL, 0.5-10 μg / mL, 0.5-5 μg / mL, for example, about 1, about 2, about 3, about 4, about 5 μg / mL;

[0159] The aforementioned step 5) adjusting the concentration of the TNF family member includes, for example, lowering the concentration of BAFF (e.g., adjusting to 0.01-0.99 times, 0.05-0.9 times, 0.05-0.5 times, 0.05-0.2 times the original concentration, for example, 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, about 0.9 times), increasing the concentration of APRIL (e.g., adjusting to 1.1-1000 times, 2-100 times, 5-50 times, 10-20 times, such as 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, about 50 times));

[0160] The aforementioned detection of B cell differentiation includes, for example, detection of plasma cells (CD27 + CD38 + )quantity;

[0161] The aforementioned test sample is an immunomodulator (eg, an immunostimulatory agent, an immunosuppressive agent) or a B cell differentiation regulator, such as a TNF pathway regulator (eg, a BAFF, APRIL pathway agonist or inhibitor).

[0162] In some embodiments, a method for screening, preparing or producing an immunomodulator is provided, comprising:

[0163] Step 1) administering CpG (e.g., CpG-A or CpG-B) or R848 or LPS to B cells;

[0164] Step 2) removing CpG (e.g., CpG-A or CpG-B) or R848 or LPS on day 24;

[0165] Step 3) adding interleukins IL-6, IL-10 and / or IL-21;

[0166] Optionally, in step 4), BAFF and APRIL are added;

[0167] Optionally, step 5) adjusting the concentrations of BAFF and APRIL;

[0168] Optionally, step 6) adding a sample to be tested to detect B cell differentiation;

[0169] Optionally, in step 7), a test sample capable of activating or inhibiting B cell differentiation is selected as an immunomodulator;

[0170] Wherein, M and N are integers ranging from 1 to 30 respectively.

[0171] In some embodiments, a method for screening, preparing or producing an immunomodulator is provided, comprising:

[0172] Step 1) administering CpG-B to B cells;

[0173] Step 2) removing CpG-B on day 24;

[0174] Step 3) adding interleukins IL-6, IL-10 and IL-21;

[0175] Optionally, step 4) adding BAFF and APRIL;

[0176] Optionally, step 5) reducing the BAFF concentration and increasing the APRIL concentration;

[0177] Optionally, step 6) adding a sample to be tested to detect B cell differentiation;

[0178] Optionally, in step 7), a test sample capable of activating or inhibiting B cell differentiation is selected as an immunomodulator;

[0179] Wherein, M and N are integers of 1-20 respectively, M is 3, 4, 5 or 6, and N is 2, 3 or 4.

[0180] In the aforementioned embodiments, the concentration of CpG-B is selected from 0.1-100 μg / mL, 0.2-20 μg / mL, 0.5-10 μg / mL, 0.5-5 μg / mL, 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, about 5 μg / mL;

[0181] The concentration of CpG-A or LPS is 0.01-10 μM, 0.05-5 μM, 0.1-2 μM, 0.2-1 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, about 3 μM;

[0182] R484 concentration is 0.1-100 μg / mL, 0.2-20 μg / mL, 0.5-10 μg / mL, 0.5-5 μg / mL, such as 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, about 4 μg / mL;

[0183] The concentration of interleukins (e.g., IL-3, IL-4, IL-6, IL-10, IL-21) is 0.1-1000 ng / mL, 1-500 ng / mL, 5-200 ng / mL, respectively, 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, about 200 ng / mL;

[0184] The concentration of the TFN pathway modulator (e.g., BAFF, APRIL) is 1-10,000 ng / mL, 10-5,000 ng / mL, 50-2,000 ng / mL, 20-200 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, about 1,000 ng / mL.

[0185] In some embodiments, a method for screening, preparing or producing an immunomodulator is provided, comprising:

[0186] Step 1) administering 0.5-10 μg / mL CpG-B to B cells;

[0187] Step 2) CpG-B removal on days 2-8;

[0188] Step 3) adding 2-100 ng / mL IL-6, 10-1000 ng / mL IL-10, and 10-1000 ng / mL IL-21;

[0189] Optionally, in step 4), 10-2000 ng / mL BAFF and 10-2000 ng / mL APRIL are added and stimulated for another 2-5 days;

[0190] Optionally, step 5) reducing the BAFF concentration and increasing the APRIL concentration;

[0191] Optionally, in step 6), a test sample is added on days 0-21 to detect B cell differentiation;

[0192] Optionally, in step 7), a test sample capable of activating or inhibiting B cell differentiation is selected as an immunomodulator.

[0193] In some embodiments, a method for screening, preparing or producing an immunomodulator is provided, comprising:

[0194] Step 1) administering 1-5 μg / mL CpG-B to B cells;

[0195] Step 2) Remove CpG-B on days 3-5;

[0196] Step 3) Add 5-50 ng / mL IL-6, 100-500 ng / mL IL-10, and 100-500 ng / mL IL-21;

[0197] Step 4) Add 200-1000 ng / mL BAFF and 20-200 ng / mL APRIL and stimulate for another 2-4 days;

[0198] Step 5) reducing the BAFF concentration to 20-200 ng / mL and increasing the APRIL concentration to 200-1000 ng / mL;

[0199] Step 6) adding the test sample at a concentration gradient from day 0 to day 14 to detect B cell differentiation;

[0200] Step 7) Select a test sample that can activate or inhibit B cell differentiation as an immunomodulator.

[0201] In some embodiments, a method for screening, preparing or producing an immunomodulator is provided, comprising:

[0202] Step 1) administering approximately 2 μg / mL CpG-B to B cells;

[0203] Step 2) removing CpG-B on day 4 or 5;

[0204] Step 3) adding about 10 ng / mL IL-6, about 50 ng / mL IL-10, and about 50 ng / mL IL-21;

[0205] Step 4) Add about 500 ng / mL BAFF and about 50 ng / mL APRIL,

[0206] Step 5) After 3 days, adjust the BAFF concentration to approximately 50 ng / mL and the APRIL concentration to approximately 500 ng / mL;

[0207] Step 6) adding the test sample at a concentration gradient from day 0 to day 14 to detect B cell differentiation;

[0208] Step 7) Select a test sample that can activate or inhibit B cell differentiation as an immunomodulator.

[0209] In the aforementioned scheme, the immunomodulator includes but is not limited to type I interferon pathway regulators, such as IFNAR1 signaling pathway regulators; the IFNAR1 regulator includes but is not limited to antibodies or antigen-binding fragments thereof, small molecule compounds, nucleic acids, aptamers, receptors, ligands, and any molecules comprising the antibodies or antigen-binding fragments thereof, small molecule compounds, nucleic acids, aptamers, receptors, ligands. Exemplary immunomodulators include but are not limited to Anifrolumab and other anti-IFNAR1 antibodies disclosed herein, and fusion proteins comprising the anti-IFNAR1 antibodies (e.g., fusion proteins comprising TACI polypeptides and BCMA polypeptides and anti-IFNAR1 antibodies, fusion proteins comprising TACI polypeptides and anti-IFNAR1 antibodies, fusion proteins comprising BCMA polypeptides and anti-IFNAR1 antibodies); and fusion proteins comprising TACI polypeptides and BCMA polypeptides disclosed herein.

[0210] In a fifth aspect, some embodiments of the present disclosure provide methods for screening, preparing or producing immunomodulators, comprising:

[0211] Induction steps: administer TLR agonists and IFN to B cells; remove TLR agonists and IFN on day M; add interleukin; add TNF family members; after adding TNF family members for N days, adjust the concentration of TNF family members;

[0212] Detection step: adding a test sample during or after the induction step to detect B cell differentiation; selecting a test sample that can activate or inhibit B cell differentiation as an immunomodulator;

[0213] Wherein, M and N are integers of 1-30, such as 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0214] In some embodiments of the present disclosure, a method for screening, preparing or producing an immunomodulator is provided, comprising steps 1), 6), and 7); optionally, further comprising steps 2)-3); optionally, further comprising steps 4)-5):

[0215] Step 1) administering TLR agonists and IFN to B cells;

[0216] Step 2) removing TLR agonists and IFN on day 24;

[0217] Step 3) adding interleukin;

[0218] Step 4) adding TNF family members;

[0219] Step 5) adjusting the concentration of TNF family members;

[0220] Step 6) adding the sample to be tested to detect B cell differentiation;

[0221] Step 7) selecting a test sample that can activate or inhibit B cell differentiation as an immunomodulator;

[0222] Wherein, M and N are integers of 1-30, such as 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0223] In some specific embodiments, the aforementioned TLR agonists include but are not limited to TLR7, TLR8, and TLR9 agonists, for example, selected from R848, LPS, or CpG; for example, selected from CpG-A, CpG-B; for example, CpG-B;

[0224] The aforementioned IFN is selected from IFNβ and IFNα;

[0225] The aforementioned interleukin is, for example, selected from IL-4, IL-3, IL-6, IL-10 and / or IL-21; for example, selected from IL-6, IL-10 and / or IL-21; for example, IL-6, IL-10 and IL-21;

[0226] The aforementioned TNF family members are molecules capable of regulating the TNF signaling pathway, for example, selected from BAFF and APRIL; for example, BAFF and APRIL;

[0227] The concentration of the aforementioned TLR agonist is selected from 0.1-100 μg / mL, 0.2-20 μg / mL, 0.5-10 μg / mL, 0.5-5 μg / mL, for example, about 1, about 2, about 3, about 4, about 5 μg / mL;

[0228] The aforementioned adjustment of the concentration of TNF family members includes, for example, lowering the concentration of BAFF (for example, adjusting it to 0.01-0.99 times, 0.05-0.9 times, 0.05-0.5 times, 0.05-0.2 times the original concentration, for example, 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 0.16, about 0.17, about 0.18, about 0.19, about 10.20, about 10.21, about 10.22, about 10.23, about 10.24, about 10.25, about 10.26, about 10.27, about 10.28, about 10.29, about 10.30, about 10.31, about 10.32, about 10.33, about 10.34, about 10.35, about 10.36, about 10.37, about 10.38, about 10.39, about 10.40, about 10.41, about 10.42, about 10.43, about 10.44, about 10.45, about 10.46, about 10.47, about 10.48, about 10.49, about 10.50, about 10.51, about 10.52, about 10.53, about 10.54, about 10.56, about 10.57, about 10.58, .2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9 times), increasing the concentration of APRIL (e.g., adjusting to 1.1-1000 times, 2-100 times, 5-50 times, 10-20 times, 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, about 50 times the original));

[0229] The aforementioned detection of B cell differentiation includes, for example, detection of plasma cells (CD27 + CD38 + )quantity;

[0230] The aforementioned test sample is an immunomodulator (eg, an immunostimulatory agent, an immunosuppressive agent) or a B cell differentiation regulator, such as a TNF pathway regulator (eg, a BAFF, APRIL pathway agonist or inhibitor).

[0231] In some embodiments, a method for screening, preparing or producing an immunomodulator is provided, comprising:

[0232] Step 1) administering CpG (e.g., CpG-A or CpG-B) or R848 or LPS, and IFNα to B cells;

[0233] Step 2) removing CpG (e.g., CpG-A or CpG-B) or R848 or LPS, and IFNα on day M;

[0234] Step 3) adding interleukins IL-6, IL-10 and / or IL-21;

[0235] Optionally, in step 4), BAFF and APRIL are added;

[0236] Optionally, step 5) adjusting the concentrations of BAFF and APRIL;

[0237] Optionally, step 6) adding a sample to be tested to detect B cell differentiation;

[0238] Optionally, in step 7), a test sample capable of activating or inhibiting B cell differentiation is selected as an immunomodulator;

[0239] Wherein, M and N are integers ranging from 1 to 30 respectively.

[0240] In some embodiments, a method for screening, preparing or producing an immunomodulator is provided, comprising:

[0241] Step 1) administering CpG-B and IFNα to B cells;

[0242] Step 2) removing CpG-B and IFNα on day M;

[0243] Step 3) adding interleukins IL-6, IL-10 and IL-21;

[0244] Optionally, step 4) adding BAFF and APRIL;

[0245] Optionally, step 5) reducing the BAFF concentration and increasing the APRIL concentration;

[0246] Optionally, step 6) adding a sample to be tested to detect B cell differentiation;

[0247] Optionally, in step 7), a test sample capable of activating or inhibiting B cell differentiation is selected as an immunomodulator;

[0248] Wherein, M and N are integers of 1-20 respectively, M is 3, 4, 5 or 6, and N is 2, 3 or 4.

[0249] In the aforementioned embodiments, the concentration of CpG-B is selected from 0.1-100 μg / mL, 0.2-20 μg / mL, 0.5-10 μg / mL, 0.5-5 μg / mL, 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, about 5 μg / mL;

[0250] The concentration of CpG-A or LPS is 0.01-10 μM, 0.05-5 μM, 0.1-2 μM, 0.2-1 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, about 3 μM;

[0251] R484 concentration is 0.1-100 μg / mL, 0.2-20 μg / mL, 0.5-10 μg / mL, 0.5-5 μg / mL, such as 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, about 4 μg / mL;

[0252] The concentration of IFN (e.g., IFNα) is 1-10,000 U / mL, 50-1,000 U / mL, 100-500 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, about 1,000 U / mL;

[0253] The concentration of interleukins (e.g., IL-3, IL-4, IL-6, IL-10, IL-21) is 0.1-1000 ng / mL, 1-500 ng / mL, 5-200 ng / mL, respectively, 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, about 200 ng / mL;

[0254] The concentration of the TFN pathway modulator (e.g., BAFF, APRIL) is 1-10,000 ng / mL, 10-5,000 ng / mL, 50-2,000 ng / mL, 20-200 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, about 1,000 ng / mL.

[0255] In some embodiments, a method for screening, preparing or producing an immunomodulator is provided, comprising:

[0256] Step 1) On day 0, administer 0.5-10 μg / mL CpG-B and 50-1000 U / mL IFNα to B cells;

[0257] Step 2) Remove CpG-B and IFNα on days 2-8;

[0258] Step 3) adding 2-100 ng / mL IL-6, 10-1000 ng / mL IL-10, and 2-30 ng / mL IL-21;

[0259] Optionally, in step 4), 10-2000 ng / mL BAFF and 10-2000 ng / mL APRIL are added and stimulated for another 2-5 days;

[0260] Optionally, step 5) reducing the BAFF concentration and increasing the APRIL concentration;

[0261] Optionally, in step 6), a test sample is added on days 0-21 to detect B cell differentiation;

[0262] Optionally, in step 7), a test sample capable of activating or inhibiting B cell differentiation is selected as an immunomodulator.

[0263] In some embodiments, a method for screening, preparing or producing an immunomodulator is provided, comprising:

[0264] Step 1) On day 0, administer 1-5 μg / mL CpG-B and 100-500 U / mL IFNα to B cells;

[0265] Step 2) Remove CpG-B and IFNα on days 3-5;

[0266] Step 3) adding 5-50 ng / mL IL-6, 100-500 ng / mL IL-10, and 5-20 ng / mL (or 5-21, 5-22, 5-23, 5-24 ng / mL) IL-21;

[0267] Step 4) Add 200-1000 ng / mL BAFF and 20-200 ng / mL APRIL and stimulate for another 2-4 days;

[0268] Step 5) reducing the BAFF concentration to 20-200 ng / mL and increasing the APRIL concentration to 200-1000 ng / mL;

[0269] Step 6) adding the test sample at a concentration gradient from day 0 to day 14 to detect B cell differentiation;

[0270] Step 7) Select a test sample that can activate or inhibit B cell differentiation as an immunomodulator.

[0271] In some embodiments, a method for screening, preparing or producing an immunomodulator is provided, comprising:

[0272] Step 1) administering approximately 2 μg / mL CpG-B and 250 U / mL IFNα to B cells;

[0273] Step 2) removal of CpG-B and IFNα on day 4 or 5;

[0274] Step 3) adding about 10 ng / mL IL-6, about 50 ng / mL IL-10, and about 15 ng / mL IL-21;

[0275] Step 4) Add about 500 ng / mL BAFF and about 50 ng / mL APRIL,

[0276] Step 5) After 3 days, adjust the BAFF concentration to approximately 50 ng / mL and the APRIL concentration to approximately 500 ng / mL;

[0277] Step 6) adding the test sample at a concentration gradient from day 0 to day 14 to detect B cell differentiation;

[0278] Step 7) Select a test sample that can activate or inhibit B cell differentiation as an immunomodulator.

[0279] In some embodiments, the present disclosure provides a cell model for screening immunomodulators, comprising: B cells induced to differentiate by any of the aforementioned methods.

[0280] In the schemes of the fourth and fifth aspects above, the immunomodulators include but are not limited to type I interferon pathway regulators (e.g., IFNAR1 signaling pathway regulators) and TNF pathway regulators (e.g., BAFF, ARRIL pathway regulators); the IFNAR1 regulators, BAFF and / or ARRIL pathway regulators include but are not limited to antibodies or antigen-binding fragments thereof, small molecule compounds, nucleic acids, aptamers, receptors, ligands, and any molecules comprising the antibodies or antigen-binding fragments thereof, small molecule compounds, nucleic acids, aptamers, receptors, ligands. Exemplary IFNAR1 regulators include but are not limited to Anifrolumab and other anti-IFNAR1 antibodies disclosed herein, and fusion proteins comprising the anti-IFNAR1 antibodies (e.g., fusion proteins comprising TACI polypeptides and BCMA polypeptides and anti-IFNAR1 antibodies, fusion proteins comprising TACI polypeptides and anti-IFNAR1 antibodies, fusion proteins comprising BCMA polypeptides and anti-IFNAR1 antibodies); and fusion proteins comprising TACI polypeptides and BCMA polypeptides disclosed herein.

[0281] The immunomodulators (e.g., BAFF and / or ARRIL pathway regulators) screened, prepared, and produced using the methods provided herein include, but are not limited to, TACI, Telitacicept, and the following TACI polypeptides, BCMA polypeptides, and fusion proteins comprising the TACI polypeptides and / or BCMA polypeptides provided herein (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, a fusion protein comprising a BCMA polypeptide and an anti-IFNAR1 antibody, and a fusion protein comprising a TACI polypeptide and a BCMA polypeptide).

[0282] For example, 1x10 5 The B cell preparation method comprises isolating B cells from PBMCs and culturing them in basal medium containing 1640 microliters of GlutaMAX supplemented with 50 μM β-mercaptoethanol, MEM non-essential amino acids (glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine), and sodium pyruvate.

[0283] In a sixth aspect, the present disclosure further provides a pharmaceutical composition comprising an immunomodulator obtained by screening using the aforementioned method, and one or more pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the amount of the immunomodulator in the pharmaceutical composition is 0.1-2000 mg, and in some specific embodiments, 1-1000 mg.

[0284] Furthermore, the present disclosure also provides the use of the immunomodulator obtained by the aforementioned method for preparing a pharmaceutical composition. Furthermore, the present disclosure also provides a method for producing or preparing a pharmaceutical composition, comprising using the immunomodulator obtained by the aforementioned method. The method for producing or preparing a pharmaceutical composition comprises the step of mixing any of the aforementioned immunomodulators with one or more pharmaceutically acceptable carriers, diluents or excipients; or, comprises the step of preparing or producing any of the aforementioned immunomodulators, and the step of mixing the obtained immunomodulator with one or more pharmaceutically acceptable carriers, diluents or excipients.

[0285] In a seventh aspect, the present disclosure provides a method for using the aforementioned immunomodulator or pharmaceutical composition to treat or improve a disease or condition, the method comprising administering the aforementioned immunomodulator or pharmaceutical composition to a subject in need thereof;

[0286] In some embodiments, the disease or condition is a B cell disorder or an autoimmune disease;

[0287] In some embodiments, the B cell disorder or autoimmune disease is a disease or condition associated with TACI and / or BCMA expression;

[0288] In some embodiments, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, insulin-dependent diabetes mellitus, Crohn's disease, rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, and psoriatic arthritis; and the B cell disorder is selected from the group consisting of tumors, chronic leukocytic leukemia, multiple myeloma, non-Hodgkin's lymphoma, post-transplant lymphoproliferative disease, and light chain gammaglobulinopathy.

[0289] In some embodiments, methods for preparing the aforementioned immunomodulators are provided. These immunomodulators can be prepared, without limitation, by conventional methods known in the art. For example, the antibody or antigen-binding fragment is expressed in a host cell, and the fusion protein is isolated from the host cell. Optionally, a purification step may be included. Optionally, a filtration or concentration step may also be included. Soluble mixtures and polymers may also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product must be immediately frozen, such as at -70°C, or lyophilized.

[0290] Furthermore, the present disclosure provides a novel fusion protein containing a TACI polypeptide, its encoding nucleic acid, vector, host cell, pharmaceutical composition, method for treating or ameliorating a disease (e.g., B cell disorder or autoimmune disease), and pharmaceutical use thereof.

[0291] Fusion protein

[0292] The present disclosure provides a fusion protein, wherein:

[0293] (1) comprising a TACI polypeptide;

[0294] (2) containing BCMA polypeptide;

[0295] (3) comprising a TACI polypeptide and a BCMA polypeptide;

[0296] (4) comprising a TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody);

[0297] (5) comprising a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody); or

[0298] (6) comprising a TACI polypeptide, a BCMA polypeptide, and an antibody (e.g., an anti-IFNAR1 antibody).

[0299] About TACI peptide in fusion protein:

[0300] In some embodiments, the TACI polypeptide comprises CRD1 and / or CRD2 of the TACI extracellular region, for example, comprises CRD2 of the TACI extracellular region.

[0301] In some embodiments, the TACI polypeptide comprises the following amino acid sequence:

[0302] (1) SEQ ID NO: 1 or an amino acid sequence having at least 90% sequence identity thereto;

[0303] (2) amino acid residues 33-67 of SEQ ID NO: 1;

[0304] (3) amino acid residues 70-104 of SEQ ID NO: 1 or a variant thereof;

[0305] (4) amino acid residues 30-110, 69-111, 69-112, 13-118, or 33-104 of SEQ ID NO: 1, or a variant thereof; or

[0306] (5) Amino acid residues 68-105, 68-106, 68-107 or 68-108 of SEQ ID NO: 1 or a variant thereof.

[0307] In some embodiments, the variant has one or more amino acid mutations selected from positions 69, 72, 73, 77, 85, 102, and 103.

[0308] In some embodiments, the variant has one or more amino acid substitutions selected from 69T or 69R, 72S, 73E or 73Q, 77E, 85T or 85A, 102A or 102R, 103Y.

[0309] In some specific embodiments, the variant has an amino acid substitution or combination of substitutions selected from any of the following: 69T; 72S; 73E; 73Q; 77E; 69R / 85T; 69R / 85A; 102A; 69R / 85T / 102R; 73E / 77E; 72S / 73E / 77E; 69T / 102A; 69T / 103Y; 69T / 102A, 103Y; 69T / 73E / 77E / 102A.

[0310] In some specific embodiments, single amino acid substitutions are linked by " / " to represent combined mutations at multiple given positions. For example, a combined mutation at positions 69T and 102A can be represented as: 69T / 102A.

[0311] In some specific embodiments, the variant has an amino acid substitution combination of 69T / 73E / 77E / 102A, for example, an amino acid substitution combination of L69T / K73E / K77E / Y102A.

[0312] The site of the amino acid mutation is the amino acid residue site numbered relative to the natural sequence of the TACI extracellular region (SEQ ID NO: 1), for example, "the variant has an amino acid mutation selected from position 69" means that the amino acid residue corresponding to position 69 of SEQ ID NO: 1 has a mutation.

[0313] In some embodiments, the amino acid sequence of the TACI polypeptide comprises an amino acid sequence selected from any one of SEQ ID NOs: 1, 2, 6-29.

[0314] In the present disclosure, "at least 90% (sequence) identity" encompasses at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity; "at least 80% (sequence) identity" encompasses at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (sequence) identity.

[0315] Regarding the BCMA peptide in the fusion protein:

[0316] In some embodiments, the BCMA polypeptide comprises the extracellular region of BCMA CRD1.

[0317] In some embodiments, the BCMA polypeptide comprises amino acid residues 7-41 of SEQ ID NO:30.

[0318] In some embodiments, the BCMA polypeptide comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 30, 67, and 68, or an amino acid sequence having at least 90% sequence identity thereto.

[0319] Regarding antibodies in fusion proteins:

[0320] In some embodiments, the antibody in the fusion protein is an anti-IFNAR1 antibody.

[0321] 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 with amino acid sequences as shown in SEQ ID NOs: 45-47, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 48-50, respectively.

[0322] In some embodiments, the amino acid sequence of the heavy chain variable region of the anti-IFNAR1 antibody is as shown in SEQ ID NO: 43 or has at least 90% sequence identity thereto, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 44 or has at least 90% sequence identity thereto.

[0323] In some embodiments, the amino acid sequence of the heavy chain of the anti-IFNAR1 antibody is as shown in SEQ ID NO: 55 or has at least 80% sequence identity thereto, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 54 or has at least 80% sequence identity thereto.

[0324] In some embodiments, the amino acid sequence of the heavy chain of the anti-IFNAR1 antibody is as shown in SEQ ID NO: 61 or has at least 80% sequence identity thereto, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 54 or has at least 80% sequence identity thereto.

[0325] In some embodiments, the present disclosure introduces Anifrolumab, sifalimumab, and the entire text of WO20062002177A, WO2009100309A (e.g., 3F11, 4G5, 11E2, 9D4), WO2020156474A (e.g., 7G4, 10C5, etc.), WO2020057541A (e.g., 8G11H and 485G1 0H), CN201610634601.7 (e.g., H19B7+L16C11, H19B7+L8C3, H15D10+L16C11, H15D10+L8C3 or anti-IFNAR1-C1), CN201510685200.X, and the anti-IFNAR1 antibodies or antigen-binding fragments thereof in WO2012162367A as the anti-IFNAR1 antibodies of the present disclosure.

[0326] About fusion proteins:

[0327] 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.

[0328] In some embodiments, the fusion protein further comprises an immunoglobulin heavy chain constant region (Fc region). In some specific embodiments, the Fc region comprises two subunits capable of associating. In some embodiments, the two subunits are the same or different first subunit and second subunit. In some embodiments, the Fc region is the Fc region of IgG, IgA, IgM, or IgD; in some embodiments, the Fc region is the Fc region of human IgG1, IgG2, IgG3, or IgG4, such as the Fc region of human IgG1, and for example, the Fc region shown in SEQ ID NO: 3.

[0329] In some embodiments, the Fc region in the fusion protein comprises one or more amino acid substitutions compared to the wild-type Fc region, and the amino acid substitutions can reduce its binding to the Fc receptor (FcR); in some embodiments, the amino acid substitutions can reduce its binding to the Fcγ receptor (FcγR); in some embodiments, the Fc region has a YTE mutation (M252Y, S254T and T256E), S228P, L234F, L235E, L234F / L235E, L234A / L235A or L234F / L235E / P331S mutation, and the mutation sites are numbered according to the EU index.

[0330] In some embodiments, the Fc region in the fusion protein comprises a first subunit and a second subunit that can associate with each other, and the first subunit and the second subunit have one or more amino acid substitutions that reduce homodimerization. In some embodiments, the first subunit has a protrusion structure according to the pestle-and-mortar technique, and the second subunit has a hole structure according to the pestle-and-mortar technique, or the first subunit has a hole structure according to the pestle-and-mortar technique, and the second subunit has a protrusion structure according to the pestle-and-mortar technique. In some embodiments, the amino acid residue substitutions of the first subunit include one or more amino acid substitutions in the sites selected from 354, 356, 358, and 366, and the amino acid residues of the second subunit include one or more amino acid substitutions in the sites selected from 349, 356, 358, 366, 368, and 407. In some embodiments, the first subunit includes one or more amino acid substitutions selected from 354C, 356E, 358M, and 366W, and the second subunit includes one or more amino acid substitutions selected from 349C, 356E, 358M, 366S, 368A, and 407V. In some embodiments, the first subunit comprises amino acid substitutions of 354C, 356E, 358M, and 366W, and the second subunit comprises amino acid substitutions of 349C, 356E, 358M, 366S, 368A, and 407V.

[0331] Regarding the fusion protein comprising the TACI polypeptide and the BCMA polypeptide:

[0332] 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.

[0333] In some embodiments, the fusion protein comprises one or more (e.g., 2, 3, 4, 5, 6) TACI polypeptides and one or more (e.g., 2, 3, 4, 5, 6) BCMA polypeptides. In some embodiments, the fusion protein comprises 1 TACI polypeptide and 1 BCMA polypeptide, or 2 TACI polypeptides and 2 BCMA polypeptides, or 3 TACI polypeptides and 3 BCMA polypeptides, or 4 TACI polypeptides and 4 BCMA polypeptides, or 2 TACI polypeptides and 1 BCMA polypeptide, or 1 TACI polypeptide and 2 BCMA polypeptides, or 3 TACI polypeptides and 1 BCMA polypeptide, or 1 TACI polypeptide and 3 BCMA polypeptides; when comprising 2 or more TACI polypeptides, the TACI polypeptides may be TACI polypeptides with the same or different sequences as those provided herein; when comprising 2 or more BCMA polypeptides, the BCMA polypeptides may be BCMA polypeptides with the same or different sequences as those provided herein.

[0334] In some embodiments, in the fusion protein, the TACI polypeptide and a subunit of the Fc region are linked in any order. Optionally, the TACI polypeptide is linked to the first or second subunit of the Fc region via a linker or directly. In some embodiments, as in any of the preceding fusion proteins, the BCMA polypeptide and a subunit of the Fc region are linked in any order. Optionally, the BCMA polypeptide is linked to the first or second subunit of the Fc region via a linker or directly. In some embodiments, the C-terminus of the TACI polypeptide is linked to the N-terminus of the first or second subunit of the Fc region via a linker or directly; or the N-terminus of the TACI polypeptide is linked to the C-terminus of the first or second subunit of the Fc region via a linker or directly. In some embodiments, the C-terminus of the BCMA polypeptide is linked to the N-terminus of the first or second subunit of the Fc region via a linker or directly; or the N-terminus of the BCMA polypeptide is linked to the C-terminus of the first or second subunit of the Fc region via a linker or directly.

[0335] In some embodiments, the fusion protein containing a TACI polypeptide and a BCMA polypeptide comprises a polypeptide chain as shown in any one of the following (I)-(VIII):

[0336] (I) [TACI polypeptide]-[Linker 1]a-[BCMA polypeptide]-[Linker 2]b-[Fc region]e;

[0337] (II) [BCMA polypeptide]-[Linker 1]a-[TACI polypeptide]-[Linker 2]b-[Fc region]e;

[0338] (III) [Fc region] e-[Linker 3] c-[TACI polypeptide]-[Linker 4] d-[BCMA polypeptide];

[0339] (IV) [Fc region] e-[Linker 3] c-[BCMA polypeptide]-[Linker 4] d-[TACI polypeptide];

[0340] (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];

[0341] (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];

[0342] (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];

[0343] (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];

[0344] wherein - represents a peptide bond, a linker is a polypeptide capable of achieving a linking function, and linker 1, linker 2, linker 3, and linker 4 may be the same or different; TACI polypeptide 1 and TACI polypeptide 2 are selected from the TACI polypeptides provided herein, and may be the same or different; BCMA polypeptide 1 and BCMA polypeptide 2 are selected from the BCMA polypeptides provided herein, and may be the same or different; a, b, c, d, and e are each independently 0 or 1, for example, in scheme (I) or (II), a and b are both 1, or a is 1 and b is 0, or a is 0 and b is 1, and e is 0 or 1; For example, in Scheme (III) or (IV), c and d are all 1, or c is 1 and d is 0, or c is 0 and d is 1, and e is 0 or 1; for example, in Scheme (V), (VI), (VII) or (VIII), a, b, c, d are all 1, or a, b, c are all 1 and d is 0, or a, b are 1 and c, d are 0, or a is 1 and b, c, d are all 0, or a is 0 and b, c, d are all 1, or a, b are 0 and c, d are 1, or a, b, c are 0 and d is 1, or a, c are 1 and b, d are 0, or a, c are 0 and b, d are 1, and e is 0 or 1.

[0345] In some embodiments, the linker is (G m S n ) h or (GGNGT) h (SEQ ID NO: 75) or (YGNGT) h (SEQ ID NO: 76) or (EPKSS) h (SEQ ID NO: 77), wherein m and n are each independently selected from an integer of 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and h is independently selected from an integer of 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, the linker has a sequence such as (G4S) y (SEQ ID NO: 78), wherein y is independently selected from an integer of 1-6 (e.g., y is 1, 2, 3, 4, 5, or 6). For example, the linker is (G x S) y A linker, wherein x is selected from an integer of 1-5, and y is selected from an integer of 1-6, such as a linker as shown in any one of SEQ ID NOs: 39-41. For example, the linker has (G4S) y (SEQ ID NO: 78), wherein y is independently selected from integers of 1-6 (e.g., y is 1, 2, 3, 4, 5, 6).

[0346] In some embodiments, a fusion protein comprising a TACI polypeptide and a BCMA polypeptide is provided, comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 31-38, 69, and 70, or an amino acid sequence having at least 90% sequence identity thereto.

[0347] In some embodiments, the fusion protein is a multimer, such as a dimer (eg, a homodimer or a heterodimer).

[0348] In some embodiments, a fusion protein containing a TACI polypeptide and a BCMA polypeptide is provided, and the TACI polypeptide may be a variant, and the variant has 1 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations; in some embodiments, a fusion protein containing a TACI polypeptide and a BCMA polypeptide is provided, and the BCMA polypeptide may be a variant, and the variant has 1 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations; the amino acid mutations may be conservative replacements, substitutions or modifications, and / or deletions or additions that do not affect the function.

[0349] In some embodiments, a protein or molecule is provided that competes with the fusion protein containing a TACI polypeptide and a BCMA polypeptide, the TACI polypeptide, or the BCMA polypeptide for binding to BAFF and / or APRIL, or blocks the binding of the fusion protein containing a TACI polypeptide and a BCMA polypeptide, the TACI polypeptide, or the BCMA polypeptide to BAFF and / or APRIL.

[0350] Regarding the fusion protein comprising the TACI polypeptide and the anti-IFNAR1 antibody:

[0351] In some embodiments, the TACI polypeptide is any of the TACI polypeptides provided herein, and the anti-IFNAR1 antibody is any of the anti-IFNAR1 antibodies provided herein.

[0352] In some embodiments, the fusion protein comprising a TACI polypeptide and an anti-IFNAR1 antibody comprises one or more (e.g., 2, 3, 4, 5, 6) TACI polypeptides. In some embodiments, the fusion protein comprising a TACI polypeptide and an anti-IFNAR1 antibody comprises one or more (e.g., 2, 3, 4) anti-IFNAR1 antibodies.

[0353] In some embodiments, the fusion protein containing the TACI polypeptide and the anti-IFNAR1 antibody comprises a polypeptide chain as shown in any one of the following (I)-(VIII):

[0354] (I) a first polypeptide chain, which, from N-terminus to C-terminus, consists of: [TACI polypeptide]-[Linker 1]a-[antibody heavy chain], and a second polypeptide chain, which is an antibody light chain;

[0355] (II) a first polypeptide chain, which comprises, from N-terminus to C-terminus: [antibody heavy chain]-[linker 2]b-[TACI polypeptide], and a second polypeptide chain, which is an antibody light chain;

[0356] (III) a first polypeptide chain, which is an antibody heavy chain, and a second polypeptide chain, which, from N-terminus to C-terminus, consists of: [TACI polypeptide]-[Linker 3]c-[Antibody light chain];

[0357] (IV) a first polypeptide chain, which is an antibody heavy chain, and a second polypeptide chain, which, from N-terminus to C-terminus, consists of: [antibody light chain]-[Linker 4]d-[TACI polypeptide];

[0358] (V) a first polypeptide chain comprising, from N-terminus to C-terminus, [TACI polypeptide 1]-[Linker 1]a-[Antibody heavy chain], and a second polypeptide chain comprising, from N-terminus to C-terminus, [Antibody light chain]-[Linker 4]d-[TACI polypeptide 2];

[0359] (VI) a first polypeptide chain comprising, from N-terminus to C-terminus, [antibody heavy chain]-[linker 2]b-[TACI polypeptide 1], and a second polypeptide chain comprising, from N-terminus to C-terminus, [TACI polypeptide 2]-[linker 3]c-[antibody light chain];

[0360] (VII) a first polypeptide chain, which, from N-terminus to C-terminus, consists of: [TACI polypeptide 1]-[Linker 1]a-[Antibody heavy chain]-[Linker 2]b-[TACI polypeptide 2], and a second polypeptide chain, which is an antibody light chain;

[0361] (VIII) a first polypeptide chain, which is an antibody heavy chain, and a second polypeptide chain, which, from N-terminus to C-terminus, consists of: [TACI polypeptide 1]-[Linker 3]c-[Antibody light chain]-[Linker 4]d-[TACI polypeptide 2];

[0362] Wherein, - represents a peptide bond, a linker is a polypeptide capable of achieving a linking function, Linker 1, Linker 2, Linker 3, and Linker 4 may be the same or different; TACI polypeptide 1 and TACI polypeptide 2 are selected from any of the aforementioned TACI polypeptides disclosed herein, TACI polypeptide 1 and TACI polypeptide 2 may be the same or different, and a, b, c, and d are each independently 0 or 1. In Scheme (I), a is 1 or 0. In Scheme (II), b is 1 or 0. In Scheme (III), c is 1 or 0. In Scheme (IV), d is 1 or 0. In scheme (V), a and d can be 0 or 1 at the same time, or a is 0 and d is 1, or a is 1 and d is 0; in scheme (VI), b and c can be 0 or 1 at the same time, or b is 0 and c is 1, or b is 1 and c is 0; in scheme (VII), a and b can be 0 or 1 at the same time, or a is 0 and b is 1, or a is 1 and b is 0; in scheme (VIII), c and d can be 0 or 1 at the same time, or c is 0 and d is 1, or c is 1 and d is 0; in the above schemes, e can independently be 0 or 1.

[0363] In some embodiments, the linker is (G m S n ) h or (GGNGT) h or (YGNGT) h or (EPKSS) h The amino acid sequence shown in FIG, wherein m and n are each independently selected from an integer of 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and h is independently selected from an integer of 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, the linker has a sequence such as (G4S) yThe amino acid sequence shown, y is independently selected from an integer of 1-6 (for example, y is 1, 2, 3, 4, 5, 6). For example, the linker is (G x S) y The linker, wherein x is selected from an integer of 1-5, and y is selected from an integer of 1-6, for example, the linker shown in any one of SEQ ID NOs: 39-41.

[0364] In some embodiments, a fusion protein comprising a TACI polypeptide and an anti-IFNAR1 antibody is provided, comprising a first polypeptide chain as shown in any one of SEQ ID NOs: 51-53, 62, or having at least 80% or at least 90% sequence identity thereto, and a second polypeptide chain as shown in SEQ ID NO: 54, or having at least 80% or at least 90% sequence identity thereto;

[0365] comprising a first polypeptide chain as shown in SEQ ID NO: 55, or having at least 80%, at least 90% sequence identity thereto, and a second polypeptide chain as shown in any one of SEQ ID NOs: 56-60, or having at least 80%, at least 90% sequence identity thereto; or

[0366] The invention also comprises a first polypeptide chain as shown in SEQ ID NO: 61 or having at least 80%, at least 90% sequence identity thereto, and a second polypeptide chain as shown in any one of SEQ ID NOs: 56-60 or having at least 80%, at least 90% sequence identity thereto.

[0367] In some embodiments, the aforementioned fusion protein comprises two identical first polypeptide chains and two identical second polypeptide chains.

[0368] In some embodiments, the present disclosure provides a fusion protein comprising a TACI polypeptide and an anti-IFNAR1 antibody, wherein the TACI polypeptide may be a variant having 1 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations; the amino acid mutations may be conservative replacements, substitutions or modifications, and / or deletions or additions that do not affect the function.

[0369] In some embodiments, a protein or molecule is provided, which competes with the aforementioned fusion protein containing a TACI polypeptide and an anti-IFNAR1 antibody for binding to BAFF and / or APRIL, or blocks its binding to BAFF and / or APRIL.

[0370] Regarding the fusion protein comprising TACI polypeptide, BCMA polypeptide and anti-IFNAR1 antibody:

[0371] In some embodiments, the TACI polypeptide is any TACI polypeptide provided herein, the BCMA polypeptide is any BCMA polypeptide provided herein, and the anti-IFNAR1 antibody is any anti-IFNAR1 antibody provided herein.

[0372] In some embodiments, the antibody fusion protein comprising a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody comprises one or more (e.g., 2, 3, 4, 5, 6) TACI polypeptides. In some embodiments, the antibody fusion protein comprising a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody comprises one or more (e.g., 2, 3, 4, 5, 6) BCMA polypeptides. In some embodiments, the antibody fusion protein comprising a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody comprises one or more (e.g., 2, 3, 4) anti-IFNAR1 antibodies. In some embodiments, the aforementioned antibody fusion protein comprising a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody comprises one aforementioned TACI polypeptide and one aforementioned BCMA polypeptide, or two aforementioned TACI polypeptides and two aforementioned BCMA polypeptides, or three aforementioned TACI polypeptides and three aforementioned BCMA polypeptides, or four aforementioned TACI polypeptides and four aforementioned BCMA polypeptides, or two aforementioned TACI polypeptides and one aforementioned BCMA polypeptide, or one aforementioned TACI polypeptide and two aforementioned BCMA polypeptides, or three aforementioned TACI polypeptides and one aforementioned BCMA polypeptide, or one aforementioned TACI polypeptide and three aforementioned BCMA polypeptides, and an anti-IFNAR1 antibody; when comprising two or more aforementioned TACI polypeptides, the TACI polypeptides may be TACI polypeptides having the same or different sequences as provided herein; when comprising two or more aforementioned BCMA polypeptides, the BCMA polypeptides may be BCMA polypeptides having the same or different sequences as provided herein.

[0373] In some embodiments, the fusion protein containing a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody as described in any of the preceding items comprises a polypeptide chain as shown in any one of the following (I)-(VIII):

[0374] (I) a first polypeptide chain, which consists, from N-terminus to C-terminus, of: [TACI polypeptide and BCMA polypeptide domains]-[Linker 1]a-[antibody heavy chain], and a second polypeptide chain, which is an antibody light chain;

[0375] (II) a first polypeptide chain, which comprises, from N-terminus to C-terminus: [antibody heavy chain]-[Linker 2]b-[TACI polypeptide and BCMA polypeptide domain], and a second polypeptide chain, which is an antibody light chain;

[0376] (III) a first polypeptide chain, which is an antibody heavy chain, and a second polypeptide chain, which, from N-terminus to C-terminus, consists of: [TACI polypeptide and BCMA polypeptide domains]-[Linker 3]c-[antibody light chain];

[0377] (IV) a first polypeptide chain, which is an antibody heavy chain, and a second polypeptide chain, which, from N-terminus to C-terminus, consists of: [antibody light chain]-[Linker 4]d-[TACI polypeptide and BCMA polypeptide domains];

[0378] (V) a first polypeptide chain comprising, from N-terminus to C-terminus, the following sequence: [TACI polypeptide and BCMA polypeptide domain 1]-[Linker 1]a-[antibody heavy chain], and a second polypeptide chain comprising, from N-terminus to C-terminus, the following sequence: [antibody light chain]-[Linker 4]d-[TACI polypeptide and BCMA polypeptide domain 2];

[0379] (VI) a first polypeptide chain comprising, from N-terminus to C-terminus, the following sequence: [antibody heavy chain]-[linker 2]b-[TACI polypeptide and BCMA polypeptide domain 1], and a second polypeptide chain comprising, from N-terminus to C-terminus, the following sequence: [TACI polypeptide and BCMA polypeptide domain 2]-[linker 3]c-[antibody light chain];

[0380] (VII) a first polypeptide chain, which, from N-terminus to C-terminus, consists of: [TACI polypeptide and BCMA polypeptide domain 1]-[Linker 1]a-[antibody heavy chain]-[Linker 2]b-[TACI polypeptide and BCMA polypeptide domain 2], and a second polypeptide chain, which is an antibody light chain;

[0381] (VIII) a first polypeptide chain, which is an antibody heavy chain, and a second polypeptide chain, which, from N-terminus to C-terminus, consists of: [TACI polypeptide and BCMA polypeptide domain 1]-[Linker 3]c-[Antibody light chain]-[Linker 4]d-[TACI polypeptide and BCMA polypeptide domain 2];

[0382] Wherein, the structures of "[TACI polypeptide and BCMA polypeptide domain]", "[TACI polypeptide and BCMA polypeptide domain 1]", and "[TACI polypeptide and BCMA polypeptide domain 2]" from N-terminus to C-terminus are: [TACI polypeptide]-[Linker 5]e-[BCMA polypeptide], or: [BCMA polypeptide]-[Linker 5]e-[TACI polypeptide], and the TACI in "TACI polypeptide and BCMA polypeptide domain 1" and "TACI polypeptide and BCMA polypeptide domain 2" may be the same or different, and the BCMA may be the same or different;

[0383] Wherein, - represents a peptide bond, a linker is a polypeptide capable of achieving a linking function, and linker 1, linker 2, linker 3, linker 4, and linker 5 may be the same or different; a, b, c, d, and e are each independently 0 or 1. a, b, c, and d are each independently 0 or 1; in scheme (I), a is 1 or 0. In scheme (II), b is 1 or 0. In scheme (III), c is 1 or 0. In scheme (IV), d is 1 or 0. In scheme (V), a and d can be 0 or 1 at the same time, or a is 0 and d is 1, or a is 1 and d is 0; in scheme (VI), b and c can be 0 or 1 at the same time, or b is 0 and c is 1, or b is 1 and c is 0; in scheme (VII), a and b can be 0 or 1 at the same time, or a is 0 and b is 1, or a is 1 and b is 0; in scheme (VIII), c and d can be 0 or 1 at the same time, or c is 0 and d is 1, or c is 1 and d is 0; in the above schemes, e can independently be 0 or 1.

[0384] In some embodiments, the linker is (G m S n ) h or (GGNGT) h or (YGNGT) h or (EPKSS) h The amino acid sequence shown in FIG, wherein m and n are each independently selected from an integer of 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and h is independently selected from an integer of 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, the linker has a sequence such as (G4S) y The amino acid sequence shown, y is independently selected from an integer of 1-6 (for example, y is 1, 2, 3, 4, 5, 6). For example, the linker is (G x S) y A linker, wherein x is selected from an integer of 1-5, and y is selected from an integer of 1-6, such as a linker as shown in any one of SEQ ID NOs: 39-41. For example, the linker has (G4S) y In the amino acid sequence shown, y is independently selected from an integer of 1-6 (eg, y is 1, 2, 3, 4, 5, 6).

[0385] In some embodiments, a fusion protein comprising a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody is provided, comprising a first polypeptide chain as set forth in SEQ ID NO: 55, or having at least 80% or at least 90% sequence identity thereto, and a second polypeptide chain as set forth in SEQ ID NOs: 63-66, 71, or 72, or having at least 80% or at least 90% sequence identity thereto;

[0386] comprising a first polypeptide chain as shown in SEQ ID NO: 61, or having at least 80%, at least 90% sequence identity thereto, and a second polypeptide chain as shown in any one of SEQ ID NOs: 63-66, 71, 72, or having at least 80%, at least 90% sequence identity thereto; or

[0387] The invention also comprises a first polypeptide chain as shown in SEQ ID NO: 73 or 74, or having at least 80%, at least 90% sequence identity thereto, and a second polypeptide chain as shown in SEQ ID NO: 54, or having at least 80%, at least 90% sequence identity thereto.

[0388] In some embodiments, the aforementioned fusion protein comprises two identical first polypeptide chains and two identical second polypeptide chains.

[0389] In some embodiments, the present disclosure provides a fusion protein comprising a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody, wherein the TACI polypeptide may be a variant having 1 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations; the amino acid mutations may be conservative replacements, substitutions, or modifications, and / or deletions or additions that do not affect the function.

[0390] In some embodiments, a protein or molecule is provided that competes with the aforementioned fusion protein containing a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody for binding to BAFF and / or APRIL, or blocks its binding to BAFF and / or APRIL.

[0391] Regarding the aforementioned fusion protein:

[0392] 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:

[0393] (a) Activity of binding to BAFF;

[0394] (b) activity binding to TALL-2 / APRIL;

[0395] (c) reducing the concentration of serum immunoglobulins (such as IgE or IgM);

[0396] (d) decreased spleen weight;

[0397] (e) inhibiting or blocking the BAFF / APRIL pathway;

[0398] (f) Reduce the number of B cells.

[0399] In some embodiments, the protein comprising a TACI polypeptide provided by the present disclosure (e.g., TACI-Fc) has one or more of the following functional activities:

[0400] a. TACI polypeptide fragments are not prone to cleavage; in some embodiments, the cleavage can be detected by mass spectrometry, such as the TACI cleavage analysis method in Example 2;

[0401] b. Blocking BAFF from binding to BAFF-R; In some embodiments, TACI-Fc blocks IC binding of BAFF to BAFF-R 50 value is less than 23 nM, less than 10 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM or less; the IC 50 The value is detected by ELISA method; in some embodiments, the IC 50 The value test method is shown in Example 1;

[0402] c. Ability to bind to BAFF; In some embodiments, the TACI-Fc binds to the EC of BAFF 50 The value is less than 5 nM, 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or less, the EC 50 The value is detected by ELISA method, such as the ELISA binding experiment (coated antigen protein) in Example 2;

[0403] d. Inhibit BAFF-induced B cell proliferation; In some embodiments, TACI-Fc inhibits BAFF-induced B cell proliferation IC 50 The IC value is less than 0.3 nM, less than 0.2 nM, less than 0.1 nM, 0.06 nM, less than 0.05 nM, less than 0.01 nM or less. 50 The value is detected by ELISA method, such as the ELISA experiment in Example 3;

[0404] e. Inhibit APRIL-induced B cell proliferation; In some embodiments, TACI-Fc inhibits APRIL-induced B cell proliferation IC 50 value is less than 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, 0.2 nM, less than 0.1 nM, less than 0.05 or less; the IC 50 The values ​​were detected by ELISA method;

[0405] f. High affinity binding to human BAFF and / or human APRIL; in some embodiments, the affinity is detected by Biacore method; in some embodiments, the KD value of the TACI-Fc fusion protein binding to human BAFF is less than 1E-10M, less than 9E-11M, 8E-11M or less; in some embodiments, the KD value of the TACI-Fc fusion protein binding to human APRIL is less than 2.3E-11M, less than 2.0E-11M, 1.9E-11M, 1.3E-11M or less;

[0406] g. Good in vivo pharmacokinetics; in some embodiments, the half-life of TACI-Fc in rats is greater than 4 days; and / or

[0407] h. Good stability; in some embodiments, the purity of the TACI-Fc fusion protein can be maintained above 94% (SEC%) after being stored at a constant temperature of 40°C for four weeks; in some embodiments, the pI value of the TACI-Fc is less than 9, less than 8, less than 7, less than 6, or even less, as determined by "18cProt / TrEMBL" system analysis.

[0408] In some embodiments, the fusion protein containing a TACI polypeptide and a BCMA polypeptide provided by the present disclosure has one or more of the following functional activities:

[0409] a. TACI polypeptide fragments are not prone to cleavage; in some embodiments, the cleavage can be detected by mass spectrometry, such as the TACI cleavage analysis method in Example 2;

[0410] b. Ability to bind to BAFF; In some embodiments, the fusion protein binds to BAFF EC 50 The value is less than 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or less, the EC 50 The value is detected by ELISA method, such as the ELISA experiment in Example 5;

[0411] c. Ability to bind to APRIL; In some embodiments, the fusion protein binds to the EC of APRIL 50 The value is less than 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or less, the EC 50 The value is detected by ELISA method, such as the ELISA experiment in Example 5;

[0412] d. Good pharmacokinetic properties in vivo;

[0413] e.Good stability.

[0414] In some embodiments, the antibody fusion protein provided by the present disclosure comprising a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody has one or more of the following functional activities:

[0415] a. TACI polypeptide fragments that are not susceptible to cleavage; in some embodiments, the cleavage can be detected by mass spectrometry, such as the TACI cleavage analysis method in Example 2;

[0416] b. Inhibition of type I interferon activity; in some embodiments, the fusion protein has an inhibition rate on IFN activity that is consistent with or similar to that of an anti-IFNAR1 antibody (e.g., Anifrolumab), and the inhibition rate is detected in vitro by an IFNα / β reporter gene method, such as the experiment in Example 10; the inhibition rate is detected in vivo by detecting the mRNA expression of IFNα downstream genes ISG in PBMCs, such as the experiment in Example 21;

[0417] c. Ability to bind to BAFF; In some embodiments, the fusion protein binds to the EC of BAFF 50 The value is less than 1.5 nM, 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or less, the EC 50 The value is detected by ELISA method, such as the ELISA experiment in Example 11;

[0418] d. Ability to bind to APRIL; In some embodiments, the fusion protein binds to the EC of APRIL 50 The value is less than 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or less, the EC 50 The value is detected by ELISA method, such as the ELISA experiment in Example 11;

[0419] e. Inhibit IFNα secretion; In some embodiments, the fusion protein has an inhibitory ability on IFNα cytokine secretion by plasmacytoid dendritic cells (pDCs) that is consistent or similar to that of an anti-IFNAR1 antibody (e.g., Anifrolumab), and the inhibitory ability is detected using, for example, the detection method in Example 15; In some embodiments, the fusion protein has an ability to inhibit IFN-α-induced B cell differentiation into plasma cells in vitro that is consistent or similar to that of an anti-IFNAR1 antibody (e.g., Anifrolumab), and the inhibitory ability is detected using, for example, the detection method in Example 16;

[0420] f. Inhibiting the differentiation of plasma cells and inhibiting BAFF-induced B cell proliferation; in some embodiments, the fusion protein inhibits BAFF-induced B cell proliferation by an inhibition rate of at least 50%, at least 40%, at least 30%, at least 20%, or at least 10%, wherein the inhibition rate is the ratio of the number of B cells after addition of the antibody fusion protein to the number of B cells after addition of the same concentration of IgG1, and is detected by FACS, such as the method in Example 17;

[0421] g. Inhibiting APRIL-induced B cell proliferation; in some embodiments, the fusion protein inhibits APRIL-induced B cell proliferation by an inhibition rate of at least 50%, at least 40%, at least 30%, at least 20%, or at least 10%, wherein the inhibition rate is the ratio of the number of B cells after addition of the antibody fusion protein to the number of B cells after addition of the same concentration of IgG1, and the inhibition rate is detected by FACS, for example, as described in Example 18;

[0422] h. inhibiting BAFF- and APRIL-induced plasma cell production; in some embodiments, the fusion protein inhibits BAFF- and APRIL-induced plasma cell production by at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, or at least 20%, wherein the inhibition rate is the ratio of the number of B cells after addition of the antibody fusion protein to the number of B cells after addition of the same concentration of IgG1, and the inhibition is detected by FACS, such as the method in Example 19;

[0423] i. Inhibits the production of IgA in vivo; in some embodiments, the fusion protein inhibits the production of IgA in vivo by at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, or at least 30%, compared to an equivalent concentration of PBS, and the inhibition is detected by ELISA, such as the method in Example 22;

[0424] j. Good pharmacokinetic properties in vivo;

[0425] k.Good stability.

[0426] In addition, in some embodiments, a product or combination is provided, comprising any of the aforementioned fusion proteins containing a TACI polypeptide and a BCMA polypeptide as disclosed herein, and further comprising an antibody. In some embodiments, a product or combination is provided, comprising any of the aforementioned TACI polypeptides as disclosed herein, and further comprising an antibody. In some embodiments, the antibody is an anti-IFNAR1 antibody, such as anifrolumab.

[0427] In some embodiments, a complex is provided comprising any of the aforementioned TACI polypeptides disclosed herein and any of the aforementioned BCMA polypeptides disclosed herein. Optionally, the TACI polypeptide and BCMA polypeptide may be operably linked directly or via any of the aforementioned linkers disclosed herein, or may be unlinked.

[0428] In some embodiments, a complex is provided, comprising any of the aforementioned TACI polypeptides disclosed herein and any of the aforementioned BCMA polypeptides disclosed herein; or any of the aforementioned TACI polypeptides disclosed herein and any of the aforementioned anti-IFNAR1 antibodies disclosed herein; or any of the aforementioned BCMA polypeptides disclosed herein and any of the aforementioned anti-IFNAR1 antibodies disclosed herein; or any of the aforementioned TACI polypeptides disclosed herein and any of the aforementioned anti-IFNAR1 antibodies disclosed herein and any of the aforementioned BCMA polypeptides disclosed herein. Optionally, the TACI polypeptide, BCMA polypeptide, and anti-IFNAR1 antibody may be operably linked directly or via a linker disclosed herein, or may be unlinked.

[0429] Polynucleotides and vectors

[0430] The present disclosure provides (isolated) polynucleotides encoding any of the aforementioned TACI polypeptides, BCMA polypeptides, fusion proteins comprising a TACI polypeptide and a BCMA polypeptide, fusion proteins comprising a TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), fusion proteins comprising a TACI polypeptide and a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), products, combinations, or complexes. The polynucleotides may be DNA or RNA (e.g., mRNA).

[0431] The nucleic acids disclosed herein may also be in the form of, present in, and / or part of, a vector, such as a plasmid, cosmid, YAC, or viral vector. The vector may particularly be an expression vector, i.e., a vector that provides for expression of a CD40-binding molecule in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). The expression vector typically comprises at least one nucleic acid disclosed herein, operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). The selection of such elements and their sequences for expression in a particular host is within the skill of the art. Regulatory and other elements useful or necessary for expression of the TACI polypeptides, BCMA polypeptides, fusion proteins comprising a TACI polypeptide and a BCMA polypeptide, fusion proteins comprising a TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), fusion proteins comprising a TACI polypeptide and a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), products, combinations, or complexes disclosed herein include, for example, promoters, enhancers, terminators, integration factors, selection markers, leader sequences, and reporter genes.

[0432] The nucleic acids of the present disclosure can be prepared or obtained by known means (eg, by automated DNA synthesis and / or recombinant DNA technology) based on the information of the amino acid sequence of the polypeptides of the present disclosure, and / or can be isolated from suitable natural sources.

[0433] host cells

[0434] The present disclosure provides recombinant host cells that express or are capable of expressing one or more TACI polypeptides, BCMA polypeptides, fusion proteins comprising a TACI polypeptide and a BCMA polypeptide, fusion proteins comprising a TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), fusion proteins comprising a TACI polypeptide and a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), products, combinations or complexes of the present disclosure, and / or contain a nucleic acid or vector of the present disclosure.

[0435] In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0436] Examples of bacterial cells include cells of gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0437] Exemplary fungal cells include cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or cells of species of the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0438] Examples of mammalian cells include HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0439] However, the present disclosure may also be used with amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins.

[0440] The cells of the present disclosure are incapable of developing into complete plants or animals.

[0441] Preparation method

[0442] The present disclosure provides a method for preparing a TACI polypeptide, a BCMA polypeptide, a fusion protein comprising a TACI polypeptide and a BCMA polypeptide, a fusion protein comprising a TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), a fusion protein comprising a TACI polypeptide and a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), a product, a combination, or a complex, comprising: expressing the aforementioned polypeptide, fusion protein, product, combination, or complex in a host cell as described above, and isolating the polypeptide, fusion protein, product, combination, or complex from the host cell. Optionally, a purification step may also be included. Optionally, a filtration or concentration step may also be included. Soluble mixtures and polymers may also be removed by conventional methods, such as molecular sieves and ion exchange. The obtained product must be immediately frozen, such as at -70°C, or lyophilized.

[0443] The polypeptide, fusion protein, product, combination or complex can be prepared and purified by conventional methods. For example, the cDNA sequence encoding the fusion protein can be cloned and recombined into an expression vector. The recombinant expression vector can stably transfect CHO cells. Mammalian expression systems can lead to glycosylation of the protein (for example, at the highly conserved N-terminus of the Fc region). The positive clones obtained are expanded in the culture medium of a bioreactor to produce the protein. The culture fluid that secretes the protein can be purified, collected, filtered and concentrated using conventional techniques. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. However, the polypeptide, fusion protein, product, combination or complex of the present disclosure can also be obtained by other methods of producing proteins known in the art, such as chemical synthesis, including solid phase or liquid phase synthesis.

[0444] Composition

[0445] The present disclosure provides compositions, e.g., pharmaceutical compositions, containing a therapeutically effective amount of a TACI polypeptide, a BCMA polypeptide, a fusion protein comprising a TACI polypeptide and a BCMA polypeptide, a fusion protein comprising a TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), a fusion protein comprising a TACI polypeptide and a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), a product, combination, complex, or polynucleotide, and one or more pharmaceutically acceptable excipients, diluents, or carriers.

[0446] In some embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of any of the aforementioned polypeptides, fusion proteins, products, combinations, complexes, or polynucleotides per unit dose. In some embodiments, the pharmaceutical composition may contain 0.1 to 2000 mg, and in some embodiments, 1 to 1000 mg, of any of the aforementioned polypeptides, fusion proteins, products, combinations, complexes, or polynucleotides per unit dose.

[0447] Reagent test kit

[0448] The present disclosure provides kits comprising the TACI polypeptides, BCMA polypeptides, fusion proteins comprising a TACI polypeptide and a BCMA polypeptide, fusion proteins comprising a TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), fusion proteins comprising a TACI polypeptide and a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), products, combinations, complexes, or polynucleotides of the present disclosure. In some embodiments, diagnostic reagents comprising the above-mentioned polynucleotides are also provided, as well as related diagnostic uses.

[0449] Methods for treating diseases and pharmaceutical uses

[0450] In some embodiments, the present disclosure provides a method for treating or ameliorating a disease or condition, comprising administering to a subject in need thereof any of the aforementioned TACI polypeptides, BCMA polypeptides, fusion proteins comprising a TACI polypeptide and a BCMA polypeptide, fusion proteins comprising a TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), fusion proteins comprising a TACI polypeptide and a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), products, combinations, complexes, polynucleotides, or pharmaceutical compositions. In some embodiments, the disease or condition is a disease or condition associated with TACI, BCMA, and / or IFN expression.

[0451] In some embodiments, methods or pharmaceutical uses for treating diseases or conditions by combining any of the aforementioned TACI polypeptides, BCMA polypeptides, and anti-IFNAR1 antibodies are provided. In some embodiments, methods or pharmaceutical uses for treating diseases or conditions by combining any of the aforementioned TACI polypeptides and anti-IFNAR1 antibodies are provided; in some specific embodiments, these methods further include use with a BCMA polypeptide. In some embodiments, methods or pharmaceutical uses for treating diseases or conditions by combining any of the aforementioned fusion proteins containing a BCMA polypeptide and an anti-IFNAR1 antibody are provided; in some specific embodiments, these methods further include use with a TACI polypeptide. For example, the anti-IFNAR1 antibody is anifrolumab.

[0452] In some embodiments, a method for treating or ameliorating a B cell disorder or an autoimmune disease is provided, comprising administering to a subject in need thereof a therapeutically effective amount of a TACI polypeptide, a BCMA polypeptide, a fusion protein comprising a TACI polypeptide and a BCMA polypeptide, a fusion protein comprising a TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), a fusion protein comprising a TACI polypeptide and a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), a product, a combination, a complex, a polynucleotide, or a pharmaceutical composition of the present disclosure.

[0453] In some embodiments, the present invention provides a TACI polypeptide, a BCMA polypeptide, a fusion protein comprising a TACI polypeptide and a BCMA polypeptide, a fusion protein comprising a TACI polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), a fusion protein comprising a TACI polypeptide and a BCMA polypeptide and an antibody (e.g., an anti-IFNAR1 antibody), a product, a combination, a complex, a polynucleotide, or a pharmaceutical composition for use in the preparation of a medicament for treating or preventing a disease.

[0454] In some embodiments, the disease or condition is a B cell disorder or an autoimmune disease.

[0455] In some embodiments, the disease or condition is associated with TACI expression, or BCMA expression, or IFN expression, or any two or three of TACI, BCMA, and IFN expression, such as overexpression or abnormal expression.

[0456] In some embodiments, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, insulin-dependent diabetes mellitus, Crohn's disease, rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, and psoriatic arthritis; in some embodiments, the B cell disorder is selected from the group consisting of cancer, chronic leukemia, multiple myeloma, non-Hodgkin's lymphoma, post-transplant lymphoproliferative disorder, and light chain gammopathy. In some embodiments, the autoimmune disease is systemic lupus erythematosus.

[0457] Definition of terms

[0458] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise explicitly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0459] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0460] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0461] Unless the context clearly requires otherwise, in the patent specification and claims, the words "comprising," "having," "including," and the like should be construed in the sense of "including but not limited to," rather than in an exclusive or exhaustive sense.

[0462] The term "cytokine" or "factor" is a general term for proteins released by one cell population that act as intercellular mediators on other cells. Examples of such cytokines include lymphokines, monokines, chemokines, and traditional polypeptide hormones. Exemplary cytokines include IL-2, IFN-γ, IL-6, TNFα, IL-17, and IL-5.

[0463] The TACI (transmembrane activator and calcium modulator and cyclophilinligand-interactor) described in the present disclosure is a membrane-bound receptor. Human TACI belongs to the TNFR superfamily and is a 293-amino acid polypeptide comprising an N-terminal region (amino acid residues 1-165, see SEQ ID NO: 1 of the present disclosure), a transmembrane region (amino acid residues 166-186), and an intracellular region (amino acid residues 187-293). The extracellular region contains two cysteine-rich pseudo-repeats (CRD1 and CRD2). The "CRD1" of TACI in the present disclosure encompasses wild-type human TACI CRD1 (e.g., amino acids 33-67 of SEQ ID NO: 1) and variants thereof. In the present disclosure, "CRD2" of TACI encompasses wild-type human TACI CRD2 (e.g., amino acids 70-104 or 69-104 of SEQ ID NO: 1) and variants thereof (including but not limited to variants of one or more amino acids at positions 69, 72, 73, 77, 85, 102, and 103 of SEQ ID NO: 1 of the present disclosure, such as variants set forth in SEQ ID NOs: 6-29). The CRD1, CRD2, and variants thereof of TACI are all capable of binding to APRIL and / or BAFF, for example, simultaneously binding to APRIL and BAFF. Methods for detecting such binding are well known in the art, for example, as provided in the Examples of the present disclosure.

[0464] The BCMA (B cell maturation antigen) described in the present disclosure is a membrane-bound receptor. The human BCMA protein is a 184-amino acid polypeptide, comprising an N-terminal region of the protein (amino acid residues 1-50, see amino acids 1-50 of SEQ ID NO: 30 of the present disclosure), a transmembrane region (amino acid residues 51-93) and an intracellular region (amino acid residues 94-184). The extracellular region of BCMA contains a CRD (or CRD1). The "CRD1" of BCMA in the present disclosure covers the CRD1 of wild-type human BCMA (e.g., amino acids 7-41 of SEQ ID NO: 30 of the present disclosure) and its variants. The CRD1 of BCMA or its variants are all capable of binding to APRIL and / or BAFF, for example, simultaneously binding to APRIL and BAFF, and the detection method of the binding is well known in the art, for example, as provided in the embodiments of the present disclosure.

[0465] In the present disclosure, “TACI extracellular region” and “TACI extracellular domain” are interchangeable, and “BCMA extracellular region” and “BCMA extracellular domain” are interchangeable.

[0466] The terms "interferon alpha", "IFNα", "IFNa", "IFNA" and "IFN alpha" are used interchangeably and shall refer to an IFNα protein encoded by a functional gene of the interferon alpha locus that has 75% or more sequence identity with IFNα1 (protein encoded by GenBank Accession No. NP_076918 or GenBank Accession No. NM_024013). Examples of IFNα subtypes include IFNα1, α2a, α2b, α4, α4bα5, α6, α7, α8, α10, α13, α14, α16, α17 and α21. "Interferon alpha" and "IFNα" shall include recombinant forms of the various IFNα subtypes, as well as naturally occurring preparations comprising IFNα proteins, such as leukocyte IFN and lymphoblastoid IFN.

[0467] The terms "interferon alpha receptor-1," "IFNAR1," "IFNAR-1," and "IFNAR-1 antigen" are used interchangeably and include variants, isoforms, germline homologs of human IFNAR-1, and analogs that share at least one common epitope with IFNAR-1. Thus, the human antibodies of the present invention may, in some embodiments, cross-react with IFNAR-1 from species other than human, or other proteins structurally related to human IFNAR-1 (e.g., human IFNAR-1 homologs). In other embodiments, the antibodies may be completely specific for human IFNAR-1 and exhibit no species or other types of cross-reactivity. The complete cDNA sequence of human IFNAR-1 has GenBank Accession No. NM_000629.

[0468] The term "and / or" is intended to include both "and" and "or". For example, the phrase "A, B and / or C" is intended to include each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0469] The term "antibody" is used in the broadest sense to encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. An antibody may refer to an immunoglobulin, a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and arrangement of the constant regions of immunoglobulins' heavy chains vary, resulting in different antigenicity. Consequently, immunoglobulins can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE, corresponding to μ, δ, γ, α, and ε chains, respectively. Within the same class, Igs can be further divided into subclasses based on differences in the amino acid composition of their hinge regions and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either kappa or lambda chains based on differences in their constant regions. Each of the five classes of Ig can have either kappa or lambda chains. The approximately 110 amino acids near the N-terminus of both the heavy and light chains of antibodies vary greatly in sequence and constitute the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region (C region). The variable region consists of three hypervariable regions (CDRs) and four framework regions (FRs) whose sequences are relatively conserved. These three hypervariable regions determine the specificity of the antibody and are also known as complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3. "Antibodies" in the present disclosure encompass "antigen-binding fragments", which include: single-chain antibodies (i.e., full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single-domain antibodies (e.g., VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody and epitope-binding fragments of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217).

[0470] For the determination or definition of CDRs, the definitive delineation of CDRs and the identification of residues comprising the binding site of the antibody can be accomplished by resolving the structure of the antibody and / or resolving the structure of the antibody-ligand complex. This can be accomplished by any of the various techniques known to those skilled in the art, such as X-ray crystallography. A variety of analytical methods can be used to identify CDRs, including but not limited to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definitions, and conformational definitions.

[0471] The Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8). The Chothia numbering system is similar to the Kabat numbering system, but takes into account the positions of certain structural loop regions. (See, e.g., Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83). The AbM numbering system uses an integrated suite of computer programs produced by the Oxford Molecular Group for modeling antibody structure (see, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd). The AbM numbering system uses a combination of knowledge databases and ab initio methods to model the tertiary structure of antibodies from primary sequences (see Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," PROTEINS, Structure, Function and Genetics Suppl., 3: 194-198). Contact definitions are based on analysis of available complex crystal structures (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 5: 732-45). In conformational definitions, CDR positions can be identified as residues that contribute to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166). Other CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened based on predictions or experimental results that a particular residue or group of residues does not significantly affect antigen binding. As used herein, CDR may refer to a CDR defined by any method known in the art (including a combination of methods). The correspondence between various numbering systems is well known to those skilled in the art, and is exemplified by the following Table 1.

[0472] Table 1. Relationships between CDR numbering systems

[0473] Unless otherwise indicated, the variable region and CDR sequences disclosed herein are numbered using the Kabat numbering system.

[0474] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of the antibody heavy chain, including native Fc regions and reconstructed Fc regions. In some embodiments, the Fc region comprises two identical or different subunits. In some embodiments, the Fc region of the human IgG heavy chain is defined as the amino acid residue at the Cys226 position or extending from Pro230 to its carboxyl terminus. Suitable Fc regions for the antibodies described herein include the Fc regions of human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. In some embodiments, the boundaries of the Fc region can also vary, such as the C-terminal lysine in the Fc region (residue 447 according to the EU numbering system) or the C-terminal glycine and lysine in the Fc region (residues 446 and 447 according to the EU numbering system) are deleted. Unless otherwise indicated, the numbering convention of the Fc region is the EU numbering system, also known as the EU index.

[0475] The terms "homology" or "identity" refer to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same nucleotide or amino acid monomer subunit, for example, if every position in two DNA molecules is occupied by the same nucleotide, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, a comparison is made when the two sequences are aligned to achieve the maximum percent homology.

[0476] The term "amino acid mutation" includes amino acid substitutions (also known as amino acid replacements), deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions, and modifications can be performed to achieve the final construct, as long as the final construct possesses the desired properties, such as reduced binding to Fc receptors. Amino acid sequence deletions and insertions include deletions and insertions at the amino and / or carboxyl termini of the polypeptide chain. Specific amino acid mutations can be amino acid substitutions. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid having different structural and / or chemical properties. Amino acid substitutions include replacement with non-naturally occurring amino acids or with derivatives of the 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like. It is anticipated that methods other than genetic engineering to alter amino acid side chain groups, such as chemical modification, may also be useful. Various names may be used herein to refer to the same amino acid mutation. Herein, the amino acid residue at a specific position can be represented by position + amino acid residue, for example, 366W means that the amino acid residue at position 366 is W. T366W means that the amino acid residue at position 366 has mutated from T to W.

[0477] The term "conservative substitution" refers to a substitution with another amino acid residue having properties similar to the original amino acid residue. For example, lysine, arginine, and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have non-polar side chains. In addition, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when substituting an amino acid residue in a group that exhibits similar properties as described above, it will not exhibit specific changes in properties.

[0478] The terms "polypeptide," "protein," or "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

[0479] The term "nucleic acid" is used interchangeably with the term "polynucleotide" in this article, and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or connections, which are synthetic, naturally occurring, and non-naturally occurring, with binding properties similar to reference nucleic acids, and metabolized in a manner similar to reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). "Isolated" nucleic acids refer to polynucleotides separated from the components of their natural environment. Isolation nucleic acids are included in the polynucleotides contained in the following cells, which cells typically contain the polynucleotides, but the polynucleotides are present outside the chromosome or are present at a chromosomal position different from their natural chromosomal position. The isolated nucleic acids encoding a polypeptide or fusion protein refer to one or more polynucleotides encoding a polypeptide or fusion protein, including such one or more polynucleotides in a single vector or a separate vector, and such one or more polynucleotides present in one or more positions in a host cell. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as explicitly indicated sequences. Specifically, as described in detail below, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues.

[0480] The term "effector function" refers to those biological activities attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence mutated Fc region) and varies with the antibody isotype. Examples of antibody effector functions include, but are not limited to, C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0481] The term "binding affinity" or "affinity" is used in this disclosure as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or portion thereof and an antigen, a ligand and a receptor). The binding affinity between two molecules can be quantified by determining the dissociation constant (KD). K can be determined by measuring the kinetics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) methods (Biacore). D The rate constants corresponding to the association and dissociation of a monovalent complex are called the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. D Through equation K D= kd / ka is related to ka and kd. The value of the dissociation constant can be determined directly by well-known methods and can be calculated even for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9:340-362). For example, K can be determined using a double filtration nitrocellulose filter binding assay such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90:5428-5432). D Other standard assays for assessing the binding ability of an antibody to a target antigen are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis, as well as other assays exemplified elsewhere in this disclosure. The binding kinetics and binding affinity of an antibody can also be determined by standard assays known in the art, such as surface plasmon resonance (SPR), for example, by using Biacore TM The K of each antibody / antigen complex can be compared by comparing the K D The K values ​​can be used to compare the binding affinities associated with different molecular interactions, for example, the binding affinities of different antibodies for a given antigen. Similarly, the specificity of an interaction can be determined and compared by determining and comparing the K values ​​of the interactions of interest (e.g., the specific interaction between an antibody and an antigen). D The K value is compared with the K value of non-target interactions (e.g., a control antibody known not to bind the target antigen). D The value is evaluated.

[0482] The term "linker" or "linker" refers to a connecting unit that connects two polypeptide fragments, which is generally flexible and does not cause loss of the original function of the protein domain. In this article, the linkers appearing in the same structure can be the same or different. The linker can be a peptide linker, which contains one or more amino acids, typically about 1-30, 2-24, or 3-15 amino acids. The linkers used in the present disclosure can be the same or different.

[0483] The term "fused" or "linked" means that the components (e.g., TACI polypeptide and BCMA polypeptide) are linked by covalent bonds, either directly or via one or more linkers. When the linker is a peptide linker, the covalent bond is a peptide bond.

[0484] "Administer," "apply," and "treat" as applied to an animal, a human, a laboratory subject, a cell, a tissue, an organ, or a biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, a human, a subject, a cell, a tissue, an organ, or a biological fluid. "Administer," "apply," and "treat" can refer to, for example, therapeutic, pharmacokinetics, diagnostics, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "apply," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. "Treatment," as applied to a human, veterinary, or research subject, refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0485] "Treatment" means administering an internal or external therapeutic agent, such as a composition comprising any of the antibodies or antigen-binding fragments thereof disclosed herein, or a fusion protein thereof, to a subject who has, is suspected of having, or is predisposed to having one or more diseases or symptoms thereof, and for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more disease symptoms in the treated subject or population, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable degree. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether the disease symptoms have been alleviated can be evaluated by any clinical detection method commonly used by doctors or other health care professionals to evaluate the severity or progression of the symptoms. Although embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may not be effective in alleviating symptoms of the target disease in a certain subject, they should alleviate symptoms of the target disease in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0486] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular subject or veterinary subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0487] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the offspring of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and offspring derived therefrom, without regard to the number of passages. Offspring may not be identical to the parent cell in nucleic acid content, but may contain mutations. Mutant offspring having the same function or biological activity as screened or selected in the initial transformed cells are included herein. Host cells include prokaryotic and eukaryotic host cells, wherein eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include humans, mice, rats, dogs, monkeys, pigs, goats, cattle, horses, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.

[0488] "Optional," "optionally," and "optionally" mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. For example, "optionally comprises 1-3 antibody heavy chain variable regions" means that an antibody heavy chain variable region of a specified sequence may but need not be present.

[0489] The term "fusion protein comprising a TACI polypeptide and a BCMA polypeptide" disclosed herein encompasses all molecules comprising a fusion protein of the TACI polypeptide and the BCMA polypeptide disclosed herein, the term "fusion protein comprising a TACI polypeptide and an anti-IFNAR1 antibody" encompasses all molecules comprising a fusion protein of the TACI polypeptide and the anti-IFNAR1 antibody (or an antigen-binding fragment thereof), and the term "fusion protein comprising a TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody (or an antigen-binding fragment thereof)" encompasses all molecules comprising a fusion protein of the TACI polypeptide, a BCMA polypeptide, and an anti-IFNAR1 antibody (or an antigen-binding fragment thereof). For example, the fusion protein may comprise one or more effector molecules, for example, by conjugation. The "effector molecule" alone can have therapeutic activity (e.g., anti-tumor activity or immune activation or inhibition activity) or detection function, and can be in any form, such as biologically active proteins (e.g., enzymes), other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof, such as DNA, RNA and fragments thereof, radionuclides (particularly radioiodides), radioisotopes, chelated metals, nanoparticles, and reporter groups (e.g., fluorescent compounds), or compounds detectable by NMR or ESR spectroscopy. The conjugation of the effector molecule to the fusion protein of the present invention can be achieved by conventional methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0490] Figure 1. Schematic diagram of the fusion protein structure of TACI and Fc.

[0491] Figure 2. Schematic diagram of the interaction between the ligands BAFF and APRIL and the receptors BAFF-R, TACI, and BCMA. BAFF is a membrane-expressed protein that functions as a soluble trimer after proteolytic cleavage. BAFF can bind to BAFF-R, TACI, and BCMA. APRIL is a soluble trimeric protein that can bind to TACI and BCMA. Thicker black arrows represent stronger interactions, while thinner black arrows represent weaker interactions.

[0492] Figure 3. Schematic diagram of different TACI and BCMA protein domains, including TACI-ECD, TACI-d2, TACI-T2, TACI-T4, and BCMA-ECD.

[0493] Figure 4. Schematic diagram of the TACI-BCMA fusion protein structure, including B575701, B575702, B575703, and B575704.

[0494] Figure 5. ELISA results of TACI-BCMA fusion proteins (B575701, B575702, B575703, B575704) binding to BAFF, using Telitacicept and IgG1 isotype as controls.

[0495] Figure 6. ELISA assay results of the binding ability of TACI-BCMA fusion proteins (B575701, B575702, B575703, B575704) to APRIL, using Telitacicept and IgG1 isotype as controls.

[0496] Figure 7. Schematic diagram of the TACI-CRD2 and TACI-19-16 domains, including TACI-CRD2 and TACI-19-16.

[0497] Figure 8. Schematic diagram of the structures of the fusion proteins of Anifrolumab and TACI-T4 (B385801, B385802, B385803, B385804, B498301, B498302) and the fusion proteins of Anifrolumab and TACI-19-16 (B606401, B606401-LALA, B805201, B805201-LALA, the LALA mutation is the L234A / L235A mutation of Fc, the same below).

[0498] Figure 9. The results of the fusion protein of Anifrolumab and TACI-T4 inhibiting IFN-β activity. Figure 9A shows the results of B385801, B385802, B385803, and B385804 inhibiting IFN-β activity; Figure 9B shows the results of B385804, B498301, and B498302 inhibiting IFN-β activity; Figure 9C shows the results of B606401 inhibiting IFN-β activity. Anifrolumab and IgG1 isotype were used as controls in Figures 9AC.

[0499] Figure 10. ELISA results of the binding ability of the fusion protein of Anifrolumab and TACI-T4 to BAFF, wherein Figure 10 A shows the binding ability of B385801, B385802, B385803, and B385804 to BAFF, using Atacicept, Telitacicept, and IgG1 isotypes as controls; Figure 10 B shows the binding ability of B385804, B498301, and B498302 to BAFF; Figure 10 C shows the binding ability of B606401 to BAFF, using Telitacicept and IgG1 isotypes as controls.

[0500] Figure 11. Schematic diagram of the fusion protein structure of Anifrolumab and TACI-BCMA, including B613301, B613302, B613303, and B613304.

[0501] Figure 12. Results of the inhibition test on IFN-β activity by anifrolumab and TACI-BCMA fusion proteins (B613301, B613302, B613303), using anifrolumab and IgG1 isotype as controls.

[0502] Figure 13. ELISA assay results for the binding ability of anifrolumab and TACI-BCMA fusion proteins (B613301, B613302, B613303) to BAFF, using Telitacicept and IgG1 isotype as controls.

[0503] Figure 14. ELISA results of the binding ability of anifrolumab and TACI-BCMA fusion proteins (B613301, B613302, B613303) to APRIL, using Telitacicept and IgG1 isotype as controls.

[0504] Figure 15. Schematic diagram 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, and B746201-LALA).

[0505] Figure 16. Results of the inhibition test on IFN-β activity by fusion proteins of anifrolumab with TACI-19-16 and BCMA (B637302, B746201), and fusion proteins of anifrolumab with TACI-19-16 (B606401, B805201), using anifrolumab and IgG1 isotype as controls.

[0506] Figure 17. ELISA results of the binding ability of B637302, B746201, B606401, and B805201 to BAFF, using Telitacicept and IgG1 isotype as controls.

[0507] Figure 18. ELISA results of the binding ability of B637302, B746201, B606401, and B805201 to APRIL, using Telitacicept and IgG1 isotype as controls.

[0508] Figure 19. pDC function assay results in PBMCs. Human PBMCs cultured in vitro were stimulated with CpG-A and treated with gradient concentrations of B637302 and B606401. Anifrolumab and IgG1 were used as controls. IFNα secretion levels in the supernatant were measured 24 hours later.

[0509] Figure 20. Results of an in vitro plasma cell differentiation experiment. B cells isolated from human PBMCs were cultured in vitro, supplemented with CpG-B and IFN-α, and treated with gradient concentrations of B637302, B746201, B606401, and B805201. Anifrolumab and IgG1 were used as controls. Flow cytometry analysis was performed after 4 days to assess the proportion of plasma cells (CD27+CD38+).

[0510] Figure 21. Results of a BAFF-induced B cell proliferation experiment in vitro. B cells were isolated from human PBMCs, labeled with CTV, and then cultured in vitro. IL-4, anti-IgM, CD40L, and IL-17 were used as basal stimulatory signals. Under these basal conditions, BAFF was added to induce B cell proliferation. Gradual concentrations of B637302, B606401, and B805201 were then added. Telitacicept, an IgG1 isotype, was used as a control. B cell proliferation signals were detected by flow cytometry on day 5. Drug concentrations ranged from 100 nM, 300 nM, and 1000 nM.

[0511] Figure 22. APRIL-induced B cell proliferation in vitro assay. B cells isolated from human PBMCs were labeled with CTV and cultured in vitro. IL-4, anti-IgM, CD40L, and IL-17 were used as basal stimulatory signals. Under these basal conditions, APRIL was added to induce B cell proliferation. B637302 and B606401 were then treated with gradient concentrations. Telitacicept and IgG1 were used as controls. B cell proliferation was detected by flow cytometry on day 5. Drug concentrations were 100 nM, 300 nM, and 1000 nM.

[0512] Figure 23. In vitro plasma cell generation induced by BAFF and APRIL. B cells isolated from human PBMCs were cultured in vitro and stimulated with CpG-B from days 1 to 4. CpG-B was removed from days 4 to 10, and culture was continued with IL-6, IL-10, and IL-21. Under these basal conditions, plasma cell generation was induced in vitro by the addition of 500 ng / mL BAFF and 50 ng / mL APRIL from days 4 to 7, and 50 ng / mL BAFF and 500 ng / mL APRIL from days 7 to 10. On days 4 to 10, B637302, B606401, B606401, and B805201 were treated at concentrations of 10, 100, and 1000 nM. Telitacicept and an IgG1 isotype were used as controls. Plasma cell numbers were assessed by flow cytometry on day 10.

[0513] Figure 24. In vitro plasma cell generation induced by the combination of IFN-α, BAFF, and APRIL. Figure 24A is a schematic diagram of the experimental process. B cells isolated from human PBMCs were cultured in vitro. CpG-B and IFN-α were added for stimulation on days 1 to 4. CpG-B was removed from days 4 to 10, and culture was continued with IL-6, IL-10, and IL-21. Under these basal conditions, 500 ng / mL BAFF and 50 ng / mL APRIL were added on days 4 to 7, and 50 ng / mL BAFF and 500 ng / mL APRIL were added on days 7 to 10 to induce plasma cell generation in vitro. From day 0 to day 10, the cells were treated with drugs B637302 and B606401 at different concentrations of 10, 100, and 1000 nM. Telitacicept and IgG1 isotype were used as controls, and the number of plasma cells was detected by flow cytometry on day 10. Figures B, C, and D show the detection results of drugs at 10 nM, 100 nM, and 1000 nM, respectively.

[0514] Figure 25. Results of a PD experiment in humanized mice induced by PEG-IFN-α. Figure 25A is a schematic diagram of the experimental process. Using B-NDG immunodeficient mice, PBMCs were injected into the tail vein for humanization and PEG-IFN-α was injected intraperitoneally for induction. In this model, mice were treated with different concentrations of drugs B637302 and B606401 via intraperitoneal injection. Anifrolumab and PBS were used as controls. Figure 25B is a schematic diagram of the downstream signaling pathway induced by IFN-α. Figure 25C shows the results of PBMC collection at 2 hours for the detection of pSTAT1 levels. Figures 25D, E, and F show the mRNA expression levels of IFN-α downstream genes detected by QPCR using PBMCs collected at 1, 3, and 7 days, respectively.

[0515] Figure 26. Results of a BAFF and APRIL-co-induced PD mouse model. Figure 26A is a schematic diagram of the experimental process. BAFF+APRIL was injected intraperitoneally in C57 / B6 mice to induce PD. The model was then treated with varying concentrations of B637302 and B606401 via intraperitoneal injection. Telitacicept and PBS were used as controls. Peripheral blood was collected on days 4 and 7, and plasma IgA levels were measured by ELISA. Figures 26B and C are graphs showing the results of plasma IgA analysis on days 4 and 7, respectively.

[0516] 21 to 26 , the statistical method used was Student's t-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0517] Figure 27. Results of B cell in vitro differentiation experiment. B cells sorted from human PBMC were cultured in vitro, and IL-3, CpG-A, IFNα and IL-21 were added to stimulate B cell differentiation. After 6 days, the cell viability and the ratio of differentiated plasma cells (CD27+CD38+) were detected. Among them, Figure 27 A is the FACS detection result of IL-3+CpG-A, IL-3+CpG-A+IL-21, and IL-3+CpG-A+IFNα, using 1640 culture medium as a control; Figure 27 B is the detection result of the corresponding live cell ratio; Figure 27 C is the corresponding plasma cell ratio detection result.

[0518] Figure 28. Results of B cell differentiation experiment in vitro. B cells sorted from human PBMC were cultured in vitro, and R848, CpG-B, IFNα, BAFF, APRIL and RA were added to stimulate B cell differentiation. After 6 days, the cell viability and the ratio of differentiated plasma cells (CD27+CD38+) were detected. Among them, Figure 28 A is the FACS detection result of CpG-B+IFNα, R484+IFNα, CpG-B+BAFF+APRIL, R484+BAFF+APRIL, CpG-B+IFNα+RA+BAFF+APRIL, R484+IFNα+RA+BAFF+APRIL, using 1640 culture medium as a control; Figure 28 B is the detection result of the corresponding live cell ratio, and Figure 28 C is the corresponding plasma cell (CD27 + CD38 + ) ratio test results.

[0519] Figure 29. Results of B cell differentiation assay in vitro. B cells isolated from human PBMC were cultured in vitro, stimulated with CpG-B and IFNα, and treated with anifrolumab as a control. Plasma cells (CD27 + CD38 + ) differentiation ratio.

[0520] Figure 30. Results of an in vitro B cell differentiation assay. B cells isolated from human PBMCs were cultured in vitro and stimulated with CpG-B and IFNα. Anifrolumab was then added to the cells for treatment. Plasma cell (CD27+CD38+) differentiation was measured 4 days later. This demonstrates that anifrolumab can stimulate plasma cell differentiation in a concentration-dependent manner under these conditions. IgG1 was used as a control.

[0521] Figure 31. Results of plasma cell generation assay. B cells isolated from human PBMC were cultured in vitro and induced to generate plasma cells using IL-3, CpG-A, IFNα, BAFF, and APRIL. Plasma cells (CD27+ CD38 + ) differentiation ratio. Figure 31 A shows the FACS detection results of IL-3+CpG-A, IL-3+CpG-A+BAFF+APRIL, IL-3+CpG-A+IFNα, and IL-3+CpG-A+IFNα+BAFF+APRIL, using 1640 culture medium as a control; Figure 31 B shows the corresponding plasma cells (CD27 + CD38 + ) ratio test results.

[0522] Figure 32. Results of plasma cell generation assay. B cells isolated from human PBMC were cultured in vitro and induced with CpG-B, IFNα, BAFF, and APRIL. Plasma cell generation was detected after 7, 10, 14, and 21 days (CD27 + CD38 + ) differentiation quantity.

[0523] Figure 33. Results of plasma cell generation assay. B cells isolated from human PBMC were cultured in vitro and stimulated with CpG-B and IFNα from day 0 to day 4. After day 4, CpG-B and IFNα were removed and IL-6, BAFF, and APRIL were added to induce plasma cell generation. Plasma cells (CD27 + CD38 + ) differentiation quantity.

[0524] Figure 34. Plasma cell generation assay results. B cells isolated from human PBMCs were cultured in vitro and stimulated with CpG-B from day 0 to day 4. CpG-B was removed after day 4, and IL-6, IL-10, IL-21 (5 ng / mL), BAFF, and APRIL were added to induce plasma cell generation. 500 ng / mL BAFF and 50 ng / mL APRIL were added from day 4 to day 7, and 50 ng / mL BAFF and 500 ng / mL APRIL were added from day 7 to day 10. Plasma cells (CD27 + CD38 + ) differentiation quantity.

[0525] Figure 35. Plasma cell production assay results. B cells isolated from human PBMCs were cultured in vitro and stimulated with CpG-B from day 0 to day 4. After day 4, CpG-B was removed and IL-6, IL-10, IL-21 (50 ng / mL), BAFF, and APRIL were added to induce plasma cell production. 500 ng / mL BAFF and 50 ng / mL APRIL were added from day 4 to day 7, and from day 7 to day 10. Plasma cells (CD27 + CD38 + ) differentiation quantity.

[0526] Figure 36. Results of plasma cell generation assay. B cells isolated from human PBMCs were cultured in vitro and stimulated with CpG-B from day 0 to day 4. CpG-B was removed after day 4 and IL-6, IL-10, IL-21 (15, 25, or 40 ng / mL), BAFF, and APRIL were added to induce plasma cell generation. 500 ng / mL BAFF and 50 ng / mL APRIL were added from day 5 to day 7, and 50 ng / mL BAFF and 500 ng / mL APRIL were added from day 7 to day 10. Telitacipep was added at a gradient concentration on day 4 as a control. Plasma cells (CD27 + CD38 + ) differentiation number. Among them, A, B, and C in Figure 36 are plasma cells (CD27 + CD38 + ) number test results. DETAILED DESCRIPTION

[0527] The present disclosure is further described below with reference to the following examples, but these examples are not intended to limit the scope of the present disclosure.

[0528] Experimental methods in the disclosed embodiments or test examples, where specific conditions are not specified, generally followed conventional conditions or those recommended by the raw material or commercial manufacturer. See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory; and Current Methods in Molecular Biology, Ausubel et al., Greene Publishing Associates, Wiley Interscience, NY. Reagents whose sources are not specified were commercially available.

[0529] Example 1. TACI sequence analysis and modification

[0530] Due to the presence of multiple protease cleavage sites in the TACI sequence, full-length TACI is prone to fragmentation after expression. In this experiment, TACI sequence fragments of varying lengths were designed. The C-terminus of TACI-ECD or its fragments was directly fused to the N-terminus of human IgG1 Fc (SEQ ID NO: 3) to construct TACI-Fc fusion proteins (including full-length TACI-ECD-Fc and TACI-9-Fc). These proteins were transfected and expressed in 293E (EBNA1 gene-modified human embryonic kidney) cells. The TACI-Fc fusion protein containing two identical polypeptide chains was purified (see Figure 1 for its structure). RCT-18 (Telitacicept) was used as a positive control.

[0531] The sequence looks like this:

[0532] >TACI-ECD (amino acid residues 1 to 165 of wild-type human TACI)

[0533] >TACI-9 (human TACI-ECD positions 68 to 108 by natural numbering)

[0534] >Human IgG1 Fc sequence

[0535] >Telitacicept (RCT-18)

[0536] >Atacicept

[0537] Note: In the Telitacicept (RCT-18) sequence, the ununderlined portion is the TACI sequence (positions 13 to 118 of the human TACI extracellular domain by natural numbering), and the underlined portion is the Fc sequence. In the Atacicept sequence, the ununderlined portion is the TACI sequence (positions 30 to 110 of the human TACI extracellular domain by natural numbering) and the linker EPKSS (in italics), and the underlined portion is the Fc sequence.

[0538] The obtained TACI-Fc fusion protein was subjected to mass spectrometry analysis. The experimental results showed that TACI-9-Fc had no cleavage fragments, but TACI cleavage fragments were detected in both the positive control RCT-18 and the full-length TACI-ECD-Fc (results not shown).

[0539] The ability of the TACI-Fc fusion protein to block BAFF-R binding was further tested as follows: the receptor protein was diluted to 2 μg / mL in PBS (Source Bio, B320) at pH 7.4 and added to a 96-well microtiter plate (Corning, 3590) at a volume of 100 μL / well. The plate was incubated overnight at 4°C. After discarding the liquid, 200 μL of 1% Casein blocking solution (Thermo, 37528) was added to each well for blocking and incubation at 37°C for 2 hours. After blocking, the blocking solution was discarded and the plate was washed three times with PBST buffer (pH 7.4 PBS containing 0.1% tween-20) before use. A fixed concentration of biotin-labeled ligand protein was mixed with serially diluted antibody or fusion protein, pre-incubated at 37°C for 30 minutes, and then added to the blocked microtiter plate and incubated at 37°C for 1.5 hours. After incubation, wash the plate three times with PBST, add 100 μL streptavidin-HRP (Invitrogen, 434323, 1:4000 dilution) to each well, and incubate at 37°C for 1 hour. Remove the supernatant, wash the plate three times with PBST, and add 100 μL TMB colorimetric substrate (KPL, 5120-0077) to each well. Incubate at room temperature for 10-15 minutes. Add 50 μL 1M H2SO4 to each well to terminate the reaction. Read the absorbance at 450 nm using a microplate reader. Use software to fit the curve of inhibition of ligand and receptor binding, and calculate the IC 50 The sources of the ligand and receptor proteins used are as follows: BAFF (Sino biological, 10056-HNCH), BAFF-R (Sino biological, 16079-H02H).

[0540] The results showed that TACI-9-Fc and RCT-18 blocked the IC of BAFF binding to BAFF-R. 50 The values ​​were 5.02 nM and 27.48 nM, respectively. The functional activity of TACI-9-Fc was significantly stronger than that of the control RCT-18. Therefore, TACI-9 is considered a potential candidate molecule.

[0541] Example 2. Analysis of TACI truncated fragments

[0542] Based on TACI-9, the sequence length of TACI was further truncated to test the functional activity of the truncated TACI fragment. The sequence of the further truncated TACI fragment is shown below:

[0543] >TACI-10 (TACI-9 C-terminus truncated with one amino acid "L") (human TACI-ECD positions 68 to 107 by natural numbering)

[0544] >TACI-11 (TACI-9 C-terminally truncated with two amino acids "KL") (human TACI-ECD positions 68 to 106 by natural numbering)

[0545] >TACI-12 (TACI-9 C-terminally truncated with 3 amino acids "NKL") (human TACI-ECD positions 68 to 105 by natural numbering)

[0546] The C-terminus of the truncated TACI fragment was fused to the N-terminus of human IgG Fc (SEQ ID NO: 3) to construct a TACI-Fc fusion protein, which was transfected and expressed in 293E cells and purified to obtain a TACI-Fc fusion protein containing two identical polypeptides (see Figure 1 for the structure).

[0547] The TACI-Fc fusion proteins constructed above were subjected to mass spectrometry analysis. The experimental results are shown in Table 2. The experimental results showed that the TACI-Fc fusion proteins constructed with TACI-9 truncated fragments did not undergo TACI polypeptide cleavage.

[0548] In addition, the activity of the constructed TACI-Fc fusion protein in binding to BAFF was detected, as well as the activity of the TACI-Fc fusion protein in blocking the binding function of BAFF and BAFF-R (see Example 1 for experimental methods).

[0549] The BAFF binding activity of TACI-Fc fusion protein was assayed as follows: BAFF (Sino Biological, 10056-HNCH) protein was diluted to 1 μg / mL in PBS (B320, pH 7.4) and added to a 96-well microtiter plate (Corning, 3590) at a volume of 100 μL / well. The plate was incubated overnight at 4°C. After discarding the liquid, 300 μL of 5% skim milk (BD, 232100) diluted in PBS was added to each well for blocking, and the plate was incubated at 37°C for 2 hours. After blocking, the blocking solution was discarded, and the plate was washed three times with PBST buffer (PBS, pH 7.4 containing 0.1% Tween-20). Then, 100 μL of serially diluted test antibody or fusion protein solution was added to each well and incubated at 37°C for 1 hour. After incubation, wash the plate three times with PBST, add 100 μL mouse anti-human IgG (H+L) (Jackson ImmunoResearch, 209-035-088, 1:8000 dilution) to each well, and incubate at 37°C for 1 hour. After washing the plate three times with PBST, add 100 μL TMB colorimetric substrate (KPL, 5120-0077) to each well and incubate at room temperature for 10-15 minutes. Add 50 μL 1M H2SO4 to each well to terminate the reaction. Read the absorbance at 450 nm with a microplate reader, and use software to fit the binding curve of the antibody and antigen to calculate the EC 50 value.

[0550] The results in Table 2 show that the TACI-10-Fc, TACI-11-Fc, and TACI-12-Fc fusion proteins constructed from the truncated fragments of TACI-9, TACI-10, TACI-11, and TACI-12, have the same binding activity to BAFF and functional activity in blocking the binding of BAFF to BAFF-R as the TACI-9-Fc fusion protein.

[0551] Table 2. TACI-Fc fusion protein functional activity and mass spectrometry fragmentation test results

[0552] Example 3. Sequence modification of TACI truncated fragments

[0553] TACI protein is prone to aggregation in neutral solutions. To further improve TACI's stability, we used MOE (Molecular Operating Environment) software to analyze the hydrophobic and ionic groups in the TACI fragment based on the TACI crystal structure (PDB ID: 1XU1). We then made amino acid substitutions at multiple amino acid residues in TACI-9, reducing the area of ​​exposed hydrophobic and ionic groups on the TACI protein, reducing TACI's aggregation potential, and improving its stability while still retaining its functional activity. The amino acid sequence of the modified TACI fragment is as follows:

[0554] >TACI-9-1 (TACI-9 with L69T replacement)

[0555] >TACI-9-2 (TACI-9 with R72S mutation)

[0556] >TACI-9-3 (TACI-9 with K73E mutation)

[0557] >TACI-9-4 (TACI-9 with K73Q mutation)

[0558] >TACI-9-5 (TACI-9 with K77E mutation)

[0559] >TACI-9-6 (TACI-9 with L69R, D85T mutations)

[0560] >TACI-9-7 (TACI-9 with L69R, D85A mutations)

[0561] >TACI-9-8 (TACI-9 with Y102A mutation)

[0562] >TACI-9-9 (TACI-9 with L69R, D85T, and Y102R mutations)

[0563] >TACI-9-10 (TACI-9 with K73E and K77E mutations)

[0564] >TACI-9-11 (TACI-9 with R72S, K73E, K77E mutations)

[0565] >TACI-9-12 (TACI-9 with L69T, Y102A mutations)

[0566] >TACI-9-13 (TACI-9 with L69T, F103Y mutations)

[0567] >TACI-9-14 (TACI-9 with L69T, Y102A, F103Y mutations)

[0568] >TACI-9-15 (TACI-9 containing L69T, K73E, K77E, and Y102A mutations)

[0569] Note: In the above sequence, the underlined part is the amino acid residue after mutation.

[0570] The C-terminus of the TACI fragment modified from TACI-9 was fused to the N-terminus of human IgG Fc (SEQ ID NO: 3) to construct a TACI-Fc fusion protein (structure shown in Figure 1). The protein was transfected and expressed in 293E cells, and then affinity purified by Protein A to obtain a TACI-Fc fusion protein containing two identical polypeptide chains.

[0571] The activity of the constructed TACI-Fc fusion protein in binding to BAFF was detected (see Example 2 for the experimental method). The experimental results are shown in Table 3:

[0572] Table 3. Binding activity of TACI-Fc fusion protein to BAFF

[0573] Note: The mutation sites in the table are amino acid residue sites (natural sequence numbering) relative to TACI-ECD (SEQ ID NO: 1). For example, "L69T" indicates that the amino acid residue at position 69 (natural sequence numbering) in SEQ ID NO: 1 is mutated from L to T.

[0574] In addition, the partially purified TACI-Fc fusion protein was replaced with a PBS solution using an ultrafiltration tube. The solution was then visually inspected for the presence of precipitation. The results showed that the TACI-Fc fusion protein constructed with the modified TACI fragment produced no precipitation, while the PBS solution of RCT-18 did produce precipitation (results not shown).

[0575] Further provided are truncated forms of TACI-9-15 (TACI-9-15a, TACI-9-15b, TACI-9-15c), the amino acid sequences of which are as follows:

[0576] >TACI-9-15a

[0577] >TACI-9-15b

[0578] >TACI-9-15c (i.e., TACI-19-16)

[0579] Example 4. Construction, expression and purification of TACI-BCMA fusion protein

[0580] As previously mentioned, both atacicept and telitacicept are BAFF / APRIL antagonists in the form of TACI-Fc molecules. As shown in Figure 2, both TACI and BCMA bind to BAFF and APRIL, with TACI binding more strongly to BAFF and less strongly to APRIL, while BCMA binds more strongly to APRIL. We designed BCMA and TACI into a fusion protein in the hope of enhancing their neutralizing activity against APRIL.

[0581] As shown in Figure 3, TACI-ECD is the TACI extracellular region containing amino acid residues 1-165, TACI-d2 is the CRD2 ligand binding domain containing amino acid residues 69-111, TACI-T2 is the CRD1 and CRD2 ligand binding domain containing amino acid residues 13-118, TACI-T4 is the CRD1 and CRD2 ligand binding domain containing amino acid residues 30-110, and BCMA-ECD is the BCMA extracellular region containing amino acid residues 1-54.

[0582] As shown in Figure 4, four forms of fusion proteins containing TACI polypeptide and BCMA polypeptide were designed, among which fusion proteins B575701 and B575702 connected TACI-d2 and BCMA-ECD sequences and fused with human wild-type IgG1-Fc; fusion proteins B575703 and B575704 connected TACI-T2 and BCMA-ECD sequences and fused with human wild-type IgG1-Fc.

[0583] The sequence of TACI-ECD is shown in SEQ ID NO: 1. The remaining sequences are as follows, where the underlined portion represents human IgG1 Fc. The last G in SEQ ID NO: 27 may be absent.

[0584] >TACI-d2 (SEQ ID NO: 27)

[0585] >TACI-T2 (SEQ ID NO: 28)

[0586] >TACI-T4 (SEQ ID NO: 29)

[0587] >BCMA-ECD (SEQ ID NO: 30)

[0588] >TACI-d2-BCMA-ECD(SEQ ID NO: 31)

[0589] >BCMA-ECD-TACI-d2(SEQ ID NO: 32)

[0590] >TACI-T2-BCMA-ECD(SEQ ID NO: 33)

[0591] >BCMA-ECD-TACI-T2(SEQ ID NO: 34)

[0592] B575701, B575702, B575703, and B575704 are human IgG1 Fc (SEQ ID NO: 3) linked to the C-terminus of TACI-d2-BCMA-ECD, BCMA-ECD-TACI-d2, TACI-T2-BCMA-ECD, and BCMA-ECD-TACI-T2, respectively. In the following sequences, the underlined Fc is shown.

[0593] >B575701 (SEQ ID NO: 35)

[0594] >B575702 (SEQ ID NO: 36)

[0595] >B575703 (SEQ ID NO: 37)

[0596] >B575704 (SEQ ID NO: 38)

[0597] The gene sequence encoding the above-mentioned protein was synthesized and subcloned into pcDNA3.4. 50 μg of expression plasmid was diluted with culture medium and mixed. 200 μL of transfection reagent was diluted with culture medium and mixed. The mixture was then incubated at 37°C for 15 minutes. The mixture was then added dropwise to HEK293 cells. After incubation at 37°C on a shaker for one week, the supernatant was centrifuged at 8000 rpm for 5 minutes and collected. A Protein A affinity chromatography column was equilibrated with 20 mL of 1× PBS at a flow rate of 1 mL / min. The protein supernatant was loaded at a flow rate of 1 mL / min. Nonspecifically bound proteins were washed away with another 20 mL of 1× PBS at a flow rate of 1 mL / min. Finally, the column was eluted with pH 3.4 citrate buffer at a flow rate of 1 mL / min. The eluted protein was transferred to a dialysis bag and dialyzed against 1× PBS, replacing the PBS storage buffer. The target protein was detected.

[0598] Example 5. In vitro binding experiment of TACI-BCMA fusion protein to BAFF and APRIL

[0599] ELISA was used to detect the binding ability of TACI-BCMA fusion proteins (B575701, B575702, B575703, B575704) to BAFF and APRIL, respectively.

[0600] The method is as follows: BAFF (PeproTech, Cat: 310-13) or ARPIL (Acro Biosystems, Cat: APL-H52D1) was diluted in PBS to 1 μg / mL and coated in 96-well plates (Costar, Cat: 3590) overnight at 4°C. The plates were washed with PBST and blocked with Blocking Buffer (PBST containing 1% BSA) at 37°C for 1 hour. The plates were tapped and the test antibody was diluted in Blocking Buffer and incubated at 37°C for 1.5 hours. The plates were washed with PBST and HRP-mouse anti-human Fc antibody (GenScript, Cat: A01854-200) was diluted in Blocking Buffer and incubated at 37°C for 40 minutes. The plate was washed with PBST, 100 μL of TMB color development solution (Biopanda, Cat: TMB-S-003) was added and color was developed at 37° C. for 3 minutes, 100 μL of ELISA stop solution (Solarbio, Cat: C1058) was added, and OD450 was detected.

[0601] 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 Telitacicept. Among them, B575702 and B575704 have the same ability to bind to BAFF. Compared with Telitacicept, EC 50B575701 and B575703 have the same ability to bind to BAFF, and compared with Telitacicept, EC 50 Reduced to 1 / 2 of its level.

[0602] Table 4. Binding of TACI-BCMA fusion protein to BAFF

[0603] As shown in Table 5 and Figure 6, the four fusion proteins can clearly bind to APRIL, and the binding ability is basically the same; the binding ability of the four fusion proteins to APRIL is stronger than that of Telitacicept, EC 50 Reduced to 1 / 4 to 1 / 3 of its level.

[0604] Table 5. Binding of TACI-BCMA fusion protein to APRIL

[0605] Example 6. Construction, expression and purification of anifrolumab and TACI fusion protein

[0606] Anifrolumab is an antagonist targeting IFNAR1, blocking type I interferon activation of cells through IFNAR1. It is used to treat systemic lupus erythematosus. As shown in Figure 7, TACI-CRD2 is a CRD2 ligand-binding domain containing amino acid residues 68-105, and TACI-19-16 is a TACI-CRD2 peptide with several amino acid mutations (L69T, K73E, K77E, and Y102A).

[0607] As shown in Figure 8, TACI-T4 or TACI-19-16 were connected to the N-terminus or C-terminus of the anifrolumab heavy chain or light chain, respectively, through linkers of different lengths. Specifically, B385801 connected TACI-T4 to the N-terminus of the anifrolumab heavy chain through linker 1 of (G4S)2; B385802 connected TACI-T4 to the C-terminus of the anifrolumab heavy chain through linker 1; B385803 connected TACI-T4 to the N-terminus of the anifrolumab light chain through linker 1; B385804 connected TACI-T4 to the C-terminus of the anifrolumab light chain through linker 1; B498301 connected TACI-T4 to the C-terminus of the anifrolumab light chain through linker 2 of (G4S)3; and B498302 connected TACI-T4 to the C-terminus of the anifrolumab light chain through linker 3 of (G4S)4. B606401 links TACI-19-16 to the C-terminus of the anifrolumab light chain via Linker 1; B805201 links TACI-19-16 to the C-terminus of the anifrolumab heavy chain via Linker 2. B606401-LALA links TACI-19-16 to the C-terminus of the anifrolumab light chain via Linker 1, and the Fc portion is a human IgG1 Fc with L234A and L235A mutations. B805201-LALA links TACI-19-16 to the C-terminus of the anifrolumab heavy chain via Linker 2, and the Fc portion is a human IgG1 Fc with L234A and L235A mutations.

[0608] TACI-19-16 is shown in SEQ ID NO: 26, and the other sequences are as follows:

[0609] >Linker 1 (SEQ ID NO: 39)

[0610] >Linker 2 (SEQ ID NO: 40)

[0611] >Linker 3 (SEQ ID NO: 41)

[0612] >TACI-CRD2 (SEQ ID NO: 42, same as SEQ ID NO: 8)

[0613] >VH of Anifrolumab (SEQ ID NO: 43)

[0614] >VL of Anifrolumab (SEQ ID NO: 44)

[0615] >HCDR1 of Anifrolumab (SEQ ID NO: 45)

[0616] >HCDR2 of Anifrolumab (SEQ ID NO: 46)

[0617] >HCDR3 of Anifrolumab (SEQ ID NO: 47)

[0618] >LCDR1 of Anifrolumab (SEQ ID NO: 48)

[0619] >LCDR2 of Anifrolumab (SEQ ID NO: 49)

[0620] >LCDR3 of Anifrolumab (SEQ ID NO: 50)

[0621] >Heavy chain of B385801 (SEQ ID NO: 51)

[0622] >Heavy chain of B385802 (SEQ ID NO: 52)

[0623] >Heavy chain of B805201 (SEQ ID NO: 53)

[0624] >Light chain of B385801, B385802, B805201, B805201-LALA (SEQ ID NO: 54)

[0625] >Heavy chain of B385803, B385804, B498301, B498302, B606401 (SEQ ID NO: 55)

[0626] >Light chain of B385803 (SEQ ID NO: 56)

[0627] >Light chain of B385804 (SEQ ID NO: 57)

[0628] >Light chain of B498301 (SEQ ID NO: 58)

[0629] >Light chain of B498302 (SEQ ID NO: 59)

[0630] >Light chain of B606401, B606401-LALA (SEQ ID NO: 60)

[0631] >Heavy chain of B606401-LALA (SEQ ID NO: 61)

[0632] >Heavy chain of B805201-LALA (SEQ ID NO: 62)

[0633] The Fc region of IgG is underlined in the heavy chain, CH1 is underlined in the light chain, and the linker is in italics.

[0634] The protein preparation method provided in Example 1 was used, and transient transfection and protein expression were performed. The protein was passed through a Protein A affinity chromatography column and eluted. After detection, the target protein was obtained.

[0635] Example 7. Inhibition of Type I Interferon Activity by Fusion Protein of Anifrolumab and TACI

[0636] The HEK-Blue IFN-α / β cell (InvivoGen, Cat: hkb-ifnab) kit was used to detect the inhibitory effect of the Anifrolumab and TACI fusion proteins B385801, B385802, B385803, B385804, B498301, B498302, and B606401 on the activity of IFN-α / β.

[0637] Method: Digest HEK-Blue TMIFN-α / β cells were resuspended to 2.8E5 / mL in DMEM (Gibco, Cat: 11995065) containing 10% FBS and 1% P / S and seeded into 96-well plates. The test protein was added and incubated at 37°C for 30 minutes. IFN-β (Sino biological, Cat: 10704-HNAS) was added to a final concentration of 0.01 ng / mL and incubated at 37°C for 24 hours. 20 μL of culture supernatant was mixed with 180 μL of Quanti-Blue (InvivoGen, Cat: rep-qbs) in a new 96-well plate and incubated at 37°C for 1 hour. OD655 was measured.

[0638] The results showed that all seven fusion proteins had inhibitory effects on the activity of IFN-α / β. Among them, the activities of B385801 and B385803 were weaker, followed by B385802, and the activities of B385804, B498301, B498302 and B606401 were basically equivalent to those of Anifrolumab (Figure 9A, Figure 9B, Figure 9C).

[0639] Example 8. In vitro binding experiment of the fusion protein of Anifrolumab and TACI to BAFF

[0640] ELISA was used to detect the binding ability of Anifrolumab and TACI fusion proteins B385801, B385802, B385803, B385804, B498301, B498302, and B606401 to BAFF.

[0641] Methods: BAFF (PeproTech, Cat: 310-13) was diluted to 1 μg / mL in PBS and coated in 96-well plates overnight at 4°C. The plates were washed with PBST and blocked with Blocking Buffer (PBST containing 1% BSA) at 37°C for 1 hour. The plates were tapped and the test antibody was diluted in Blocking Buffer and incubated at 37°C for 1.5 hours. The plates were washed with PBST and HRP-mouse anti-human Fc antibody (GenScript, Cat: A01854-200) was diluted in Blocking Buffer and incubated at 37°C for 40 minutes. The plates were washed with PBST and developed with 100 μL of TMB colorimetric solution (Biopanda, Cat: TMB-S-003) at 37°C for 3 minutes. 100 μL of ELISA stop solution (Solarbio, Cat: C1058) was added and the OD450 was measured.

[0642] The results, as shown in Figures 10A to 10C and Table 6, show that the seven fusion proteins significantly bind to BAFF. B385801, B385803, and B606401 have the strongest binding to BAFF, exceeding that of Telitacicept and Atacicept. B385802 has weaker binding, while B385804, B498301, and B498302 have similar binding abilities, but slightly weaker than Telitacicept. Linker length does not affect the binding of TACI to BAFF.

[0643] Table 6. Binding of Anifrolumab and TACI fusion protein to BAFF

[0644] Example 9. Construction, expression and purification of anifrolumab and TACI-BCMA fusion protein

[0645] Referring to Figure 11 , the TACI-BCMA fusion proteins described in Example 4 were linked to the C-terminus of the anifrolumab antibody light chain via Linker 1. B613301 and B613302 are fusion proteins of TACI-d2 and BCMA-ECD linked to the C-terminus of the anifrolumab antibody light chain via Linker 1; and B613303 and B613304 are fusion proteins of TACI-T2 and BCMA-ECD linked to the C-terminus of the anifrolumab antibody light chain via Linker 1.

[0646] The heavy chains of B613301, B613302, B613303, and B613304 are all the full-length heavy chains of Anifrolumab (SEQ ID NO: 55). The specific sequences of the light chains are as follows, with the light chain constant region being underlined:

[0647] >Light chain of B613301 (SEQ ID NO: 63)

[0648] >Light chain of B613302 (SEQ ID NO: 64)

[0649] >Light chain of B613303 (SEQ ID NO: 65)

[0650] >Light chain of B613304 (SEQ ID NO: 66)

[0651] The protein preparation method provided in Example 4 was used, and transient transfection and protein expression were performed. The protein was passed through a Protein A affinity chromatography column and eluted. After detection, the target protein was obtained.

[0652] Example 10. Inhibitory effect of anifrolumab and TACI-BCMA fusion protein on type I interferon activity

[0653] The HEK-Blue IFN-α / β cell kit (InvivoGen, Cat: hkb-ifnab) was used to detect the inhibitory effect of the Anifrolumab and TACI-BCMA fusion proteins B613301, B613302, and B613303 on IFN-α / β activity in Example 9.

[0654] Method: Digest HEK-Blue TM IFN-α / β cells were resuspended to 2.8E5 / mL in DMEM medium (Gibco, Cat: 11995065) containing 10% FBS and 1% P / S and seeded into 96-well plates (Costar, Cat: 3599). The test antibody was added and the cells were incubated at 37°C for 30 minutes. IFN-β (Sino biological, Cat: 10704-HNAS) was added at a final concentration of 0.01 ng / mL and incubated at 37°C for 24 hours. Twenty microliters of culture supernatant was mixed with 180 microliters of Quanti-Blue (InvivoGen, Cat: rep-qbs) in a new 96-well plate and incubated at 37°C for 1 hour. OD655 was measured.

[0655] The results are shown in Figure 12, showing that all three fusion proteins were able to inhibit IFN-α / β activity, with comparable activity and consistent with that of anifrolumab. This suggests that the fusion of TACI-BCMA to anifrolumab, particularly at the C-terminus of the light chain, has little effect on anifrolumab's activity.

[0656] Example 11. In vitro binding experiment of anifrolumab and TACI-BCMA fusion protein to BAFF and APRIL

[0657] ELISA was used to detect the binding ability of Anifrolumab and TACI-BCMA fusion proteins B613301, B613302, and B613303 to BAFF and APRIL.

[0658] Method: BAFF (PeproTech, Cat: 310-13) or ARPIL (Acro Biosystems, Cat: APL-H52D1) was diluted in PBS to 1 μg / mL and coated in 96-well plates (Costar, Cat: 3590) overnight at 4°C. The plates were washed with PBST and blocked with Blocking Buffer (PBST containing 1% BSA) at 37°C for 1 hour. The plates were then tapped and the test antibody was diluted in Blocking Buffer and incubated at 37°C for 1.5 hours. The plates were then washed with PBST and HRP-mouse anti-human Fc antibody (GenScript, Cat: A01854-200) was diluted in Blocking Buffer and incubated at 37°C for 40 minutes. The plate was washed with PBST, 100 μL of TMB color development solution (Biopanda, Cat: TMB-S-003) was added and color was developed at 37° C. for 3 minutes, 100 μL of ELISA stop solution (Solarbio, Cat: C1058) was added, and OD450 was detected.

[0659] As shown in Figure 13 and Table 7, the results show that all three fusion proteins bind to BAFF significantly. B613302 has a significantly stronger ability to bind to BAFF than Telitacicept, EC 50 The level of TACI was reduced to 1 / 8 to 1 / 7 of that of BCMA, indicating that placing TACI at the C-terminus of BCMA (such as in the case of B613302) may be more conducive to maintaining the binding ability to BAFF.

[0660] Table 7. Binding of Anifrolumab and TACI-BCMA fusion protein to BAFF

[0661] As shown in Figure 14 and Table 8, the results show that all three fusion proteins bind to APRIL significantly. B613301, B613302 and B613303 bind to APRIL in a similar manner. Compared with Telitacicept, EC 50 The results showed that the BCMA-fused fusion protein indeed enhanced the binding to APRIL.

[0662] Table 8. Binding of Anifrolumab and TACI-BCMA fusion protein to APRIL

[0663] Example 12. Construction, expression and purification of anifrolumab and TACI-19-16-BCMA fusion protein

[0664] BCMA-ECD-1 (amino acid residues 1-43) and BCMA-ECD-2 (amino acid residues 1-41) remove risk points such as glycosylation sites (amino acid residues 42-44, NAS) and deamidation sites (amino acid residues 47-48, NS) that may exist in the BCMA non-CRD domain. Removing risk points can make the fusion protein more uniform.

[0665] As shown in Figure 15, B637301 fuses TACI-19-16 to the N-terminus of BCMA-ECD-1, and then connects TACI-19-16-BCMA-ECD-1 to the C-terminus of the light chain of the anifrolumab antibody through linker 1; B637302 fuses TACI-19-16 to the C-terminus of BCMA-ECD-2, and then connects BCMA-ECD-2-TACI-19-16 to the C-terminus of the light chain of the anifrolumab antibody through linker 1; B746201 fuses TACI-19-16 to the C-terminus of BCMA-ECD-2, and then connects BCMA-ECD-2-TACI-19-16 to the C-terminus of the heavy chain of the anifrolumab antibody through linker 2. B637302-LALA fuses TACI-19-16 to the C-terminus of BCMA-ECD-2, which is then linked to the C-terminus of the anifrolumab light chain via Linker 1. The Fc portion of the anifrolumab antibody is a human IgG1 Fc with L234A and L235A mutations. B746201-LALA fuses TACI-19-16 to the C-terminus of BCMA-ECD-2, which is then linked to the C-terminus of the anifrolumab heavy chain via Linker 2. The Fc portion of the anifrolumab antibody is a human IgG1 Fc with L234A and L235A mutations (see Figure 15).

[0666] >BCMA-ECD-1 (SEQ ID NO: 67)

[0667] >BCMA-ECD-2 (SEQ ID NO: 68)

[0668] >TACI-19-16-BCMA-ECD-1(SEQ ID NO: 69)

[0669] >BCMA-ECD-2-TACI-19-16(SEQ ID NO: 70)

[0670] The heavy chains of B637301 and B637302 are both the heavy chain of Anifrolumab SEQ ID NO: 55, and the sequences of the light chains are as follows:

[0671] >Light chain of B637301 (SEQ ID NO: 71)

[0672] >Light chain of B637302, B637302-LALA (SEQ ID NO: 72)

[0673] The B637302-LALA heavy chain is SEQ ID NO: 61.

[0674] >Heavy chain of B746201 (SEQ ID NO: 73)

[0675] The light chain of B746201 and B746201-LALA is the light chain of Anifrolumab, SEQ ID NO: 54.

[0676] >Heavy chain of B746201-LALA (SEQ ID NO: 74)

[0677] The B637301, B637302, and B746201 fusion proteins were expressed and purified, and SDS-PAGE analysis revealed that the heavy and light chains of the antibodies linked to TACI-BCMA were single bands (results not shown). This result suggests that removing risky glycosylation sites in the BCMA protein sequence can make the fusion proteins more homogeneous.

[0678] Example 13. Inhibitory effect of anifrolumab and TACI-19-16-BCMA fusion protein on type I interferon activity

[0679] The HEK-Blue IFN-α / β cell kit (InvivoGen, Cat: hkb-ifnab) was used to detect the inhibitory effect of anifrolumab and TACI-19-16-BCMA fusion proteins (B606401, B637302, B746201 and B805201) on IFN-α / β activity.

[0680] Method: Digest HEK-Blue TMIFN-α / β cells were resuspended to 2.8E5 / mL in DMEM medium (Gibco, Cat: 11995065) containing 10% FBS and 1% P / S and seeded into 96-well plates (Costar, Cat: 3599). The test antibody was added and the cells were incubated at 37°C for 30 minutes. IFN-β (Sino biological, Cat: 10704-HNAS) was added at a final concentration of 0.01 ng / mL and incubated at 37°C for 24 hours. Twenty microliters of culture supernatant was mixed with 180 microliters of Quanti-Blue (InvivoGen, Cat: rep-qbs) in a new 96-well plate and incubated at 37°C for 1 hour. OD655 was measured.

[0681] Referring to FIG. 16 and Table 9 , the results showed that all four fusion proteins were able to inhibit the activity of IFN-α / β, and the activities of the four fusion proteins were comparable and consistent with those of Anifrolumab.

[0682] Table 9. Fusion proteins of Anifrolumab and TACI-19-16-BCMA

[0683] Inhibition of type I interferon activity

[0684] Example 14. In vitro binding experiment of the fusion protein of Anifrolumab and TACI-19-16-BCMA to BAFF and APRIL

[0685] ELISA was used to detect the binding ability of B606401, B637302, B746201 and B805201 fusion proteins to BAFF and APRIL.

[0686] The method is as follows: BAFF (PeproTech, Cat: 310-13) or ARPIL (Acro Biosystems, Cat: APL-H52D1) was diluted in PBS to 1 μg / mL and coated in 96-well plates overnight at 4°C. The plates were washed with PBST and blocked with Blocking Buffer (PBST containing 1% BSA) at 37°C for 1 hour. The plates were tapped and the test antibody was diluted in Blocking Buffer and incubated at 37°C for 1.5 hours. The plates were washed with PBST and HRP-mouse anti-human Fc antibody (GenScript, Cat: A01854-200) was diluted in Blocking Buffer and incubated at 37°C for 40 minutes. The plates were washed with PBST and developed with 100 μL of TMB colorimetric solution (Biopanda, Cat: TMB-S-003) at 37°C for 3 minutes. 100 μL of ELISA stop solution (Solarbio, Cat: C1058) was added and the OD450 was measured.

[0687] As shown in Figure 17 and Table 10, all four fusion proteins bind to BAFF significantly, and their ability to bind to BAFF is stronger than that of Telitacicept. B606401 and B637302 have similar abilities to bind to BAFF. Compared with Telitacicept, EC 50 The levels of B746201 and B805201 were both reduced to 1 / 4 to 1 / 3 of their levels; B746201 and B805201 had similar abilities to bind to BAFF, and compared with Telitacicept, EC 50 were reduced to 1 / 2 of their levels.

[0688] Table 10. Binding of Anifrolumab and TACI-19-16-BCMA Fusion Protein to BAFF

[0689] As shown in Figure 18 and Table 11, all four fusion proteins bind to APRIL significantly. The ability of B637302 to bind to APRIL is significantly higher than that of Telitacicept. 50 B746201 and B805201 have similar binding abilities to APRIL, compared to the EC of Telitacicept. 50 When reduced to 1 / 3 of its level, B606401's ability to bind APRIL was comparable to that of Telitacicept.

[0690] Table 11. Binding of Anifrolumab and TACI-19-16-BCMA Fusion Protein to APRIL

[0691] Example 15. Plasmacytoid dendritic cell (pDC) function experiment

[0692] To evaluate the in vitro efficacy of anifrolumab and TACI-19-16-BCMA fusion protein, we established an in vitro assay for testing bioactive IFNAR1 antagonists. The functional assay for pDCs was as follows: freshly isolated PBMCs from healthy individuals were cultured in 1640GlutaMAX (sugar concentration = 11 mM) basal medium. PBMCs were plated at 3x10 6The cells were seeded at a density of 100 μg / mL in a 96-well plate and stimulated with 0.5 μM CpG-A ODN2216 (InvivoGen cat: tlrl-2216). Then, the cells were treated with gradient concentrations of anifrolumab, B637302, B606401, and IgG1 isotype. After 24 hours, the IFN-α cytokine secretion level in the cell culture supernatant was detected using a human IFN-α kit (Cisbio cat: 62HIFNAPEG).

[0693] The results showed that B637302, B606401 and the control antibody Anifrolumab were able to inhibit the production of IFN-α in a dose-dependent manner, and the inhibitory activities of the three were comparable ( FIG. 19 ).

[0694] Example 16. IFN-α-induced plasma cell differentiation in vitro functional experiment

[0695] To evaluate the antagonistic biological activity of anifrolumab and TACI-19-16-BCMA fusion protein against IFNAR1, we established a functional experimental method for in vitro differentiation of plasma cells. The method was as follows: human B cells were isolated from fresh healthy human PBMC using a B cell sorting kit (Stemcell cat: 17954) and cultured in vitro in 1640GlutaMAX (Gibco cat: 72400-47) basal medium. B cells were cultured at a density of 1×10 5 The cells were seeded at a density of 100 μg / mL 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). Anifrolumab, B637302, B746201, B606401, B805201, and IgG1 isotype control were added. After 4 days, plasma cells (CD27 + CD38 + ) differentiation ratio.

[0696] Referring to FIG20 , the results showed that B637302, B746201, B606401, B805201 and the control antibody Anifrolumab were able to effectively inhibit the in vitro differentiation of B cells into plasma cells induced by IFN-α in a dose-dependent manner. 50 They were 10.75nM, 1.895nM, 17.45nM, 6.208nM and 12.66nM respectively, indicating that the inhibitory activity of the fusion protein was comparable to that of the control antibody Anifrolumab.

[0697] Example 17. BAFF-induced B cell proliferation function experiment

[0698] To evaluate the in vitro efficacy of anifrolumab and TACI-19-16-BCMA fusion protein, we established an in vitro assay for testing TACI-BCMA bioactive molecules. The BAFF-induced B cell proliferation assay was performed as follows: Human B cells were isolated from fresh healthy human PBMC using a B cell sorting kit (Stemcell cat: 17954) and labeled with CTV (Invitrogen cat: C34557). B cells were cultured in vitro in 1640GlutaMAX (Gibco cat: 72400-47) basal medium at a density of 1×10 5 The cells were seeded at a density of 100 μg / mL / well in a 96-well plate and stimulated with 20 μg / mL anti-human IgM antibody (Jacksonimmuno cat:109-006-129), 10 ng / mL recombinant human IL-4 (PeproTech cat:AF-200-04-20), 100 ng / mL CD40L (R&D cat:2706-CL-025 / CF), 1.5 μg / mL anti-His antibody (R&D cat:MAB050-500), 1 ng / mL recombinant human IL-17 (SinoBiological cat:12047-HNAS), and 200 ng / mL recombinant human BAFF (R&D cat:7537-BF-025 / CF). Under these conditions, gradient concentrations of Telitacicept, B637302, B606401, B80521, and IgG1 isotype were added, and B cell proliferation signals were detected by flow cytometry 5 days later.

[0699] The results showed, as shown in Table 12, that B637302, B606401, B805201, and the control, Telitacicept, all effectively inhibited BAFF-induced B cell proliferation in vitro in a dose-dependent manner. As shown in Figure 21, B637302 and B805201 exhibited significantly stronger inhibitory effects than Telitacicept. At a low drug concentration of 10 nM, Telitacicept exhibited no inhibitory activity, while B637302, B606401, and B805201 all demonstrated significant inhibitory activity against plasma cell differentiation.

[0700] Table 12. Results of BAFF-induced B cell proliferation

[0701] Example 18. APRIL-induced B cell proliferation function experiment

[0702] To evaluate the in vitro efficacy of anifrolumab and TACI-19-16-BCMA fusion protein, we established an in vitro assay for testing TACI-BCMA bioactive molecules. The APRIL-induced B cell proliferation assay was performed as follows: Human B cells were isolated from fresh healthy human PBMC using a B cell sorting kit (Stemcell cat: 17954), labeled with CTV (Invitrogen cat: C34557), and cultured in vitro in 1640GlutaMAX (Gibco cat: 72400-47) basal medium. B cells were plated at 1×10 5 The cells were seeded at a density of 100 μg / mL / well in a 96-well plate and stimulated with 20 μg / mL anti-human IgM antibody (Jacksonimmuno cat:109-006-129), 10 ng / mL recombinant human IL-4 (PeproTech cat:AF-200-04-20), 100 ng / mL CD40L (R&D cat:2706-CL-025 / CF), 1.5 μg / mL anti-His antibody (R&D cat:MAB050-500), 1 ng / mL recombinant human IL-17 (SinoBiological cat:12047-HNAS), and 20 ng / mL recombinant human ARPIL (R&D cat:5860-AP-010 / CF). Under these conditions, gradient concentrations of Telitacicept, B637302, B606401, and IgG1 isotype were added, and B cell proliferation signals were detected by flow cytometry 5 days later.

[0703] The results showed that, as shown in Table 13 and Figure 22, B637302, B606401 and the control Telitacicept were all able to effectively inhibit APRIL-induced B cell proliferation in vitro in a dose-dependent manner, and the efficacy of B637302 was significantly better than that of Telitacicept; under the condition of 1000nM drug treatment, the efficacy of B606401 was also significantly better than that of the control Telitacicept.

[0704] Table 13. Results of APRIL-induced B cell proliferation

[0705] Example 19. Experimental study on the function of BAFF+APRIL-induced plasma cell production

[0706] To evaluate the biological activity of TACI-BCMA in the fusion protein of Anifrolumab and TACI-19-16-BCMA, we established an in vitro experimental method for BAFF+APRIL-induced plasma cell generation. The specific process is as follows: human B cells were isolated from fresh healthy human PBMC using a B cell sorting kit (Stemcell cat: 17954) and cultured in vitro in 1640GlutaMAX (Gibco cat: 72400-47) basal medium. B cells were cultured at a density of 1×10 5 Cells were seeded at a density of 100 μg / well in a 96-well plate and stimulated with 2 μg / mL CpG-B 2006 (InvivoGen, cat: tlrl-2006). On day 4, the medium was replaced with 10 ng / mL recombinant human IL-6, 50 ng / mL recombinant human IL-10 (Peprotech, cat: 200-10), 50 ng / mL recombinant human IL-21 (Peprotech, cat: AF-200-21-10), 500 ng / mL recombinant human BAFF (R&D, cat: 7537-BF-025 / CF), and 50 ng / mL recombinant human ARPIL (R&D, cat: 5860-AP-010 / CF). Seven days later, half of the medium was replaced, and the concentrations of BAFF and ARPIL were adjusted to 50 ng / mL and 500 ng / mL, respectively. The concentrations of the remaining stimulatory signals remained unchanged. During this process, Telitacicept, B637302, B746201, B606401, B805201, and IgG1 isotype were added from day 4 to day 10, and plasma cells (CD27 + CD38 + )number.

[0707] As shown in Table 14, the results of the flow cytometry assay for plasma cell counts on day 10 showed that BAFF and ARPIL were able to effectively induce plasma cell generation in vitro. As shown in Figure 23, under 10 nM drug treatment conditions, Telitacicept was unable to inhibit plasma cell generation, while B637302, B746201, B606401, and B805201 were able to significantly inhibit plasma cell generation, indicating that B637302, B746201, B606401, and B805201 were more effective than Telitacicept in inhibiting BAFF and ARPIL. Under 100 nM drug treatment conditions, all four test drugs and Telitacicept significantly inhibited plasma cell generation. Under 1000 nM drug treatment conditions, B637302 had the strongest activity in inhibiting plasma cell generation, surpassing Telitacicept.

[0708] Table 14. Results of BAFF+APRIL-induced plasma cell production

[0709] Example 20. Experiment on the function of IFN-α+BAFF+APRIL inducing plasma cell production

[0710] To evaluate the synergistic biological activity of Anifrolumab and TACI-19-16-BCMA in the fusion protein of Anifrolumab and TACI-BCMA in the differentiation and generation of B cells into plasma cells, we established an in vitro experimental method for inducing plasma cell generation by IFN-α+BAFF+APRIL. The specific process is as follows: human B cells were isolated from fresh healthy human PBMC using a B cell sorting kit (Stemcell cat: 17954) and cultured in vitro in 1640GlutaMAX (Gibco cat: 72400-47) basal medium. B cells were cultured at a density of 1x10 5 Cells were seeded at a density of 1 / well in a 96-well plate and stimulated with 2 μg / mL CpG-B 2006 (InvivoGen, cat: tlrl-2006) and 250 U / mL IFN-α (Biolegend, cat: 592704). On day 4, the medium was replaced with 10 ng / mL recombinant human IL-6, 50 ng / mL recombinant human IL-10 (Peprotech, cat: 200-10), 15 ng / mL recombinant human IL-21 (Peprotech, cat: AF-200-21-10), 500 ng / mL recombinant human BAFF (R&D, cat: 7537-BF-025 / CF), and 50 ng / mL recombinant human ARPIL (R&D, cat: 5860-AP-010 / CF). Seven days later, half of the medium was replaced, and the concentrations of BAFF and ARPIL were adjusted to 50 ng / mL and 500 ng / mL, respectively. The concentrations of the remaining stimulatory signals remained unchanged. During this process, Anifrolumab, Telitacicept, B637302, B606401, and IgG1 isotype were added at gradient concentrations from day 0 to day 10, and plasma cells (CD27 + CD38 + ) number (A in Figure 24).

[0711] In an in vitro synergistic experiment on plasma cell generation, after optimizing experimental conditions, we found that under IL-21 15 ng / mL conditions, IFN-α and BAFF+APRIL synergistically promoted plasma cell differentiation and generation, with comparable promoting effects. Therefore, we chose to compare the inhibitory activity of various protein drugs at different concentrations under these conditions. The results showed that B637302, B606401, Telitacicept, and Anifrolumab all inhibited plasma cell generation in a dose-dependent manner (see Table 15 and Figures 24B to D). At a low drug concentration of 10 nM, Telitacicept or Anifrolumab alone had no significant inhibitory activity on plasma cell generation, while B637302 and B606401 showed significant inhibitory activity (see Figure 24B).

[0712] Under conditions of drug treatment at a concentration of 100 nM, Anifrolumab alone had no significant inhibitory activity, Telitacicept alone had some activity, and the activity of B637302 was significantly better than Telitacicept, as shown in Figure 24 C. Under conditions of drug treatment at a concentration of 1000 nM, Anifrolumab alone had some activity, and the activity of the B637302 and B606401 fusion proteins was also significantly better than Anifrolumab, as shown in Figure 24 D.

[0713] Table 15. Results of IFN-α+BAFF+APRIL-induced plasma cell production

[0714] Example 21. PEG-IFN-α induced PD experiment in PBMC humanized mice

[0715] To test the in vivo efficacy of anifrolumab and TACI-19-16-BCMA fusion protein, we used B-NDG immunodeficient mice and injected 2×10 7 PBMC cells were humanized and 0.6 μg PEG-IFN-α ( Roche) for induction. In this model, mice were treated with intraperitoneal injections of various concentrations of the drugs anifrolumab, B637302, and B606401, as well as PBS. PBMCs were collected 2 h later and pSTAT1 (#9167, Cell Signaling Technology) levels were detected by flow cytometry. Peripheral blood was collected on days 1 and 3 to isolate PBMCs, and the mRNA expression levels of IFN-α downstream genes (such as ISG-15, IFI13, MX-1, HERC5, etc.) were detected by QPCR, as shown in Figures 25A and 25B.

[0716] Referring to Figure 25C, flow cytometry analysis of pSTAT1 in PBMCs collected 2 hours after induction revealed that B637302 and B606401 exhibited comparable hIFN-α inhibitory activity as anifrolumab. Referring to Figures 25D and 25E, QPCR analysis of ISG mRNA expression levels on days 1, 3, and 7 also revealed similar results, consistent with the results of the previous in vitro reporter assays and biological activity experiments.

[0717] Example 22. BAFF+APRIL-induced PD in mice

[0718] To detect the in vivo efficacy of anifrolumab and the TACI-19-16-BCMA end of the TACI-19-16-BCMA fusion protein, we used C57 / B6 mice and induced them by intraperitoneal injection of 3 μg BAFF (BAF-H52D4-1 mg, AcroBio) and 0.5 μg APRIL (APL-H52D1-1 mg, AcroBio). In this model, mice were treated with different concentrations of drugs such as Telitacicept, B637302, and B606401, as well as PBS, by intraperitoneal injection. Peripheral blood was collected on days 4 and 7, and plasma IgA levels were detected by ELISA (Figure 26A).

[0719] The results showed that there was no significant difference in IgA between the PBS and drug-treated groups on days 0 and 2 (results not shown), while on days 4 and 7, B637302, B606401, and Telitacicept significantly inhibited IgA production compared to the PBS control group, as shown in Figures 26B and 26C. Furthermore, the results on day 7 showed that under equimolar dosing conditions, the efficacy of B637302 > B606401 > Telitacicept, as shown in Figure 26C. This is consistent with the results of previous in vitro BAFF and APRIL ELISA binding experiments and in vitro biological activity experiments.

[0720] Example 23. Establishment of B cell differentiation conditions in vitro

[0721] Existing functional assays for B cells primarily use 3H-labeled thymidine (TdR) methods. 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 the basic stimulation conditions that effectively induce B cell differentiation in vitro.

[0722] B cell sorting and in vitro culture conditions are as follows: human B cells were isolated from fresh healthy human PBMC using B cell sorting kit (Stemcell cat: 17954) and cultured in vitro in 1640GlutaMAX (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 GlutaMAXTM (Gibco cat: 35050061).

[0723] 1) B cell differentiation conditions in vitro

[0724] B cells were cultured at 1x10 5 The cells were seeded at a density of 10 ng / mL recombinant human IL-3, 0.5 μM CpG-A, and stimulated with 1000 U / mL IFNα or 33 ng / mL recombinant human IL-21. After 6 days, plasma cells (CD27 + CD38 + ) differentiation ratio and cell viability.

[0725] The results showed that IL-3 and CpG-A as the basic stimulation could induce the differentiation of some B cells. After adding IL-21 or IFNα, both could further stimulate B cell differentiation (Figure 27A and B), but IL-3, CpG-A and IL-21 could not increase the overall cell viability, while the cell viability increased after adding IFNα (Figure 27C).

[0726] 2) Basic B cell stimulation conditions in vitro

[0727] B cells were cultured at 1x10 5 The cells were seeded at a density of 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) for stimulation. After 6 days, the plasma cells (CD27 + CD38+ ) differentiation ratio.

[0728] The results showed that CpG-B / R848+IFNα effectively stimulated B cell differentiation (Figure 28A and C). However, CpG-B / R848+BAFF+APRIL did not effectively stimulate B cell differentiation under these conditions and only increased cell viability (Figure 28B). CpG-B / R848+IFNα+BAFF+APRIL+RA showed a trend to further promote B cell differentiation on the basis of CpG-B / R848+IFNα (Figure 28A and 28C).

[0729] 3) B cell in vitro differentiation detection time and detection window

[0730] Take CpG-B+IFNα induced differentiation as an example. B cells were cultured at a rate of 1×10 5 The cells were seeded at a density of 1 μg / mL CpG-B / well in a 96-well plate and stimulated with 500 U / mL IFNα for 4 days. During this process, 100 nM Anifrolumab or control IgG was added from day 0 to day 4. After 4 days, plasma cells (CD27 + CD38 + ) differentiation ratio.

[0731] The results showed that when the induction time was set to 4 days, IFNα could not induce B cell differentiation alone, but could promote B cell differentiation based on CpG-B induction. At the same time, the control antibody Anifrolumab could inhibit the effect of IFNα, and there was a detection window with an inhibition rate of 75% (Figure 29).

[0732] Example 24. Evaluation Method for Screening IFNAR1 Antagonists by In Vitro B Cell Differentiation Assay

[0733] Take CpG-B+IFNα induced differentiation as an example. B cells were cultured at a rate of 1×10 5 The cells were seeded at a density of 100 μg / mL in 96-well plates and stimulated with 2 μg / mL CpG-B and 250 U / mL IFNα for 4 days. During this process, gradient concentrations of Anifrolumab were added from day 0 to day 4 for treatment. On day 4, plasma cells (CD27 + CD38 + Repeated testing showed that the method had good repeatability and stability. Table 16 shows the results of two of the tests, Experiment 1 and Experiment 2.

[0734] The results showed that, in this screening method, anifrolumab was able to inhibit the differentiation of B cells into plasmablasts in a dose-dependent manner (Figure 30), indicating that this screening method can be used to screen for molecules such as IFNAR1 antagonists. It also shows that the concentrations of CpG-B and IFNα in this screening method are adjustable within a certain range.

[0735] Table 16. Results of IFNα-induced B cell differentiation

[0736] Example 25. Evaluation Method for Screening BAFF / ARPIL Antagonists by In Vitro B Cell Differentiation Assay

[0737] To clarify the conditions for BAFF / ARPIL-induced plasma cell generation, we established a functional experimental method for plasma cell generation in vitro.

[0738] 1) IL-3+CpG-A+IFNα

[0739] B cells were cultured at 1×10 5 The cells were seeded at a density of 100 μg / mL in a 96-well plate and stimulated with 10 ng / mL recombinant human IL-3, 0.5 μM CpG-A, 1000 U / mL IFNα or 4 nM recombinant human BAFF and 3 nM recombinant human APRIL. After 6 days, plasma cells (CD27 + CD38 + ) differentiation ratio.

[0740] The results showed that IL-3 and CpG-A as a primary stimulus induced some B cells to differentiate into plasma cells, but the proportion was low. Adding IFNα to this basal stimulus further stimulated B cell differentiation (Figure 31A and B). However, the addition of BAFF+APRIL to IL-3, CpG-A, or IL-3, CpG-A, and IFNα significantly reduced the ability to induce B cell differentiation (Figure 31B).

[0741] 2) CpG-B + IFNα

[0742] B cells were cultured at 1×10 5 The cells were seeded at a density of 100 μg / mL in a 96-well plate and stimulated with 2 μg / mL CpG-B for 4 days. On the 4th day, 250 U / mL IFNα or 500 ng / mL recombinant human BAFF and 50 ng / mL recombinant human APRIL were added for 3 days. On the 7th, 10th, 14th, and 18th days, half of the culture 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. The plasma cells (CD27 + CD38 + )quantity.

[0743] The results showed that under these conditions, CpG-B + BAFF + ARPIL could not induce plasma cell production in vitro. CpG-B + IFNα could induce plasma cell production in vitro, but BAFF + APRIL could not further induce plasma cell production on this basis (Figure 32). Furthermore, the window for detecting plasma cell production was larger before day 10 and narrower after day 14.

[0744] 3) CpG-B + IFNα is the early stimulation condition, and IL-6 is the late stimulation condition

[0745] BAFF / APRIL acts on the late stage of plasma cell production. B cells were cultured at a rate of 1×10 5 The cells were seeded in a 96-well plate at a density of 1 / well. B cells were stimulated according to any of the following conditions a)-d) and plasmablasts (CD27 + CD38 + )quantity.

[0746] a) Stimulate with 2 μg / mL CpG-B and 250 U / mL IFNα for 4 days. On day 4, remove CpG-B and IFNα and add 10 ng / mL recombinant human IL-6, 500 ng / mL recombinant human BAFF, and 50 ng / mL recombinant human APRIL for 3 days. On days 7 and 11, replace half of the medium with recombinant human BAFF and recombinant human APRIL adjusted to 50 ng / mL and 500 ng / mL, respectively.

[0747] b) Add 2 μg / mL CpG-B and stimulate for 4 days. On the 4th day, remove CpG-B and add 10 ng / mL recombinant human IL-6 to continue stimulation.

[0748] c) Add 2 μg / mL CpG-B and 250 U / mL IFNα for stimulation for 4 days. On the 4th day, remove CpG-B and IFNα and add 10 ng / mL recombinant human IL-6 to continue stimulation.

[0749] d) Stimulate with 2 μg / mL CpG-B for 4 days. On day 4, remove CpG-B and stimulate with 10 ng / mL recombinant human IL-6, 500 ng / mL recombinant human BAFF, and 50 ng / mL recombinant human APRIL for 3 days. On days 7 and 11, half of the culture medium was replaced with recombinant human BAFF and recombinant human APRIL adjusted to 50 ng / mL and 500 ng / mL, respectively.

[0750] The results showed that when CpG-B was used as the initial basal stimulus, IL-6+BAFF+APRIL could not induce plasma cell production alone in the later stage under this condition, nor could it further induce plasma cell production based on the initial induction of IFNα ( FIG. 33 ).

[0751] 4) CpG-B is the early stimulation condition, and IL-6+IL-10+IL-21 is the late stimulation condition

[0752] B cells were cultured at 1×10 5 The cells were seeded at a density of 100 μg / mL at 400 μg / well in a 96-well plate and stimulated with 2 μg / mL CpG-B (Invivogen cat: tlrl-2006) for 4 days. On the 4th day, CpG-B was removed and 10 ng / mL recombinant human IL-6, 50 ng / mL recombinant human IL-10, 5 ng / mL recombinant human IL-21, 500 ng / mL recombinant human BAFF and 50 ng / mL recombinant human APRIL were added for 3 days. On the 7th day, half of the culture medium was replaced with the culture medium containing recombinant human BAFF and recombinant human APRIL at concentrations of 50 ng / mL and 500 ng / mL, respectively. On the 10th day, the plasmablasts (CD27 + CD38 + )quantity.

[0753] The results showed that when CpG-B was used as the initial basal stimulation, IL-6+IL-10+BAFF+APRI and 5 ng / mL IL-21 could not induce plasma cell production in the later stage ( FIG. 34 ).

[0754] 5) CpG-B is the early stimulation condition, and IL-6+IL-10+IL-21 is the late stimulation condition

[0755] B cells were cultured at 1×10 5 The cells were seeded at a density of 100 μg / mL / well in a 96-well plate and stimulated with 2 μg / mL CpG-B for 5 days. On the 5th day, CpG-B was removed and 10 ng / mL recombinant human IL-6, 50 ng / mL recombinant human IL-10, 50 ng / mL recombinant human IL-21, 500 ng / mL recombinant human BAFF and 50 ng / mL recombinant human APRIL were added for 2 days. On the 7th day, half of the culture 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. The plasmablasts (CD27 + CD38 + )quantity.

[0756] The results showed that when CpG-B was used as the initial basal stimulation, IL-6+IL-10+BAF+APRIL and 50 ng / mL IL-21 were able to induce plasma cell production in the later stage, and there was a detection window from the ninth to the eleventh day, and the detection window showed a gradually increasing trend (Figure 35).

[0757] 6) CpG-B is the early stimulation condition, and IL-6+IL-10+IL-21 is the late stimulation condition

[0758] B cells were expressed at 1×10 5 The cells were seeded at a density of 100 μg / mL / well in a 96-well plate and stimulated with 2 μg / mL CpG-B for 4 days. On the 4th day, CpG-B was removed and 10 ng / mL recombinant human IL-6, 50 ng / mL recombinant human IL-10, 50 ng / mL recombinant human IL-21, 500 ng / mL recombinant human BAFF and 50 ng / mL recombinant human APRIL were added for 3 days. On the 7th day, half of the culture medium was replaced with the recombinant human BAFF and recombinant human APRIL at concentrations of 50 ng / mL and 500 ng / mL respectively. On the 10th day, plasmablasts (CD27 + CD38 + )quantity.

[0759] We used this in vitro cell model to screen for candidate fusion proteins. Specifically, Telitacicept was added at varying concentrations from day 4 to day 10. The results are shown in Figure 23 . These results demonstrate that Telitacicept inhibited plasma cell generation in vitro in a dose-dependent manner using this screening method ( Figure 23 ), suggesting that this screening method can be used to screen for drugs targeting BAFF / APRIL, such as the TACI-Fc molecule or other similar drugs (e.g., Telitacicept, B637302, and B606401).

[0760] Repeated tests showed that the method had good repeatability and stability. Table 17 shows the test results of two of them, Experiment 1 and Experiment 2.

[0761] Table 17. BAFF+APRIL-induced plasma cell production data

[0762] 7) CpG-B + IFNα is the early stimulation condition, IL-6 + IL-10 + IL-21 is the late stimulation condition

[0763] B cells were cultured at 1×10 5The cells were seeded at a density of 100 μg / mL at 400 μg / well in a 96-well plate and stimulated with 2 μg / mL CpG-B and 250 U / mL IFNα for 5 days. On the 5th day, CpG-B and IFNα were removed and 10 ng / mL recombinant human IL-6, 50 ng / mL recombinant human IL-10, different concentrations (15, 25 or 40 ng / mL) of recombinant human IL-21, 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 the 7th day, half of the culture 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 the 10th day, the plasmablasts (CD27 + CD38 + )quantity.

[0764] The results showed that when CpG-B+IFNα was used as the initial basal stimulation, IL-6+IL-10+BAF+APRIL and 15 ng / mL IL-21 could synergistically induce plasma cell production, while 25 or 40 ng / mL IL-21 could not synergistically induce plasma cell production ( FIG. 36 ).

[0765] The reagent information used above is: IL-3 (R&D cat: 203-IL-010 / CF), CpG-A (Invivogen cat: tlrl-2216), CpG-B (Invivogen cat: tlrl-2006), IFNα (Biolegend 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), retinoic acid (RA, Sigma-Aldrich cat: R2625).

[0766] Although specific embodiments of the present disclosure have been described above, those skilled in the art will appreciate that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present disclosure. Therefore, the scope of protection of the present disclosure is defined by the appended claims.

Claims

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

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

3. The method according to claim 1 or 2, further comprising: Step 7) selecting a test sample that can activate or inhibit B cell differentiation as an immunomodulator; optionally, step 6) includes detecting CD27 + CD38 + The percentage of B cells was used to determine the degree of B cell differentiation.

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

5. The method according to any one of claims 1 to 3, wherein when the TNF family member is BAFF and APRIL, adjusting the concentration of the TNF family member comprises reducing the concentration of BAFF and / or increasing the concentration of APRIL; Preferably, the BAFF concentration in step 4) is reduced to 0.01-0.9 times the concentration of BAFF added in step 3), more preferably 0.02-0.5 times, 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, about 0.9 times; Preferably, the concentration of APRIL in step 4) is increased to 1.1-100 times, more preferably 5-50 times, and 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).

6. A method according to any one of the preceding claims, wherein: Step 1) administering 0.1-10 μg / mL CpG-B or CpG-A or R848 to B cells; culturing for 2-8 days, Step 2) removing CpG-B or CpG-A or R848; Step 3) Add 2-100 ng / mL IL-6, 10-1000 ng / mL IL-10, and 10-200 ng / mL IL-21; as well as 10-2000 ng / mL BAFF and 10-2000 ng / mL APRIL; culture for 2-5 days; Step 4) reducing BAFF concentration and increasing APRIL concentration; Step 5) adding a test sample on any day during the culture of the B cells to detect B cell differentiation; Step 6) selecting a test sample that can activate or inhibit B cell differentiation as an immunomodulator; Preferably, Step 1) On day 0, administer 0.5-5 μg / mL CpG-B to B cells; Step 2) Remove CpG-B on days 3-5; Step 3) Add 10-50 ng / mL IL-6, 20-200 ng / mL IL-10, and 20-200 ng / mL IL-21; and add 200-1000 ng / mL BAFF and 20-200 ng / mL APRIL, and stimulate for another 2-4 days; Step 4) reducing the BAFF concentration to 20-200 ng / mL and increasing the APRIL concentration to 200-1000 ng / mL; Step 5) adding the test sample on days 0-21 to detect B cell differentiation; Step 6) selecting a test sample that can activate or inhibit B cell differentiation as an immunomodulator; More preferably, Step 1) On day 0, administer 1-2 μg / mL 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 BAFF concentration to about 50 ng / mL and the APRIL concentration to about The total is about 500ng / mL; Step 5) adding the test sample on days 0-21 to detect B cell differentiation; Step 6) Select a test sample that can activate or inhibit B cell differentiation as an immunomodulator.

7. A method according to any one of the preceding claims, wherein: Step 1) administering 0.1-10 μg / mL CpG-B or CpG-A or R848 and 50-1000 U / mL IFNα to B cells; culturing for 2-8 days; Step 2) removing CpG-B or CpG-A or R848 and removing IFNα; Step 3) Add 2-100 ng / mL IL-6, 10-1000 ng / mL IL-10, and 10-200 ng / mL IL-21; as well as 10-2000 ng / mL BAFF and 10-2000 ng / mL APRIL; culture for 2-5 days; Step 4) reducing BAFF concentration and increasing APRIL concentration; Step 5) adding the test sample on days 0-21 to detect B cell differentiation; Step 6) selecting a test sample that can activate or inhibit B cell differentiation as an immunomodulator; Preferably, Step 1) On day 0, 0.5-5 μg / mL CpG-B and 100-500 U / mL IFNα are administered to B cells to stimulate B cells; Step 2) Remove CpG-B and IFNα on days 3-5; Step 3) Add 10-50 ng / mL IL-6, 20-200 ng / mL IL-10, and 5-22 ng / mL IL-21; and add 200-1000 ng / mL BAFF and 20-200 ng / mL APRIL, and stimulate for another 2-4 days; Step 4) reducing the BAFF concentration to 20-200 ng / mL and increasing the APRIL concentration to 200-1000 ng / mL; Step 5) adding the test sample on days 0-21 to detect B cell differentiation; Step 6) selecting a test sample that can activate or inhibit B cell differentiation as an immunomodulator; More preferably, Step 1) On day 0, administer approximately 1-2 μg / mL CpG-B and approximately 250-500 U / mL IFNα to B cells; Step 2) After 4 or 5 days, remove CpG-B and IFNα; 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 BAFF concentration to about 50 ng / mL and the APRIL concentration to about 500 ng / mL; Step 5) adding the test sample on days 0-21 to detect B cell differentiation; Step 6) Select a test sample that can activate or inhibit B cell differentiation as an immunomodulator.

8. Methods for screening immunomodulators, including: 1) administering TLR agonists and interferon to B cells; 2) Add the sample to be tested on any day from day 0 to day 21 and continue culturing; 3) determine the degree of B cell differentiation; Preferably, the method further comprises: 4) Selecting a test sample that can activate or inhibit B cell differentiation as an immunomodulator.

9. The method according to claim 8, wherein: The TLR agonist is selected from one or more of TLR7, TLR8 and TLR9 agonists; preferably, the TLR agonist is selected from one or more of 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.

10. The method according to claim 8 or 9, wherein: 1) administering 0.1-20 μg / mL CpG-B and 50-1000 U / mL IFNα to B cells; 2) Add the sample to be tested on any day from day 0 to day 21 and continue culturing; 3) determine the degree of B cell differentiation; Preferably, the method further comprises: 4) Selecting a test sample that can activate or inhibit B cell differentiation as an immunomodulator; Preferably, step 1) administering 0.5-5 μg / mL CpG-B and 100-800 U / mL IFNα to B cells; More preferably, step 1) administers about 1-2 μg / mL CpG-B and 250-500 U / mL IFNα to the B cells.

11. The method according to any one of the preceding claims, wherein the immunomodulator is a type I interferon pathway modulator and / or a TNF pathway modulator; Preferably, the type I interferon pathway regulator is an IFNAR1 signaling pathway regulator; Preferably, the TNF pathway regulator is a BAFF and / or ARRIL pathway regulator.

12. The method according to any one of the preceding claims, wherein the activation or inhibition of B cell differentiation is achieved by detecting the number or proportion of B cells that differentiate into plasma cells, wherein the plasma cells are preferably CD27 + CD38 + of B cells.

13. The method according to any one of the preceding claims, wherein the concentration of the B cells is 1 x 10 5 / hole.

14. The method according to any one of the preceding claims, wherein the B cells are obtained by: 1) Isolation of B cells from PBMCs, and 2) Culture in 1640 medium supplemented with 50 μM β-mercaptoethanol, 100 μM MEM non-essential amino acids, sodium pyruvate, and GlutaMAX.

15. The method according to any one of the preceding claims, wherein the immunomodulator 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; the protein or polypeptide is preferably an antibody or antigen-binding fragment thereof, a cytokine, a receptor, or a ligand; Preferably, the immunomodulator is used to treat B cell disorders or autoimmune diseases; 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 the group consisting of systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, insulin-dependent diabetes mellitus, Crohn's disease, rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis and psoriatic arthritis; and the B cell disorder is selected from the group consisting of tumors, chronic leukocytic leukemia, multiple myeloma, non-Hodgkin's lymphoma, post-transplant lymphoproliferative disease and light chain gammaglobulinopathy.

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

19. A method for treating or ameliorating a disease or condition using the immunomodulator according to any one of claims 1 to 16 or the pharmaceutical composition according to claim 17, the method comprising administering the immunomodulator according to any one of claims 1 to 16 or the pharmaceutical composition according to claim 17 to a subject in need thereof; 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 the group consisting of systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, insulin-dependent diabetes mellitus, Crohn's disease, rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis and psoriatic arthritis; and the B cell disorder is selected from the group consisting of tumors, chronic leukocytic leukemia, multiple myeloma, non-Hodgkin's lymphoma, post-transplant lymphoproliferative disease and light chain gammaglobulinopathy.

20. A method for inducing B cell differentiation, comprising the step of administering a TLR agonist and IFN to B cells; 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.

21. The method according to claim 19, comprising: administration of CpG-A and IFNα to B cells; or a step of administering CpG-B or R484, and IFNα to B cells; Optionally, the method comprises: A step of administering CpG-A and IFNα to B cells; or administering CpG-B or R484, and IFNα to B cells; as well as, A step of simultaneously administering IL-3 and IL-21 to B cells.

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

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

24. A cell model for screening immunomodulators, comprising: B cells induced to differentiate by the method according to any one of claims 20 to 23.

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