Use of multispecific antibody for treating autoimmune disease

Abstract

The present invention relates to the use of a trispecific antibody that specifically binds to CD19, CD3 and CD28 in the preparation of a drug for treating an autoimmune disease, wherein the autoimmune disease is an autoimmune disease based on a B cell, in particular an autoimmune disease based on an autoreactive B cell.

Description

Use of multispecific antibodies for treating autoimmune diseases

[0001] This application claims priority to Chinese patent application 2024110888395 with a filing date of 2024 / 8 / 8, Chinese patent application 2025104279248 with a filing date of 2025 / 4 / 7 and Chinese patent application 2025107778661 with a filing date of 2025 / 6 / 11. This application incorporates the entire contents of the above-mentioned Chinese patent applications. TECHNICAL FIELD

[0002] The present application relates to the field of biological medicine, in particular to the use of a trispecific antibody in treating autoimmune diseases. BACKGROUND

[0003] Autoimmune diseases (AID) are chronic inflammatory diseases caused by the body attacking its own antigens and producing immune responses, which damage its own tissues and organs. It is currently believed that the development of autoimmune diseases requires the participation of both genetic and environmental factors. Autoimmune diseases are a heterogeneous disease caused by the dysfunction of B cells and T cells, leading to the formation of autoreactive T cells and B cells and autoantibodies. Autoimmune diseases are a heterogeneous disease, and the common disease basis is the impairment of immune tolerance. Autoimmune diseases are the third largest disease threatening human health after cardiovascular diseases and cancer. Autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, myasthenia gravis, pemphigus, multiple sclerosis, etc. Autoimmune diseases affect about 10% of the global population, with a higher incidence in women than in men, and the incidence increases with age, causing a serious burden to patients and society. At present, for autoimmune diseases, anti-inflammatory treatment is usually used in clinic, such as using glucocorticoids and other drugs to relieve inflammatory symptoms, or using immunosuppressive agents to inhibit abnormal reactions of the immune system. Although these drugs have strong anti-inflammatory and immunosuppressive effects, which help the immune system to return to normal, there are still problems in clinic such as long treatment cycle, some patients cannot tolerate or have poor efficacy, increase of infection and relapse after drug withdrawal, etc. In addition, non-targeted treatment also brings a series of unavoidable side effects.

[0004] B cell over-activation driven immune imbalance is the core pathogenesis of many autoimmune diseases. When the self-reactive B cells attacking self-antigens are over-activated in the body, a large amount of autoantibodies and immune complexes are produced. These antibodies and immune complexes can cause damage to different tissues and organs, leading to the occurrence of various autoimmune diseases, such as Sjogren's syndrome, idiopathic inflammatory myopathy (IIM), systemic sclerosis, systemic vasculitis, rheumatoid arthritis, and the like, and the production of pathological antibodies such as anti-CCP antibodies in rheumatoid arthritis, anti-SSA antibodies in Sjogren's syndrome, and the like. In recent years, self-reactive B cells have become a therapeutic target for many autoimmune diseases. Targeting self-reactive B cells therapy can inhibit the activation and proliferation of self-reactive B cells and reduce the production of autoantibodies. The B cell depletion therapies (BCDT) born from this, such as the drug Rituximab which directly targets the B cell surface antigen CD20, has played a role in autoimmune diseases. Rituximab is a chimeric anti-CD20 antibody that can effectively and reliably deplete CD20-positive B cells. Two large-scale phase III randomized controlled trials, including moderate to severe lupus but non-renal systemic lupus erythematosus (SLE) patients (EXPLORER) and lupus nephritis III or IV (LUNAR) patients, were conducted to evaluate the efficacy of Rituximab in the treatment of SLE. However, compared with placebo, neither of the two trials was able to achieve the primary endpoint of significantly reducing disease activity, but there were some beneficial treatment effects, such as statistically significant serological improvement, and improvement in proteinuria in subjects in the LUNAR study.

[0005] In addition to Rituximab, the currently marketed Belimumab targeting B cell stimulating factor (BLyS) and Atacicept targeting B cell stimulating factor (BLyS) and proliferation-inducing ligand (APRIL) can also be used for the treatment of autoimmune diseases. Like Rituximab, Belimumab and Atacicept also aim to reduce B cell-mediated autoimmune responses to treat autoimmune diseases. These two drugs work by inhibiting abnormal B cell activation and have limited down-regulation of B cells and autoantibodies.

[0006] Therefore, there is still a need in the art for effective and safe treatment of autoimmune diseases. SUMMARY

[0007] CD19 is a marker molecule on the surface of B cells. Compared with the target of rituximab, CD20, CD19 is expressed in a wider range of B cell lines, from pro-B cells to most plasma cells, while CD20 is not expressed in pro-B cells and plasma cells. Therefore, CD19 has a broader spectrum of advantages in targeting B cells than CD20. In addition, most CD19 in the human body is expressed in B lineage cells, and other cells rarely express CD19, while CD20 is also expressed in CD4 + T cells. Therefore, CD19 has a more specific advantage in targeting B cells than CD20. As can be seen, direct depletion of CD19 + B cells can more effectively deplete autoreactive B cells and thus deplete autoantibodies. More importantly, after B cells are activated, they gradually differentiate into plasmablasts, which are precursors of plasma cells. Plasmablasts can directly participate in autoimmune diseases and have long-term effects on diseases. Part of the plasmablasts transform into short-lived antibody-secreting plasma cells and thus participate in autoimmune diseases, and part of the plasmablasts form long-lived plasma cells and survive in the body for a long time. Long-lived plasma cells are not affected by hormones, traditional immunosuppressants and cytotoxic drugs; due to the lack of expression of CD20, they can even escape the attack of B cell targeted drugs. Long-lived plasma cells can continuously secrete autoimmune antibodies for a long period of time, and trigger memory B cells to differentiate into plasmablasts again, and then form short-lived plasma cells and secrete a large amount of autoimmune antibodies, causing disease recurrence.

[0008] Two activation signals are required for the synergistic effect of initial T lymphocytes from quiescence to complete activation. Among them, the first signal is transmitted by CD3 after the antigen / MHC complex is recognized by TCR. This signal is not enough to completely activate T cells, and the signal generated by the co-stimulatory molecule is needed as a supplement. Lack of co-stimulatory signal is easy to lead to T cell anergy or activation-induced T cell apoptosis. Among the currently discovered T cell co-activation molecules, the CD28 and B7 families are the most important class of co-stimulatory molecules, which can bind to ligands CD80 (B7-1) and CD86 (B7-2) and amplify the first signal transmitted by TCR / CD3, maintain T cell survival, and promote cytokine-induced T cell proliferation and differentiation.

[0009] The applicant of the present application has developed a recombinant Tri-specific T cell engager (TriTE) antibody drug, which is formed by three single-chain variable fragments (scFv) recognizing human CD19, CD3 and CD28 antigens in series. The Tri-specific antibody mediates T cell proliferation and activation by giving the first signal (TCR / CD3) and co-stimulatory signal (CD28) required for T cell activation while promoting the formation of a targeted bridge between T cells and CD19-positive B cells, enhancing the immune response. Compared with a bispecific T cell engager antibody containing only a single CD3 signal, the Tri-specific antibody more fully activates T cells, effectively avoids T cell anergy or activation-induced T cell apoptosis, and safely and efficiently eliminates autoreactive B cells, thereby having better therapeutic effects in treating autoimmune diseases.

[0010] Compared with belimumab and etanercept, the Tri-specific antibody also contains a domain targeting activated T cells, so it can directly bridge T lymphocytes and CD19-positive B lymphocytes, form an immunological synapse, mediate T cell proliferation and activation, directly kill CD19-positive B lymphocytes, and achieve depletion of B lymphocytes, providing a possibility for treating autoimmune diseases. The present application hopes to provide a new idea for treating patients with autoimmune diseases, i.e., directly killing B lymphocytes based on the principle of T cell double-signal activation system, reducing the production of autoantibodies, and providing better treatment options for patients with autoimmune diseases, especially those based on B cell-driven autoimmune diseases, and alleviating the suffering of patients.

[0011] The inventors found that the Tri-specific antibody developed previously, which can simultaneously bind CD19, CD3 and CD28, has better therapeutic effects in treating autoimmune diseases when exploring new uses of the Tri-specific antibody. Therefore, the inventors believe that the Tri-specific antibody has a use in treating autoimmune diseases and can be used to prepare a drug for treating autoimmune diseases. Because the Tri-specific antibody specifically targets B cells and produces an effect of eliminating autoreactive B cells, the Tri-specific antibody has a significant progress in treating B cell-based autoimmune diseases, especially autoreactive B cell-based autoimmune diseases.

[0012] In a first aspect, the present application provides a use of a Tri-specific antibody in the preparation of a medicament for autoimmune diseases. The pharmaceutical use is based on the advantages of the Tri-specific antibody in treating autoimmune diseases.

[0013] In a specific embodiment, the Tri-specific antibody simultaneously binds CD19, CD3 and CD28, and comprises:

[0014] (1) a first binding domain specifically binding to CD19;

[0015] (2) a first linker sequence;

[0016] (3) a second binding domain that specifically binds CD3;

[0017] (4) a second linker sequence; and

[0018] (5) a third binding domain that specifically binds CD28.

[0019] In preferred embodiments, the trispecific antibody comprises:

[0020] (1) a first binding domain that specifically binds CD19 comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising a heavy chain complementarity determining region 1 (HCDR1) as set forth in SEQ ID NO: 1, a heavy chain complementarity determining region 2 (HCDR2) as set forth in SEQ ID NO: 2, and a heavy chain complementarity determining region 3 (HCDR3) as set forth in SEQ ID NO: 3, the VL comprising a light chain complementarity determining region 1 (LCDR1) as set forth in SEQ ID NO: 4, a light chain complementarity determining region 2 (LCDR2) as set forth in SEQ ID NO: 5, and a light chain complementarity determining region 3 (LCDR3) as set forth in SEQ ID NO: 6;

[0021] (2) a second binding domain that specifically binds CD3 comprising a VH and a VL, the VH comprising a HCDR1 as set forth in SEQ ID NO: 12, a HCDR2 as set forth in SEQ ID NO: 13, and a HCDR3 as set forth in SEQ ID NO: 14, the VL comprising a LCDR1 as set forth in SEQ ID NO: 15, a LCDR2 as set forth in SEQ ID NO: 16, and a LCDR3 as set forth in SEQ ID NO: 17;

[0022] (3) a third binding domain that specifically binds CD28 comprising a VH and a VL, the VH comprising a HCDR1 as set forth in SEQ ID NO: 23, a HCDR2 as set forth in SEQ ID NO: 24, and a HCDR3 as set forth in SEQ ID NO: 25, the VL comprising a LCDR1 as set forth in SEQ ID NO: 26, a LCDR2 as set forth in SEQ ID NO: 27, and a LCDR3 as set forth in SEQ ID NO: 28.

[0023] In more preferred embodiments, the trispecific antibody comprises:

[0024] (1) a first binding domain that specifically binds to CD19, comprising a VH comprising an amino acid sequence as set forth in SEQ ID NO: 7, or comprising an amino acid sequence that has at least 90% sequence homology to the amino acid sequence as set forth in SEQ ID NO: 7, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 8, or comprising an amino acid sequence that has at least 90% sequence homology to the amino acid sequence as set forth in SEQ ID NO: 8;

[0025] (2) a first linker sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36;

[0026] (3) a second binding domain that specifically binds to CD3, comprising a VH comprising an amino acid sequence as set forth in SEQ ID NO: 18, or comprising an amino acid sequence that has at least 90% sequence homology to the amino acid sequence as set forth in SEQ ID NO: 18, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 19, or comprising an amino acid sequence that has at least 90% sequence homology to the amino acid sequence as set forth in SEQ ID NO: 19;

[0027] (4) a second linker sequence comprising an amino acid sequence as set forth in SEQ ID NO: 37; and

[0028] (5) a third binding domain that specifically binds to CD28, comprising a VH comprising an amino acid sequence as set forth in SEQ ID NO: 29, or comprising an amino acid sequence that has at least 90% sequence homology to the amino acid sequence as set forth in SEQ ID NO: 29, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 30, or comprising an amino acid sequence that has at least 90% sequence homology to the amino acid sequence as set forth in SEQ ID NO: 30.

[0029] In preferred embodiments, the trispecific antibody is a single chain fusion protein comprising: a scFv that specifically binds to CD19 as the first binding domain; a scFv that specifically binds to CD3 as the second binding domain; and a scFv that specifically binds to CD28 as the third binding domain.

[0030] In more preferred embodiments, in the trispecific antibody,

[0031] (1) the first binding domain comprises an amino acid sequence as set forth in SEQ ID NO: 11, or comprises an amino acid sequence that has at least 90% sequence homology to the amino acid sequence as set forth in SEQ ID NO: 11;

[0032] (2) the second binding domain comprises an amino acid sequence as shown in SEQ ID NO: 22, or an amino acid sequence with at least 90% sequence identity to the amino acid sequence as shown in SEQ ID NO: 22; and / or

[0033] (3) the third binding domain comprises an amino acid sequence as shown in SEQ ID NO: 33, or an amino acid sequence with at least 90% sequence identity to the amino acid sequence as shown in SEQ ID NO: 33.

[0034] In a still more preferred embodiment, the trispecific antibody comprises an amino acid sequence as shown in SEQ ID NO: 38, or an amino acid sequence with at least 90% sequence identity to the amino acid sequence as shown in SEQ ID NO: 38.

[0035] In a further embodiment of the first aspect, the autoimmune disease is preferably a B cell- based autoimmune disease, more preferably an autoimmune disease based on autoreactive B cells.

[0036] In a specific embodiment, the autoimmune disease based on autoreactive B cells is selected from the group consisting of systemic lupus erythematosus (SLE), lupus nephritis, type 1 diabetes, systemic sclerosis, idiopathic inflammatory myopathy (IIM), Sjogren’s syndrome, systemic vasculitis, rheumatoid arthritis, anti-synthetase syndrome, MDA5+ dermatomyositis, MDA5+ dermatomyositis interstitial lung fibrosis, primary membranous nephropathy, limbic encephalitis, scleromyositis, anti-phospholipid syndrome, autoimmune hemolytic anemia, IgG4-related disease, Wegener’s granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, polyarteritis nodosa, chronic autoimmune hepatitis, primary biliary cirrhosis, myasthenia gravis (MG), neuromyelitis optica spectrum disorder (NMOSD), multiple sclerosis (MS), Lambert-Eaton syndrome, Hashimoto’s thyroiditis, Graves’ disease, and paraneoplastic cerebellar syndrome.

[0037] In a preferred embodiment, the autoimmune disease based on autoreactive B cells is selected from the group consisting of systemic lupus erythematosus, systemic sclerosis, IgG4-related disease, anti-synthetase syndrome, MDA5+ dermatomyositis interstitial lung fibrosis, autoimmune hemolytic anemia, rheumatoid arthritis, and idiopathic inflammatory myopathy.

[0038] For example, the autoimmune disease based on autoreactive B cells is systemic lupus erythematosus (SLE).

[0039] For example, the autoimmune disease based on self-reactive B cells is systemic sclerosis.

[0040] For example, the autoimmune disease based on self-reactive B cells is rheumatoid arthritis.

[0041] For example, the autoimmune disease based on self-reactive B cells can be anti-synthetase syndrome.

[0042] For example, the autoimmune disease based on self-reactive B cells can be IgG4-related disease.

[0043] For example, the autoimmune disease based on self-reactive B cells can be autoimmune hemolytic anemia.

[0044] For example, the autoimmune disease based on self-reactive B cells can be MDA5+ dermatomyositis interstitial pulmonary fibrosis.

[0045] For example, the autoimmune disease based on self-reactive B cells is idiopathic inflammatory myopathy (IIM).

[0046] For example, the autoimmune disease based on self-reactive B cells is myasthenia gravis (MG).

[0047] For example, the autoimmune disease based on self-reactive B cells is immune thrombocytopenia (ITP).

[0048] In a further implementation form of the first aspect, the medicament comprising the trispecific antibody has different administration modes.

[0049] In a second aspect, the present application provides a pharmaceutical composition comprising the aforementioned trispecific antibody binding CD19, CD3 and CD28 simultaneously and / or a pharmaceutically acceptable carrier.

[0050] The trispecific antibody of the present application has at least the following advantages in treating autoimmune diseases:

[0051] (1) The application of CD19xCD3xCD28 trispecific antibody in treating autoimmune diseases is developed

[0052] The present application develops the use of the trispecific antibody in treating autoimmune diseases, which is the first use of a specific trispecific antibody in treating autoimmune diseases, and has important significance in developing new drugs for treating autoimmune diseases.

[0053] (2) More targeted targeting of B cells with a wider lineage

[0054] The pathogenesis of many autoimmune diseases is associated with abnormal proliferation and abnormal activation of autoreactive B cells, and thus the clearance of B cells is an important direction for the treatment of autoimmune diseases. There are two important targets for B cells, CD19 and CD20, and targeting CD19 is more specific and targets more types of B cells than targeting CD20. This is because: (a) CD19 is expressed on most B lineage cells, including pre-B cells and plasma cells, while the expression spectrum of CD20 is limited and does not express on pre-B cells and plasma cells; (b) CD19 is mostly expressed on B lineage cells in the human body, and other cells rarely express CD19, while CD20 is also expressed on CD4 + T cells.

[0055] (3) Advantages based on trispecific T cell engager

[0056] The trispecific antibody of the present application can simultaneously activate both CD3 and CD28 T cell stimulation signals, and such a "double signal" activation mechanism can better regulate the TCR / CD3 signal to produce a long-term effective immune response in the body, i.e., by inducing activated T cells rather than the drug itself to act in the body, thereby reducing the frequency of administration even in the case of a shorter half-life of the drug (relative to antibodies containing Fc regions), and maintaining long-term activation of T cells by the drug without the need for frequent continuous infusion.

[0057] Other aspects and advantages of the present application will be readily apparent to those skilled in the art from the detailed description that follows, simply indicating and describing illustrative embodiments of the present application. As will be realized, the present application is capable of modifications in various obvious aspects, without departing from the spirit and scope of the inventive concepts presented herein. Accordingly, the drawings and descriptions are to be regarded as illustrative in nature and not restrictive. BRIEF DESCRIPTION OF DRAWINGS

[0058] The specific features of the present application are shown in the appended claims. The features and advantages of the present application can be better understood by referring to the detailed description of the exemplary embodiments and the accompanying drawings. The drawings are briefly described as follows:

[0059] Figure 1 is a double binding of CC312 in vitro mediated T cells and B cells in systemic lupus erythematosus (SLE) PBMC in Example 1.

[0060] Figures 2, 3 and 4 are the activation effect of CC312 in vitro mediated T cells in systemic lupus erythematosus (SLE) PBMC in Example 2.

[0061] Figure 5 is a graph showing the results of CC312-mediated cytokine release in SLE patient PBMCs in vitro in Example 3.

[0062] Figure 6 is a graph showing the results of CC312-mediated T cell proliferation in SLE patient PBMCs in vitro in Example 4.

[0063] Figures 7 and 8 are graphs showing the results of CC312-mediated autologous B cell depletion in SLE patient PBMCs in vitro in Example 5.

[0064] Figure 9 is a graph showing the results of CC312-mediated autologous B cell depletion by T cells isolated from SLE patient PBMCs in vitro in Example 6.

[0065] Figures 10A-10C are graphs showing the results of CC312-mediated plasma cell depletion in SLE patient bone marrow in vitro in Example 7.

[0066] Figure 11 is a graph showing the results of CC312-mediated T cell activation in rheumatoid arthritis (RA) patient PBMCs in vitro in Example 8.

[0067] Figure 12 is a graph showing the results of CC312-mediated T cell proliferation in RA patient PBMCs in vitro in Example 9.

[0068] Figures 13 and 14 are graphs showing the results of CC312-mediated autologous B cell depletion in RA patient PBMCs in vitro in Example 10.

[0069] Figure 14 is a graph showing the results of CC312 compared to Blincyto in mediating autologous B cell depletion in RA patient PBMCs in vitro in Example 11.

[0070] Figure 15 is a graph showing the results of CC312-mediated T cell activation in anti-synthetase syndrome patient PBMCs in vitro in Example 12.

[0071] Figure 16 is a graph showing the results of CC312-mediated T cell proliferation in anti-synthetase syndrome patient PBMCs in vitro in Example 13.

[0072] Figure 17 is a graph showing the results of CC312-mediated autologous B cell depletion in anti-synthetase syndrome patient PBMCs in vitro in Example 14.

[0073] Figure 18 is a graph showing the results of CC312-mediated T cell activation in MDA5+ dermatomyositis interstitial fibrosis patient PBMCs in vitro in Example 15.

[0074] Figure 19 is a graph showing the results of CC312-mediated T cell proliferation in MDA5+ dermatomyositis interstitial fibrosis patient PBMCs in vitro in Example 16.

[0075] Figure 20 is a graph showing the results of CC312-mediated depletion of autologous B cells from PBMC of MDA5+ PM / IF patients in vitro in Example 17.

[0076] Figure 21 is a graph showing the results of CC312-mediated activation of T cells in PBMC of systemic sclerosis (SSc) patients in vitro in Example 18.

[0077] Figure 22 is a graph showing the results of CC312-mediated proliferation of T cells in PBMC of systemic sclerosis (SSc) patients in vitro in Example 19.

[0078] Figure 23 is a graph showing the results of CC312-mediated depletion of autologous B cells from PBMC of systemic sclerosis (SSc) patients in vitro in Example 20.

[0079] Figure 24 is a graph showing the results of CC312-mediated activation of T cells in PBMC of IgG4-related disease (IgG4 RD) patients in vitro in Example 21.

[0080] Figure 25 is a graph showing the results of CC312-mediated proliferation of T cells in PBMC of IgG4-related disease (IgG4 RD) patients in vitro in Example 22.

[0081] Figure 26 is a graph showing the results of CC312-mediated depletion of autologous B cells from PBMC of IgG4-related disease (IgG4 RD) patients in vitro in Example 23.

[0082] Figure 27 is a graph showing the results of CC312-mediated depletion of autologous B cells from PBMC of autoimmune hemolytic anemia (AIHA) patients in vitro in Example 24.

[0083] Figure 28 is a graph showing the results of CC312-mediated activation of T cells in PBMC of myasthenia gravis (MG) patients in vitro in Example 25.

[0084] Figure 29 is a graph showing the results of CC312-mediated proliferation of T cells in PBMC of myasthenia gravis (MG) patients in vitro in Example 26.

[0085] Figure 30 is a graph showing the results of CC312-mediated depletion of autologous B cells from PBMC of myasthenia gravis (MG) patients in vitro in Example 27.

[0086] Figure 31 is a graph showing the results of CC312-mediated activation of T cells in PBMC of immune thrombocytopenia (ITP) patients in vitro in Example 28.

[0087] Figure 32 is a graph showing the results of CC312-mediated proliferation of T cells in PBMC of immune thrombocytopenia (ITP) patients in vitro in Example 29.

[0088] Figure 33 is the results of CC312 in vitro mediating the clearance of autologous B cells by PBMCs from patients with immune thrombocytopenia (ITP) in Example 30.

[0089] Figure 34 is the change in body weight of female NCG mice after intraperitoneal inoculation of patient-derived PBMCs from patients with systemic lupus erythematosus in Example 31, 8 mice per group.

[0090] Figure 35 is the change in graft versus host disease (GVHD) score of female NCG mice after intraperitoneal inoculation of patient-derived PBMCs from patients with systemic lupus erythematosus in Example 31, 8 mice per group.

[0091] Figure 36 is the level of antibodies in the peripheral blood of mice in each group at the end of the study in Example 31, 8 mice per group; Figure 30A shows the total amount of human IgG; Figure 30B shows the level of anti-dsDNA antibodies; *P<0.05, ****P<0.0001, analysis method: independent sample T test.

[0092] Figure 37 is the number of cells in the peripheral blood of mice in each group at the end of the study in Example 31, 8 mice per group; Figure 37A shows the number of human CD20 + B cells in the peripheral blood; Figure 37B shows the number of human CD38 + CD138 + cells (i.e. plasma cells) in the peripheral blood; *P<0.05, analysis method: independent sample T test.

[0093] Figure 38 is the statistical results of immunofluorescence detection of kidney tissue of mice in each group at the end of the study in Example 31; ****: P<0.0001. G1 vs each group, G2 vs G3, analysis method: One-way ANOVA / Dunnet’s.

[0094] Figure 39 is the change in SLEDAI-2K score of patients with SLE after administration in the clinical trial in Example 32.

[0095] Figure 40 is the change in PGA score of patients with SLE after administration in the clinical trial in Example 32.

[0096] Figure 41 is the 24-hour proteinuria detection results of patients with SLE after administration in the clinical trial in Example 32.

[0097] Figure 42 is the anti-dsDNA antibody detection results of patients with SLE after administration in the clinical trial in Example 32.

[0098] Figure 43 is the change in the number of CD19 + B cells after administration in the clinical trial of patients with SLE in Example 32.

[0099] Figure 44 is a graph showing the depletion of B cell subpopulations in SLE patients in Example 32 after administration of the antibody.

[0100] Figure 45 is a graph showing the cytokine levels in SLE patients in Example 32 before and after administration of the antibody. DETAILED DESCRIPTION

[0101] The present application will be readily understood by the following detailed description in conjunction with the accompanying drawings, and the foregoing information. Those skilled in the art will readily appreciate that the summary and abstract are provided herein for illustrative purposes only, and are not intended to limit the scope of the application.

[0102] Definitions

[0103] The term "antibody" is interpreted in a broad sense in the context of the present application, i.e. not only includes traditional IgG class antibodies, but also antibody analogs and derivatives, such as scFv. For example, IgG class antibodies are immunoglobulins of Y-shaped structure produced by the immune system in response to foreign substances, such as pathogens. Antibodies of classical structure are homodimers, each monomer comprising one heavy chain and one light chain connected by disulfide bonds. The light chain consists of a variable region (VL) and a constant region (CL), while the heavy chain consists of a variable region (VH) and three constant regions (CHI, CH2 and CH3). Each variable region contains three "complementarity determining regions" (CDRs), which constitute the antigen binding site responsible for complementarity with antigens. The antigen binding fragment consisting of VL, CL, VH and CHI is referred to as "Fab". The remaining part, the "trunk" part below the Y-shaped structure, is referred to as the "Fc" region, which consists of the constant regions CH2 and CH3 domains of the antibody heavy chain. The "hinge region" is the part between CHI and CH2 in IgG, IgA and IgG class immunoglobulin molecules, which connects the Fab and Fc regions.

[0104] The term "trispecific antibody" refers to an antibody or antibody analog capable of simultaneously and specifically binding to three targets. In the context of the present application, trispecific antibody specifically refers to a single chain fusion protein formed by three domains having binding specificity for different antigen targets in series, such as three different scFvs in series.

[0105] A“T cell engager” or“T cell engager (class) antibody” is generally a class of artificial bispecific or multispecific antibodies based on antibodies or antibody fragments, which can link T cells in vivo with cells expressing specific tumor targeting related proteins. In certain embodiments, the TCE is a bispecific antibody, also known as“bispecific T cell engager” or BiTE, which has two different target antigen binding domains, one side of which can recognize a therapeutic target related antigen (such as CD19, CD33, BCMA, etc.), and the other side of which is usually specific to CD3 / T cell receptor (TCR) complex, so as to engage T cells and target cells, causing T cell activation and killing of tumor target cells. The term“CD3 + A“T cell engager” or“T cell engager (class) antibody” is generally a class of artificial bispecific or multispecific antibodies based on antibodies or antibody fragments, which can link T cells in vivo with cells expressing specific tumor targeting related proteins. In certain embodiments, the TCE is a bispecific antibody, also known as“bispecific T cell engager” or BiTE, which has two different target antigen binding domains, one side of which can recognize a therapeutic target related antigen (such as CD19, CD33, BCMA, etc.), and the other side of which is usually specific to CD3 / T cell receptor (TCR) complex, so as to engage T cells and target cells, causing T cell activation and killing of tumor target cells. The term“CD3 + A“T cell engager” or“T cell engager (class) antibody” is generally a class of artificial bispecific or multispecific antibodies based on antibodies or antibody fragments, which can link T cells in vivo with cells expressing specific tumor targeting related proteins. In certain embodiments, the TCE is a bispecific antibody, also known as“bispecific T cell engager” or BiTE, which has two different target antigen binding domains, one side of which can recognize a therapeutic target related antigen (such as CD19, CD33, BCMA, etc.), and the other side of which is usually specific to CD3 / T cell receptor (TCR) complex, so as to engage T cells and target cells, causing T cell activation and killing of tumor target cells. The term“CD3

[0106] “Specifically binds” or“specifically binds to” means that an antibody exhibits preferential binding to a particular target as compared to other proteins, but such specificity need not be absolute binding specificity. An antibody is considered to have“specificity” for its intended target if its binding is determinative for the presence of the target protein in a sample, e.g., does not produce an undesired result, e.g., false positives. Preferably, an antibody or antigen-binding fragment thereof of the application will bind to a target protein with an affinity that is at least 2-fold greater, more preferably at least 10-fold greater, even more preferably at least 20-fold greater, and most preferably at least 100-fold greater than its affinity for a non-target protein. Alternatively or additionally, an antibody or antigen-binding fragment thereof of the application will have a binding affinity for its target protein, as determined by a Kd of less than 1 x 10 -7 M, 1 x 10-8 M, less than 1x 10 -9 M(1nM), less than 1 x 10 -10 M, less than 1 x 10 -11 M, or even less than 1 x 10 -12M (1pM) K D The value represents the specific binding of a polypeptide containing a given amino acid sequence (e.g., human CD19, human CD3, human CD28) to a protein lacking that sequence.

[0107] In the context of this article concerning multispecific antibodies, the term "binding domain" or "antigen-binding domain" refers to a polypeptide structure that can specifically recognize and bind to a target; it is sometimes simply referred to as a "domain."

[0108] In the context of this invention, "connector sequence" refers to a peptide segment used to connect two binding domains, or a peptide segment within a binding domain used to connect VH and VL. "First connector sequence" specifically refers to a connector used to connect a first binding domain and a second binding domain, and "second connector sequence" specifically refers to a connector used to connect a second binding domain and a third binding domain. Both the first and second connector sequences are connectors used to connect different binding domains, and are therefore collectively referred to as "inter-domain connectors." If a connector also exists between VH and VL within a domain, it is called an "intra-domain connector."

[0109] "T-cell adaptor antibodies" refer to antibodies that can specifically bind to two types of cells, bringing them closer together and "adapting" them in a desired manner, wherein at least one of the two types of cells contains a T cell.

[0110] In the context of this invention, "percentage homology" or "% homology" is used to describe the degree of similarity between two nucleotide sequences or two amino acid sequences, and has the same meaning as "percentage identity". The percentage homology of two sequences can be calculated as follows: after aligning the two sequences, divide the number of identical residue positions by the total length of the aligned sequence and then multiply by 100%. Methods and tools for aligning two amino acid sequences or nucleotide sequences are well known in the art, such as the BLAST kit available on the NCBI website (Altschul, SF et al. (1990) J. Mol. Biol. 215: 403-410).

[0111] "Conservative substitution" of amino acids is well known in the art and generally refers to the alteration of one amino acid residue with another amino acid residue having a structurally or functionally similar side chain. For example, an exemplary list of conservative substitutions is provided in the table below.

[0112] An "autoimmune disease" or "autoimmune disorder" refers to a condition in which the body's immune response attacks its own normal cells. At least 80 such diseases are currently known. They can occur almost anywhere in the body. Common symptoms include fever and feeling tired. Symptoms often come and go.

[0113] "Autoreactive B cells" refer to B cells that recognize self-antigens and become activated and proliferate. Upon activation, the autoreactive B cells secrete autoantibodies that target self-antigens and cause autoimmune diseases.

[0114] "B cell-based autoimmune disease" refers to an autoimmune disease that is associated with or caused by over-activation of B cells. In certain embodiments, "B cell-based autoimmune disease" can also be referred to as "B cell-driven autoimmune disease," "B cell-associated autoimmune disease," or "B cell-promoted autoimmune disease."

[0115] An "autoimmune disease based on self-reactive B cells" refers to an autoimmune disease that is associated with or caused by the over-activation of self-reactive B cells. In certain embodiments, an "autoimmune disease based on self-reactive B cells" can also be referred to as an "abnormal B cell-driven autoimmune disease," an "abnormal B cell-associated autoimmune disease," an "abnormal B cell-based autoimmune disease," an "abnormal B cell proliferation-associated autoimmune disease," an "abnormal B cell activation-associated autoimmune disease," an "abnormal B cell proliferation-based autoimmune disease," an "abnormal B cell activation-based autoimmune disease," or an "autoimmune disease driven by self-reactive B cells." In specific embodiments, the self-reactive B cells can or can not be age-associated B cells. When self-reactive B cells are over-activated, a large amount of autoantibodies and their immune complexes can be produced, which can be pathogenic self-reactive antibodies. These autoantibodies and immune complexes can cause damage to different tissues and organs, leading to the development of various autoimmune diseases. The over-activation of B cells is also a central pathogenesis of various autoimmune diseases. An "autoimmune disease based on self-reactive B cells" includes, but is not limited to, the following diseases: systemic lupus erythematosus (SLE), lupus nephritis, type 1 diabetes, systemic sclerosis, idiopathic inflammatory myopathy (IIM), Sjogren's syndrome, systemic vasculitis, rheumatoid arthritis, anti-synthetase syndrome, MDA5+ dermatomyositis, MDA5+ dermatomyositis pulmonary fibrosis, primary membranous nephropathy, limbic encephalitis, scleromyositis, anti-phospholipid syndrome, autoimmune hemolytic anemia, IgG4-related disease, Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, polyarteritis nodosa, chronic autoimmune hepatitis, primary biliary cirrhosis, myasthenia gravis (MG), neuromyelitis optica spectrum disorder (NMOSD), multiple sclerosis (MS), Lambert-Eaton syndrome, Hashimoto's thyroiditis, Graves' disease, and paraneoplastic cerebellar syndrome.

[0116] "Treat" or "treatment" means that a disease or its symptoms are improved, alleviated, or eliminated (i.e., cured). In some cases, treatment includes prophylactic treatment.

[0117] A "drug" or "pharmaceutical composition" generally refers to a compound or composition that is capable of inducing a desired therapeutic effect when properly administered to a patient. Preferably, a pharmaceutical composition comprises a drug of the present application (e.g., a trispecific antibody) or a variant, prodrug, or other biologically active effective form thereof, and a carrier, diluent, or pharmaceutical excipient, such as a buffer, a preservative, and a tonicity adjusting agent.

[0118] A "pharmaceutically acceptable carrier" generally refers to one or more formulation materials which are suitable for achieving or enhancing delivery of a binding protein.

[0119] "and / or" should be understood to mean either one or both of the elements so conjoined, dependent from another; separation of elements for purposes of

[0120] "comprising" or "including" generally means including, but not limited to, the specified features.

[0121] "selected from" generally means selected from the group consisting of the listed objects, and all combinations thereof. For example, "selected from A, B, and C" means A, B, C, all combinations of A, B, and C, such as, for example, A, B, C, A+B, A+C, B+C, or A+B+C.

[0122] "about" generally means within 0.5-10% of the indicated value, such as within 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of the indicated value.

[0123] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Methods and materials are described herein for use in the present application; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0124] DETAILED DESCRIPTION

[0125] Trispecific antibodies

[0126] In one aspect, the present application provides the use of a trispecific antibody that can simultaneously bind CD19, CD3 and CD28, also referred to herein as a CD19xCD3xCD28 antibody, for the treatment of an autoimmune disease and / or the use of said trispecific antibody for the manufacture of a medicament for the treatment of an autoimmune disease.

[0127] Specifically, the trispecific antibody of the present application comprises three binding domains, respectively, a first binding domain capable of specifically binding to CD19, a second binding domain capable of specifically binding to and activating T cell surface CD3 molecule, and a third binding domain capable of specifically binding to and activating T cell surface CD28 molecule. In addition, the trispecific antibody further comprises a first linker between the first and second binding domains, and a second linker between the second and third binding domains.

[0128] Human CD19 antigen is a transmembrane glycoprotein of 95 kDa in size, a member of immunoglobulin superfamily, expressed on B cell surface, including normal B cells, abnormal autoreactive B cells and B cell malignancies, and can also be age-related B cells. In the absence of specific instructions, the CD19 specifically bound by the first binding domain in the context of the present application is human CD19.

[0129] CD3 is a transmembrane protein, the transmembrane region of which is connected to the transmembrane region of two peptide chains of TCR through a salt bridge, forming a TCR-CD3 complex, which participates in the recognition of antigen by T cells and transduces the activation signal generated to the inside of T cells together. In the absence of specific instructions, the CD3 specifically bound by the second binding domain in the context of the present application is human CD3.

[0130] CD28 is a dimeric transmembrane glycoprotein, expressed on the surface of about 80% of human CD4 + T cells and 50% of human CD8 + T cells. CD28 is a T cell costimulatory molecule that binds to ligands CD80 (B7-1) and CD86 (B7-2) and amplifies the first signal of TCR / CD3 conduction, maintains T cell survival, and promotes cytokine-induced T cell proliferation and differentiation. In the absence of specific instructions, the CD28 specifically bound by the third binding domain in the context of the present application is human CD28.

[0131] Therefore, for autoimmune diseases, the first binding domain in the trispecific antibody of the present application can specifically bind to CD19 on the surface of autoreactive B cells, and the second and third binding domains can bind to T cell surface receptors CD3 and CD28, respectively, thereby "linking" autoreactive B cells and T cells together, and simultaneously activating the CD3 signaling pathway and the CD28 costimulatory signaling pathway of T cells, so that the activated immune cells can exert a killing effect on target cells.

[0132] In a preferred embodiment, the trispecific antibody of the application is a single chain fusion protein comprising: a scFv that specifically binds to CD19 as a first binding domain; a scFv that specifically binds to CD3 as a second binding domain; and a scFv that specifically binds to CD28 as a third binding domain.

[0133] The scFv sequences as first, second and third binding domains can be derived from anti-CD19, anti-CD3 and anti-CD28 antibodies or antibody analogs, derivatives known in the art, respectively. For example, each binding domain can be a scFv comprising a VH and a VL derived from a known corresponding antibody. The antibody can be an animal-derived antibody such as a murine-derived antibody, a chimeric antibody such as a human-murine chimeric antibody, or a human antibody or a humanized antibody.

[0134] In a preferred embodiment, the scFv that specifically binds to CD19 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0135] the VH comprises a HCDR 1 as set forth in SEQ ID NO: 1, a HCDR2 as set forth in SEQ ID NO: 2 and a HCDR3 as set forth in SEQ ID NO: 3, and the VL comprises a LCDR 1 as set forth in SEQ ID NO: 4, a LCDR2 as set forth in SEQ ID NO: 5 and a LCDR3 as set forth in SEQ ID NO: 6.

[0136] In a preferred embodiment, the scFv that specifically binds to CD19 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0137] the VH comprises an amino acid sequence as set forth in SEQ ID NO: 7, or can comprise an amino acid sequence having at least 80% sequence homology (such as at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) to the amino acid sequence as set forth in SEQ ID NO: 7 and all amino acid differences are in amino acid sequences of non-CDR regions, or consists of such an amino acid sequence; and / or

[0138] the VL comprises an amino acid sequence as set forth in SEQ ID NO: 8, or can comprise an amino acid sequence having at least 80% sequence homology (such as at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) to the amino acid sequence as set forth in SEQ ID NO: 8 and all amino acid differences are in amino acid sequences of non-CDR regions, or consists of such an amino acid sequence.

[0139] In a further preferred embodiment, the scFv specifically binding to CD19 comprises an amino acid sequence as set forth in SEQ ID NO: 11, or an amino acid sequence which has at least 80% homology, such as at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology, and all amino acid differences are in the amino acid sequence of the non-CDR regions; or consists of such an amino acid sequence.

[0140] Preferably, the above-mentioned amino acid differences are conservative substitutions of amino acids.

[0141] In one embodiment, in the scFv specifically binding to CD19, the VH is located C-terminally or N-terminally to the VL. In a preferred embodiment, in the scFv specifically binding to CD19, the VH is located C-terminally to the VL, i.e. is connected in the order VL-linker sequence-VH.

[0142] In some embodiments, in the scFv specifically binding to CD19, the VH and the VL can be connected by an intradomain linker or directly. The intradomain linker is a linker comprising (GGGGS)n or (GGS)n, or is (GGGGS)n or (GGS)n, and n is an integer from 1 to 10, preferably an integer from 1 to 5, such as 1, 2, 3, 4 or 5. In a particular embodiment, in the scFv specifically binding to CD19, the intradomain linker between the VH and the VL is selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, preferably SEQ ID NO: 34.

[0143] In a preferred embodiment, the scFv specifically binding to CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0144] the VH comprises a HCDR 1 as set forth in SEQ ID NO: 12, a HCDR2 as set forth in SEQ ID NO: 13 and a HCDR3 as set forth in SEQ ID NO: 14, and the VL comprises a LCDR 1 as set forth in SEQ ID NO: 15, a LCDR2 as set forth in SEQ ID NO: 16 and a LCDR3 as set forth in SEQ ID NO: 17.

[0145] In a preferred embodiment, the scFv specifically binding to CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0146] the VH comprises an amino acid sequence as shown in SEQ ID NO: 18, or can comprise an amino acid sequence having at least 80% sequence identity (such as at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 96%, at least 97%, at least 98% or at least 99% identity) with the amino acid sequence shown in SEQ ID NO: 18 and all amino acid differences are in amino acid sequences which are not CDR regions, or consists of such an amino acid sequence; and / or

[0147] the VL comprises an amino acid sequence as shown in SEQ ID NO: 19, or can comprise an amino acid sequence having at least 80% sequence identity (such as at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 96%, at least 97%, at least 98% or at least 99% identity) with the amino acid sequence shown in SEQ ID NO: 19 and all amino acid differences are in amino acid sequences which are not CDR regions, or consists of such an amino acid sequence.

[0148] In a further preferred embodiment, the scFv specifically binding to CD3 comprises an amino acid sequence as shown in SEQ ID NO: 22, or an amino acid sequence having at least 80% identity (such as at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 96%, at least 97%, at least 98% or at least 99% identity) with the amino acid sequence shown in SEQ ID NO: 22 and all amino acid differences are in amino acid sequences which are not CDR regions; or consists of such an amino acid sequence.

[0149] Preferably, the above amino acid differences are conservative substitutions of amino acids.

[0150] In one embodiment, in the scFv specifically binding to CD3, the VH is located C-terminally or N-terminally to the VL. In a preferred embodiment, in the scFv specifically binding to CD3, the VH is located C-terminally to the VL, i.e. is connected in the order VL-linker sequence-VH.

[0151] In some embodiments, in the scFv that specifically binds to CD3, the VH and VL can be connected by an intradomain linker or directly connected. The intradomain linker is a linker containing (GGGGS)n or (GGS)n, or is (GGGGS)n or (GGS)n, and n is an integer from 1 to 10, preferably an integer from 1 to 5, for example 1, 2, 3, 4 or 5. In a particular embodiment, in the scFv that specifically binds to CD3, the intradomain linker between the VH and VL is selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, preferably SEQ ID NO: 36.

[0152] In a preferred embodiment, the scFv that specifically binds to CD28 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0153] the VH comprises a HCDR1 as depicted in SEQ ID NO: 23, a HCDR2 as depicted in SEQ ID NO: 24 and a HCDR3 as depicted in SEQ ID NO: 25, and the VL comprises a LCDR1 as depicted in SEQ ID NO: 26, a LCDR2 as depicted in SEQ ID NO: 27 and a LCDR3 as depicted in SEQ ID NO: 28.

[0154] In a preferred embodiment, the scFv that specifically binds to CD28 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0155] the VH comprises an amino acid sequence as depicted in SEQ ID NO: 29, or can comprise an amino acid sequence having at least 80% sequence identity (such as at least 85% identity, preferably at least 90% identity, more preferably at least 95% identity, even more preferably at least 96%, at least 97%, at least 98% or at least 99% identity) to the amino acid sequence as depicted in SEQ ID NO: 29 and all amino acid differences are in amino acid sequences of non-CDR regions, or consists of such an amino acid sequence; and / or

[0156] the VL comprises an amino acid sequence as depicted in SEQ ID NO: 30, or can comprise an amino acid sequence having at least 80% sequence identity (such as at least 85% identity, preferably at least 90% identity, more preferably at least 95% identity, even more preferably at least 96%, at least 97%, at least 98% or at least 99% identity) to the amino acid sequence as depicted in SEQ ID NO: 30 and all amino acid differences are in amino acid sequences of non-CDR regions, or consists of such an amino acid sequence.

[0157] In a further preferred embodiment, the scFv specifically binding CD28 comprises an amino acid sequence as set forth in SEQ ID NO: 33, or an amino acid sequence which is at least 80% homologous (such as at least 85% homologous, preferably at least 90% homologous, more preferably at least 95% homologous, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homologous) to the amino acid sequence as set forth in SEQ ID NO: 33 and all amino acid differences are in the amino acid sequence of the non-CDR regions; or consists of such an amino acid sequence.

[0158] Preferably, the above-mentioned amino acid differences are conservative substitutions of amino acids.

[0159] In one embodiment, in the scFv specifically binding CD28, the VH is located C-terminally or N-terminally to the VL. In a preferred embodiment, in the scFv specifically binding CD28, the VH is located C-terminally to the VL, i.e. is connected in the order VL-linker sequence-VH.

[0160] In some embodiments, in the scFv specifically binding CD28, the VH and the VL can be connected by an intradomain linker or directly. The intradomain linker is a linker comprising (GGGGS)n or (GGS)n, or is (GGGGS)n or (GGS)n, and n is an integer from 1 to 10, preferably an integer from 1 to 5, such as 1, 2, 3, 4 or 5. In a particular embodiment, in the scFv specifically binding CD28, the intradomain linker between the VH and the VL is selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, preferably SEQ ID NO: 34.

[0161] It is understood by the skilled person that the determination of the CDR regions can be determined using any of the numbering systems of Kabat, IMGT, Chothia antibody numbering system. When using different numbering systems, the amino acid sequences for the same variable region can demarcate different CDR regions. Regardless of which numbering system is used to determine the CDR regions, it falls within the scope of the present application. Unless otherwise stated, the specific amino acid sequences of the CDR regions recited in the present application are determined according to the Kabat antibody numbering system.

[0162] The linker sequences of the application can be derived from naturally occurring sequences or modified variants thereof, or can be artificial sequences designed de novo. The linker sequences can be derived from naturally occurring multi-domain proteins, for example, can be a linker sequence contained in a naturally occurring multi-domain protein or a variant thereof. An example of a linker sequence derived from a naturally occurring sequence can be a linker sequence from the IgD hinge region, which can be used in the application as a second linker sequence. Examples of artificial linker sequences are, for example, a linker sequence based on (GGGGS)n, (GGGGS)nGGGG, (SGGGG)n, GGGG(SGGGG)n, (GGS)n, (GGS)nGG, (SGG)n, or GG(SGG)n, which can be used in the application as a first inter-domain linker, or as an intra-domain linker. Another example of an artificial linker sequence is a linker sequence comprising (EAAAK)n, such as A(EAAAK)nA, A(EAAAK)nALEA(EAAAK)nA, which can be used in the application as a second linker sequence.

[0163] The rigidity / flexibility of the linker is a factor to be considered. A flexible linker allows the polypeptide sequences connected at its two ends to move within a certain range. A flexible linker is usually composed of small-sized amino acids such as glycine (G), serine (S), threonine (T), and it is these small-sized amino acids that confer flexibility to the linker. In cases where it is desirable to retain the possibility of some relative movement between the domains at the two ends of the linker, a flexible linker is preferably used. However, in some cases, a flexible linker can not allow the domains connected at its two ends to be fixed, so that the multi-domain molecule cannot be expressed or function properly. In this regard, for a multi-functional molecule, for example, a trispecific antibody that needs to bind to three targets, a rigid linker can provide better structural and functional stability than a flexible linker.

[0164] In particular to the trispecific antibodies of the application, the first domain targets a tumor cell, while the second and third domains target an immune cell, that is, the adjacent first and second domains will necessarily bind to different cells. In this case, it is desirable to have some ability for relative movement between the first and second domains to allow easier binding by adjusting the position when binding to different cells. Therefore, in a preferred embodiment, the first linker sequence of the application is a flexible linker, for example, a linker composed of one or more of glycine (G), serine (S), threonine (T), and lysine (K), such as a GS-rich linker, for example, a linker containing (GGGGS) n , (SGGGG) n , (GGS) n , (SGG) n , for example, (GGGGS) n , (GGGGS) nGGGG, (SGGGG) n GGGG(SGGGG) n (GGS) n (GGS) n GG, (SGG) n or GG(SGG) n wherein n is a positive integer, preferably 1-10. The linker for connecting the VH and VL in each binding domain is also preferably such a flexible linker.

[0165] On the other hand, as a linker antibody, it is desirable to bring the two cells being linked as close as possible, so that the distance of the immunological synapse formed between the cells is closer to the distance of the immunological synapse naturally formed in the physiological state, which is more conducive to the killing effect of the immune cells. The distance of the two cells being brought closer is affected by the length of the first linker sequence. For example, the length of the first linker sequence is not more than 30 amino acids, preferably not more than 25 amino acids, more preferably not more than 20 amino acids, and still more preferably not more than 15 amino acids. For example, if the first linker sequence is any of (GGGGS) n (SGGGG) n GGGG(SGGGG) n , then preferably n is a positive integer not more than 6, preferably a positive integer not more than 5, for example 1, 2, 3, 4, or 5; if the first linker sequence is any of (GGS) n (SGG) n or (GGS) n GG, then preferably n is a positive integer not more than 10, preferably a positive integer not more than 7, for example 1, 2, 3, 4, 5, 6, or 7. In a specific embodiment, the first linker sequence is (GGGGS)3(SEQ ID NO: 34), GGGGS (SEQ ID NO: 35), or (GGS)4GG (SEQ ID NO: 36), preferably the amino acid sequence shown in SEQ ID NO: 35.

[0166] The second linker sequence connects the second and third domains, both of which bind to receptors on the surface of a T cell. To prevent steric hindrance effects from the binding of one domain affecting the binding of the other domain to the same T cell, a slightly longer second linker sequence is preferred. Thus, in preferred embodiments, the second linker sequence is longer than the first linker sequence. For example, the second linker sequence is at least 1.25 times, at least 1.5 times, at least 2 times, at least 2.25 times, at least 2.5 times or longer in length than the first linker sequence. For example, the second linker sequence is at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids or longer in length.

[0167] In a particular embodiment, the second linker sequence of the trispecific antibody of the application is a fragment from the hinge region of immunoglobulin IgD or a variant thereof, preferably the amino acid sequence of SEQ ID NO: 37.

[0168] In preferred embodiments, the linker sequence for connecting the VH and VL in each binding domain of the trispecific antibody of the application, i.e. the intra-domain linker, is a (GGGGS)n or (GGS)n containing linker, or is (GGGGS)n or (GGS)n and n is a positive integer of no more than 10, preferably a positive integer of no more than 5, e.g. 1, 2, 3, 4 or 5. In some embodiments, the linker sequence for connecting the VH and VL in each binding domain is the same. In some embodiments, the linker sequence for connecting the VH and VL in each binding domain is different. For example, the intra-domain linker sequence is any of (GGGGS) n , (SGGGG) n , GGGG(SGGGG) n , wherein preferably n is a positive integer of no more than 6, preferably a positive integer of no more than 5, e.g. 1, 2, 3, 4 or 5. For example, the intra-domain linker is any of (GGS) n , (SGG) n or (GGS) n GG, wherein preferably n is a positive integer of no more than 10, preferably a positive integer of no more than 7, e.g. 1, 2, 3, 4, 5, 6 or 7.

[0169] In a specific embodiment, the intra-domain linker is selected from the amino acid sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 35 or SEQ ID NO: 36. In a more specific embodiment, the three intra-domain linkers are (GGGGS)3as set forth in SEQ ID NO: 34, (GGS)4GG as set forth in SEQ ID NO: 36 and (GGGGS)3as set forth in SEQ ID NO: 34, respectively.

[0170] In the case that the antigen specificity and the arrangement order of each antigen binding domain are determined, the specific sequence of each binding domain is not particularly limited. The three binding domains can be derived from the corresponding antibody or antibody analog known in the art.

[0171] In a preferred embodiment, the trispecific antibody of the present application is capable of specifically binding to human CD19, human CD3 and human CD28, and comprises the sequence of SEQ ID NO: 37 as the linker sequence. Preferably, the amino acid sequence of SEQ ID NO: 37 is the linker sequence connecting the binding domain specifically binding to CD3 and the binding domain specifically binding to CD28.

[0172] In a specific embodiment, the trispecific antibody of the present application is the trispecific antibody with the amino acid sequence as set forth in SEQ ID NO: 38 or a variant thereof; the variant has at least 80% homology with the amino acid sequence as set forth in SEQ ID NO: 38, and is capable of specifically binding to CD19, CD3 and CD28, and comprises the sequence of SEQ ID NO: 37 as the linker sequence. Preferably, the amino acid sequence of the variant has at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, at least 99% homology with the amino acid sequence as set forth in SEQ ID NO: 38.

[0173] Autoimmune diseases

[0174] On the other hand, CD19 is a good B cell target, and the trispecific antibody of the present application can achieve the purpose of treating autoimmune diseases by targeting both the self-reactive B cells and T cells, and activating the T cells to kill the self-reactive B cells through the CD28 co-stimulatory signal.

[0175] In preferred embodiments, the autoimmune disease is a B cell-based autoimmune disease, more preferably a self-reactive B cell-based autoimmune disease. The self-reactive B cell-based autoimmune disease has self-reactive B cell hyperactivation as a core pathogenesis, and can present with production of large amounts of autoantibodies. The autoantibody refers to an antibody produced by the immune system against the individual's own proteins, nucleic acids, etc. The self-reactive B cell-based autoimmune disease can include, but is not limited to, autoimmune diseases presenting with or diagnosed by production of autoantibodies. For example, the self-reactive B cell-based autoimmune disease can be selected from systemic lupus erythematosus (SLE), lupus nephritis, type 1 diabetes, systemic sclerosis, idiopathic inflammatory myopathy (IIM), Sjogren's syndrome, systemic vasculitis, rheumatoid arthritis, anti-synthetase syndrome, MDA5+ dermatomyositis, MDA5+ dermatomyositis interstitial lung fibrosis, primary membranous nephropathy, limbic encephalitis, scleromyositis, anti-phospholipid syndrome, autoimmune hemolytic anemia, IgG4-related disease, Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, Takayasu's arteritis, chronic autoimmune hepatitis, primary biliary cirrhosis, myasthenia gravis (MG), neuromyelitis optica spectrum disorder (NMOSD), multiple sclerosis (MS), Lambert-Eaton syndrome, Hashimoto's thyroiditis, Graves' disease, and paraneoplastic cerebellar syndrome.

[0176] In further preferred embodiments, the self-reactive B cell-based autoimmune disease is selected from systemic lupus erythematosus, systemic sclerosis, IgG4-related disease, anti-synthetase syndrome, MDA5+ dermatomyositis interstitial lung fibrosis, autoimmune hemolytic anemia, rheumatoid arthritis, and idiopathic inflammatory myopathy.

[0177] For example, the self-reactive B cell-based autoimmune disease can be systemic lupus erythematosus (SLE).

[0178] For example, the self-reactive B cell-based autoimmune disease can be systemic sclerosis.

[0179] For example, the self-reactive B cell-based autoimmune disease can be rheumatoid arthritis.

[0180] For example, the self-reactive B cell-based autoimmune disease can be anti-synthetase syndrome.

[0181] For example, the self-reactive B cell-based autoimmune disease can be IgG4-related disease.

[0182] For example, the autoimmune disease based on self-reactive B cells can be autoimmune hemolytic anemia.

[0183] For example, the autoimmune disease based on self-reactive B cells can be MDA5+ dermatomyositis pulmonary fibrosis.

[0184] For example, the autoimmune disease based on self-reactive B cells can be idiopathic inflammatory myopathy (IIM). Preferably, the idiopathic inflammatory myopathy is selected from the group consisting of dermatomyositis, polymyositis, rheumatoid disease overlap syndrome myositis, anti-synthetase syndrome myositis, immune-mediated necrotizing myopathy, and inclusion body myositis.

[0185] Systemic lupus erythematosus (SLE) is a chronic autoimmune disease in which the immune system mistakenly attacks multiple parts of the body, including the skin, joints, kidneys, heart, lungs, and brain. SLE is most common in women between the ages of 15 and 44, but it can occur in almost anyone. Symptoms of SLE vary widely and can include fever, fatigue, rashes, joint pain, headaches, kidney problems, and more. Common treatments include the use of nonsteroidal anti-inflammatory drugs, antimalarials such as hydroxychloroquine, corticosteroids, and immunosuppressive agents. In some cases, plasma exchange or intravenous immunoglobulin may be required. However, SLE is a chronic disease that often requires long-term treatment and management, and there is currently no cure. The limitations of treating SLE are that current treatments may not completely control symptoms in all patients, and long-term use of immunosuppressive drugs can increase the risk of infection and other complications. In addition, the heterogeneity of SLE means that different patients may respond differently to treatment, requiring individualized treatment plans.

[0186] Systemic sclerosis is a rare chronic autoimmune connective tissue disease characterized by degenerative changes and scarring of multiple organs, often involving the skin, joints, and internal organs, as well as the perivascular tissue. The disease is four times more common in women than in men, most commonly occurring in people aged 20-50, and rarely in children. About 90% of cases occur in women aged 40 to 60, but can start at any age. There is currently no specific treatment, and treatment is mainly aimed at complications.

[0187] Rheumatoid arthritis is a chronic autoimmune disease characterized by symmetrical joint pain, stiffness, and swelling. It usually affects middle-aged and elderly women. Although existing treatments such as non-steroidal anti-inflammatory drugs, traditional anti-rheumatic drugs, and hormone drugs can relieve symptoms and control inflammation, there are limitations such as high recurrence rate, drug side effects, and individual response differences.

[0188] Idiopathic inflammatory myopathies are a group of rare chronic autoimmune diseases characterized by muscle inflammation and weakness, and can be accompanied by involvement of other organs such as skin, heart, lungs and joints. These diseases include various subtypes, such as polymyositis (PM), dermatomyositis (DM), inclusion body myositis (IBM), etc. Idiopathic inflammatory myopathies can occur at any age, but certain subtypes such as dermatomyositis are more common in children, while inclusion body myositis is more common in the elderly. Women may be more susceptible to certain types of inflammatory myopathy than men. There is no cure for idiopathic inflammatory myopathy, and currently immunosuppressive drugs are usually used to reduce inflammation and muscle damage, such as corticosteroids (such as prednisone) and various immunosuppressants (such as methotrexate, azathioprine). In some cases, biologics or intravenous immunoglobulin (IVIG) can be used.

[0189] Most autoimmune diseases represented by the above four diseases have the problems of no cure and limited drug options, so the introduction of the trispecific antibody described in the present application as a new choice for treating autoimmune diseases may become a cure for the above diseases.

[0190] In a specific embodiment, the therapeutic effect of the trispecific antibody on the autoimmune disease based on autoreactive B cells can be manifested by the clearance efficiency of autologous B cells and / or plasma cells.

[0191] In a more specific embodiment, the clearance efficiency of the trispecific antibody against autologous B cells and / or plasma cells can be represented by EC50. The EC50 can be the concentration (e.g., molar concentration) of the trispecific antibody when the clearance rate reaches 50% for a certain subject. The EC50 may

[0192] In a further specific embodiment, the clearance efficiency against autologous B cells can be represented by the EC50 against autologous B cells. The EC50 against autologous B cells is preferably between 0.1-10 pM, more preferably between 0.3-5.0 pM, and still more preferably between 0.8-1.6 pM.

[0193] In another more specific embodiment, the efficiency of the trispecific antibody for the clearance of autologous B cells and / or plasma cells can be represented by the killing efficiency. The killing efficiency refers to the proportion of autologous B cells and / or plasma cells that are cleared after administration to a subject in vivo or in vitro. The killing efficiency can be calculated by the following formula: killing efficiency (%) = (1 - number of cells in administration group / number of cells in non-administration group) x 100%, wherein the non-administration group can be administered with a non-treatment functional agent (e.g., solvent, placebo, etc.) or without any agent.

[0194] For example, the killing efficiency of the trispecific antibody for autologous B cells can reach 100%.

[0195] For example, the killing efficiency of the trispecific antibody for plasma cells can reach 85%.

[0196] In another specific embodiment, the therapeutic effect of the trispecific antibody for the treatment of the autoimmune disease based on the self-reactive B cells can be represented by the clearance efficiency of disease-specific autoantibodies. Examples of the autoantibodies include, but are not limited to, anti-double stranded DNA antibody in systemic lupus erythematosus; anti-centromere antibody, anti-topoisomerase antibody in systemic sclerosis; anti-MDA5 antibody, anti-SRP antibody in idiopathic inflammatory myopathy.

[0197] For example, the clearance efficiency of the anti-double stranded DNA antibody after treatment with the trispecific antibody can reach 100%.

[0198] Pharmaceutical composition and therapeutic method

[0199] In another aspect, the present application provides a pharmaceutical composition comprising any one of the aforementioned trispecific antibodies, which is used for the treatment of autoimmune diseases, preferably autoimmune diseases based on self-reactive B cells.

[0200] In another aspect, the present application also provides the use of any one of the aforementioned trispecific antibodies in the treatment of autoimmune diseases.

[0201] In another aspect, the present application also provides a therapeutic method for preventing and / or treating autoimmune diseases, which comprises administering any one of the aforementioned trispecific antibodies to a patient.

[0202] The trispecific antibodies described herein can be administered in a therapeutically effective amount. A "therapeutically effective amount" refers to the amount of an antibody that, when administered to a subject or patient for treatment of a disease, or at least one clinical symptom of a disease or condition, is sufficient to effect such treatment for the disease, condition, or symptom. The "therapeutically effective amount" can vary with the antibody, the disease, condition, and / or symptom of the disease or condition, the severity of the disease, condition, and / or symptom of the disease or condition, the age of the subject or patient to be treated, and / or the body weight of the subject or patient to be treated.

[0203] Without wishing to be bound by any theory, the examples below are merely to illustrate the fusion proteins, the preparation methods and uses of the present application, and are not intended to limit the scope of the present application.

[0204] Examples

[0205] The effect of a trispecific antibody CC312 in treating autoimmune diseases is exemplarily described in Examples 1-3. The amino acid sequence of CC312 is shown as SEQ ID NO: 38, and the sequences of different components in CC312 and the corresponding ID numbers are shown in Table 1.

[0206] Table 1. Sequences of trispecific antibody CC312

[0207] Note: CDR sequences are shown in bold with underlining

[0208] Example 1

[0209] CC312 mediates dual binding of T cells and B cells in PBMCs of systemic lupus erythematosus (SLE) patients in vitro

[0210] In this example, the ability of CC312 to mediate dual binding of T cells and B cells in PBMC system was detected.

[0211] PBMCs used in the experiment were from SLE patients (the subjects were from Shanghai Renji Hospital).

[0212] After counting the PBMC of SLE patients, T cells and B cells were sorted by cell sorting kit (BioLegend) respectively. After sorting, the density of T cells and B cells was adjusted to 1.11E6 / mL by RPMI 1640 complete medium (Gibco), and CC312 was gradient diluted by RPMI 1640 complete medium. 90 μL of T cell suspension labeled with cell trace violet, 90 μL of B cell suspension labeled with Far red and 20 μL of antibody dilution were added to each well of a U-bottom 96-well plate, and the final concentration of CC312 was 0, 11.9, 119, 1190 pM. After mixing the antibody drug with the cells uniformly, it was incubated in a 2-8°C refrigerator for 2 hours. After 2 hours, the percentage of T cells and B cells double positive was detected by flow cytometry.

[0213] The results are shown in Figure 1. CC312 can mediate the double combination of T cells and B cells of patients, and the ability of double combination is higher than that of Blincyto.

[0214] Example 2

[0215] CC312 mediates the activation of T cells in PBMC of systemic lupus erythematosus (SLE) patients in vitro

[0216] In this example, the activity of CC312 in mediating the activation of T cells in PBMC system was detected.

[0217] The PBMC used in the test was from SLE patients (the subject was from Shanghai Renji Hospital).

[0218] After counting the PBMC of SLE patients, the density was adjusted to 1.11E6 / mL by RPMI 1640 complete medium (Gibco). CC312 and Blinatumomab, or control molecules were gradient diluted by RPIM 1640 complete medium. 180 μL of PBMC cell suspension and 20 μL of antibody dilution were added to each well of a U-bottom 96-well plate. After mixing the drug with PBMC uniformly, it was incubated in a 37°C, 5% CO2 incubator for 16 hours. After 16 hours, the cells in each well were labeled with Pacific Blue anti-human CD4 antibody (BioLegend), Brilliant Violet 605 TManti-human CD8 antibody (BioLegend), APC anti-human CD25 antibody (BioLegend), PE anti-human CD69 antibody (BioLegend) and Near-IR fluorescent reactive dye (BioLegend), and then the proportion of each of the following target cell subpopulations in each well of the 96-well plate was detected with a Thermo Fisher Attune NxT flow cytometer: CD4 + CD25 + , CD4 + CD69 + , CD8 + CD25 + , CD8 + CD69 + .

[0219] The results are shown in Figures 2A-D. CC312 exhibited good effector cell activation capacity compared with Blincyto. Figure 3 shows that the control molecule CC312 (CD3 null) did not activate CD3-independent T cells up to 10 nM when exposed to SLE donor PBMCs, indicating that CC312 conditionally binds to CD28, which requires co-binding of CD3. Figure 4 results show that the control molecule CC312 (CD19 null) did not activate target-independent T cells up to 1 nM when exposed to SLE donor PBMCs, indicating that CD3 / CD28 activity did not cause off-target T cell activation.

[0220] Example 3

[0221] CC312 mediates cytokine release in PBMCs from SLE patients in vitro

[0222] In this example, the cytokine release mediated by CC312 in PBMC systems was detected.

[0223] PBMCs used in the test were from SLE patients (test subjects were from Shanghai Renji Hospital).

[0224] After counting the PBMC of SLE patients, the density was adjusted to 1.11E6 / mL with RPMI 1640 complete medium (Gibco). CC312 and Blincyto were gradient diluted with RPIM 1640 complete medium. 180μL PBMC cell suspension and 20μL antibody diluent were added to each well of a U-bottom 96-well plate, and the final molar concentration of CC312 was 0, 0.01193, 0.1193, 1.193, 11.93, 119.3, 1193pM. After mixing the drug with PBMC evenly, it was placed in a constant temperature incubator at 37℃, 5% CO2 for 16 hours, and the supernatant was collected. The HTRF method was used to determine the IL-2, IL-6, IL-10, and IL-1beta levels in the culture supernatant.

[0225] The results are shown in Figure 5. Compared with Blincyto, CC312 at 11.93, 119.3, 1193pM caused a small amount of release of IL-2 and IL-6. CC312 showed good effector cell activation ability.

[0226] Example 4

[0227] CC312 mediates the proliferation of T cells in PBMC of SLE patients in vitro

[0228] In this example, the proliferation activity of CC312 mediated T cells in PBMC system was detected.

[0229] The PBMC used in the test came from SLE patients (the test subject came from Shanghai Renji Hospital).

[0230] After counting the PBMC of SLE patients, the PBMC was labeled with tracer dye CellTrace Violent at a final concentration of 5uM in a 37℃ incubator for 20min, and then washed 2-3 times with complete medium to adjust the cell density of PBMC to 1.11E6 / ml. CC312 and Blincyto were gradient diluted with RPIM 1640 complete medium. 180μL PBMC cell suspension and 20μL antibody diluent were added to each well of a U-bottom 96-well plate, and the final molar concentration of CC312 was 0, 0.01193, 0.1193, 0.358, 3.58, 11.93, 119.3pM. After mixing the drug with PBMC evenly, it was placed in a constant temperature incubator at 37℃, 5% CO2 for 120 hours. After 120 hours, the cells in each well were labeled with Pacific Blue anti-human CD4 antibody (BioLegend), Brilliant Violet 605 TManti-human CD8 antibody (BioLegend), and Near-IR fluorescent reactive dye (BioLegend), and then the number of CD4+ and CD8+ T cells in each well of the 96-well plate was detected with a Thermo Fisher Attune NxT flow cytometer, and the T cell proliferation ratio was calculated. Proliferation ratio (T cell Proliferation Ratio) = T cell number in sample well / T cell number in vehicle control well.

[0231] The results are shown in Figure 6, and CC312 can mediate the proliferation of CD4 + T cells and CD8 + T cells far more than Blincyto at the same molar concentration.

[0232] Example 5

[0233] CC312 mediates the clearance of autologous B cells from PBMC of SLE patients in vitro

[0234] In this example, PBMC from three SLE patients (donors from Shanghai Renji Hospital) were used to evaluate the CC312-mediated clearance of autologous B cells.

[0235] After counting the PBMC of SLE patients, the cell density was adjusted to 1.11E6 / mL with RPMI 1640 complete medium (Gibco). CC312 and Blincyto were gradient diluted with RPMI 1640 complete medium. 180 μL of PBMC cell suspension and 20 μL of antibody diluent were added to each well of a U-bottom 96-well plate, and the final concentration of CC312 was 0, 0.01193, 0.1193, 0.358, 1.193, 3.58, 11.93, 35.8, 119.3, 1193 pM. After the antibody drug was mixed evenly with the PBMC, it was incubated in a constant temperature incubator at 37°C and 5% CO2 for 72 hours. After 72 hours, the cells in each well were stained with FITC anti-human CD20 antibody (BD Pharmingen) and 7-AAD (BioLegend), and the proportion and number of CD20 + cells were detected by flow cytometry, and the killing efficiency was calculated. Killing efficiency (%) = (1 - sample well cell number / vehicle control well cell number) x 100%.

[0236] The results are shown in Figure 7, and CC312 can dose-dependently mediate the effective clearance of autologous B cells from the PBMC of three patients, with a maximum killing efficiency of 96.1%. As shown in Figure 8, compared with Blincyto, CC312 showed higher clearance ability. The EC50 The values (0.115 pM vs 0.3195 pM) and the highest killing efficiency (89% vs 67%) are both superior to Blincyto at equimolar concentration.

[0237] Example 6

[0238] CC312 mediates autologous B cell clearance from PBMC of SLE patients in vitro

[0239] In this example, CC312-mediated autologous B cell clearance was evaluated using T cells from SLE patients (subjects from Shanghai Renji Hospital).

[0240] After counting the PBMC of SLE patients, T cells and B cells were sorted by cell sorting kit (BioLegend) respectively. After sorting, the density of T cells and B cells were adjusted to 1.11E5 / mL and 1.11E6 / mL respectively by RPMI 1640 complete medium (Gibco), and B cells were labeled with fluorescence and fixed in 8-well glass plates (IBIDI, cat: 80807) with 90 μL B cells per well. After overnight, 90 μL T cell suspension and 20 μL antibody diluent were added to the 8-well glass plate, and the final concentration of CC312 was 0, 23.8 pM. After mixing the antibody drug with the cells uniformly, they were incubated in a constant temperature incubator at 37°C, 5% CO2 for 2.5 days. After 2.5 days, the labeled fluorescence B cells were scanned in the 8-well plate by confocal microscope (Nikon A1R), and then the number of B cells in each well was analyzed and counted by Image J software. Killing efficiency (%) = (1 - sample well cell number / vehicle control well cell number) x 100%.

[0241] The results are shown in Figure 9, CC312 can mediate the effective clearance of autologous B cells by T cells from patients, and the killing ability is stronger than that of Blincyto at equimolar concentration.

[0242] Example 7

[0243] CC312 mediates autologous B cell clearance from bone marrow of SLE patients in vitro

[0244] In this example, CC312-mediated autologous B cell clearance was evaluated using T cells from SLE patients (subjects from Shanghai Renji Hospital).

[0245] After counting the bone marrow cells of SLE patients, the cell density was adjusted to 1.11E6 / mL with RPMI 1640 complete medium (Gibco). CC312 and Blincyto were gradient diluted with RPMI 1640 complete medium. 180 μL of PBMC cell suspension and 20 μL of antibody diluent were added to each well of a U-bottom 96-well plate, and the final concentration of CC312 was 0, 0.05, 0.15, 0.44, 1.33, 3.98, 11.9, 36.2 pM. After the antibody drug was mixed evenly with the PBMC, it was placed in a constant temperature incubator at 37°C, 5% CO2 for 72 hours. After 72 hours, the cells in each well were stained with FITC Mouse Anti-Human CD19 (BD Pharmingen), PE anti-human CD138 Antibody (BioLegend), Brilliant Violet 421 TM anti-human BCMA Antibody (BioLegend) and Near-IR fluorescent reactive dye (BioLegend), and the proportion and number of plasma cells were detected by flow cytometry, and the killing efficiency was calculated. Killing efficiency (%) = (1 - sample well cell number / vehicle control well cell number) x 100%.

[0246] The results are shown in Figures 10A-10C. CC312 can mediate effective clearance of plasma cells in bone marrow in a dose-dependent manner, and CC312 shows higher clearance capacity compared with Blincyto.

[0247] Example 8

[0248] CC312 mediates activation of T cells in PBMC of rheumatoid arthritis patients (RA) in vitro

[0249] In this example, the proliferation activity of CC312 in mediating T cells in PBMC system was detected.

[0250] The PBMC used in the test was from a RA patient (the subject was from Shanghai Renji Hospital).

[0251] After counting the PBMC of RA patients, the density was adjusted to 1.11E6 / mL with RPMI 1640 complete medium (Gibco). CC312 and Blincyto were gradient diluted with RPIM 1640 complete medium. 180μL PBMC cell suspension and 20μL antibody dilution were added to each well of U-bottom 96-well plate, and the final molar concentration of CC312 was 0, 0.0119, 0.119, 0.358, 3.58, 11.90, 119pM. After the drug was mixed evenly with PBMC, it was placed in a constant temperature incubator at 37℃, 5% CO2 for 72 hours. After 72 hours, the cells in each well were labeled with Pacific Blue anti-human CD4 antibody (BioLegend), Brilliant Violet 605 TM anti-human CD8 antibody (BioLegend), APC anti-human CD25 antibody (BioLegend), PE anti-human CD69 antibody (BioLegend) (and Near-IR fluorescent reactive dye (BioLegend), and then the proportion of the following various target cell subpopulations in each well of the 96-well plate was detected with a Thermo Fisher Attune N×T flow cytometer: CD4 + CD25 + , CD4 + CD69 + , CD8 + CD25 + , CD8 + CD69 + .

[0252] The results are shown in Figure 11. CC312 exhibited good effector cell activation ability compared with Blincyto.

[0253] Example 9

[0254] CC312 mediates T cell proliferation in PBMC of RA patients in vitro

[0255] In this example, the proliferation activity of CC312 in mediating T cells in PBMC system was detected.

[0256] The PBMC used in the test was from a RA patient (the subject was from Shanghai Renji Hospital).

[0257] After counting the PBMC of SLE patients, PBMC were labeled with CellTrace Violent at a final concentration of 5uM for 20min at 37C incubator, then washed 2-3 times with complete medium, and adjusted the PBMC cell density to 1.11E6 / ml. CC312 and Blincyto were gradient diluted with RPIM 1640 complete medium. 180uL PBMC cell suspension and 20uL antibody dilution were added into each well of U-bottom 96-well plate, the final molar concentration of CC312 was 0, 0.01193, 0.1193, 0.358, 3.58, 11.93, 119.3pM. After mixing the drug with PBMC evenly, the plate was incubated in 37C, 5% CO2 incubator for 120 hours. After 120 hours, the cells in each well were labeled with Pacific Blue anti-human CD4 antibody (BioLegend), Brilliant Violet 605 TM anti-human CD8 antibody (BioLegend), and Near-IR fluorescent reactive dye (BioLegend), then the number of CD4+ and CD8+ T cells in each well of 96-well plate was detected by Thermo Fisher Attune NxT flow cytometer, and the T cell proliferation ratio was calculated. Proliferation ratio (T cell Proliferation Ratio) = T cell number in sample well / T cell number in vehicle control well.

[0258] The results are shown in Figure 12. CC312 had a much stronger effect on the proliferation of CD4 + T cells and CD8 + T cells in PBMC than Blincyto at 3.58, 11.93pM.

[0259] Example 10

[0260] CC312 mediates autologous B cell depletion in RA patients in vitro

[0261] In this example, PBMC from three RA patients (donors from Shanghai Renji Hospital) were used to evaluate CC312-mediated autologous B cell depletion.

[0262] PBMCs from RA patients were counted and the cell density was adjusted to 1.11E6 / mL with RPMI 1640 complete medium (Gibco). CC312 was gradient diluted with RPMI 1640 complete medium. 180 μL of PBMC cell suspension and 20 μL of antibody dilution were added to each well of a U-bottom 96-well plate, and the final concentration of CC312 was 0, 0.01193, 0.1193, 0.358, 3.58, 11.93, 119.3, 1193 pM. After the antibody drug was mixed evenly with PBMCs, it was placed in a constant temperature incubator at 37°C, 5% CO2 for 72 hours. After 72 hours, the cells in each well were labeled with FITC anti-human CD20 antibody (BD Pharmingen) and 7-AAD (BioLegend), and the proportion and number of CD20 + The killing efficiency was calculated. Killing efficiency (%) = (1 - sample well cell number / vehicle control well cell number) x 100%.

[0263] The results are shown in Figure 13. CC312 can mediate the effective clearance of autologous B cells by PBMCs from three patients in a dose-dependent manner, and the highest killing efficiency can reach 93%.

[0264] Example 11

[0265] CC312 mediates the clearance of autologous B cells by PBMCs from RA patients in vitro

[0266] In this example, T cells from RA patients (the subjects were from Shanghai Renji Hospital) were used to evaluate the CC312-mediated clearance of autologous B cells.

[0267] PBMCs from RA patients were counted and T cells and B cells were sorted by cell sorting kit (BioLegend), respectively. After sorting, the cell density was adjusted with RPMI 1640 complete medium (Gibco), and CC312 was gradient diluted with RPMI 1640 complete medium. 90 μL of T cell suspension, 90 μL of B cell suspension and 20 μL of antibody dilution were added to each well of a U-bottom 96-well plate, and the final concentration of CC312 was 0, 2.38, 23.8 pM. After the antibody drug was mixed evenly with the cells, it was placed in a constant temperature incubator at 37°C, 5% CO2 for 120 hours. After 120 hours, the cells in each well were labeled with FITC anti-human CD20 antibody (BD Pharmingen) and Near-IR fluorescent reactive dye (BioLegend), and the proportion and number of CD20 + The killing efficiency was calculated. Killing efficiency (%) = (1 - sample well cell number / vehicle control well cell number) x 100%.

[0268] Results are shown in Figure 14. CC312 can mediate effective T cell clearance of autologous B cells in patients, with much higher killing ability than Blincyto at equimolar concentration.

[0269] Example 12

[0270] CC312 mediates T cell activation in PBMC of anti-synthetase syndrome patients in vitro

[0271] In this example, the T cell activation activity mediated by CC312 in PBMC system was detected.

[0272] PBMC of anti-synthetase syndrome patients (subject from Shanghai Renji Hospital) were counted and adjusted to a density of 1.11E6 / mL with RPMI 1640 complete medium (Gibco). CC312 and Blincyto were gradient diluted with RPIM 1640 complete medium. 180μL of PBMC cell suspension and 20μL of antibody dilution were added to each well of a U-bottom 96-well plate, and the final molar concentration of CC312 was 0, 0.0119, 0.119, 1.19, 11.9, 119, 1190, 11900pM. After the drug was mixed evenly with PBMC, it was placed in a constant temperature incubator at 37℃, 5% CO2 for 24 hours. After 24 hours, the cells in each well were stained with Pacific Blue anti-human CD4 antibody (BioLegend), Brilliant Violet 605 TM anti-human CD8 antibody (BioLegend), APC anti-human CD25 antibody (BioLegend), PE anti-human CD69 antibody (BioLegend) and Near-IR fluorescent reactive dye (BioLegend), and then the proportion of the following various target cell subpopulations in each well of the 96-well plate was detected by Thermo Fisher Attune N×T flow cytometer: CD4 + CD25 + , CD4 + CD69 + , CD8 + CD25 + , CD8 + CD69 + .

[0273] Results are shown in Figure 15. CC312 exhibited good effector cell activation ability compared with Blincyto.

[0274] Example 13

[0275] CC312 mediates T cell proliferation in PBMC of anti-synthetase syndrome patients in vitro

[0276] In this example, the T cell proliferation activity mediated by CC312 in PBMC system was detected.

[0277] After counting the PBMC of anti-synthetase syndrome patients (the subject was from Shanghai Renji Hospital), the PBMC was labeled with a tracer dye CellTrace Violent at a final concentration of 5 uM in a 37°C incubator for 20 min, then washed 2-3 times with complete culture medium, and the PBMC cell density was adjusted to 1.11E6 / ml. CC312 and Blincyto were gradiently diluted with RPIM 1640 complete culture medium. 180 uL of PBMC cell suspension and 20 uL of antibody dilution were added to each well of a U-bottom 96-well plate, and the final molar concentration of CC312 was 0, 0.119, 0.358, 3.58, 11.93, and 119.3 pM. After the drug was mixed uniformly with the PBMC, it was placed in a 37°C, 5% CO2 incubator for incubation for 120 hours. After 120 hours, the cells in each well were labeled with FITC anti-human CD4 antibody (BioLegend), PE anti-human CD8 antibody (BioLegend), and Near-IR fluorescent reactive dye (BioLegend), and then the number of CD4+ and CD8+ T cells in each well of the 96-well plate was detected by Thermo Fisher Attune N×T flow cytometry, and the T cell proliferation ratio was calculated. Proliferation ratio (T cell Proliferation Ratio) = T cell number in sample well / T cell number in vehicle control well.

[0278] Results are shown in Figure 16. CC312 had a much stronger ability to proliferate CD4 + T cells and CD8 + T cells in PBMC than Blincyto at 3.58 and 11.93 pM.

[0279] Example 14

[0280] CC312 mediates the clearance of autologous B cells by PBMC of anti-synthetase syndrome patients in vitro

[0281] In this example, PBMCs from anti-synthetase syndrome patients (subjected from Shanghai Renji Hospital) were used to evaluate the CC312-mediated autologous B cell depletion.

[0282] After counting the PBMCs from anti-synthetase syndrome patients, the cell density was adjusted to 1.11E6 / mL with RPMI 1640 complete medium (Gibco). CC312 and Blincyto were gradient diluted with RPMI 1640 complete medium. 180 μL of PBMC cell suspension and 20 μL of antibody diluent were added to each well of a U-bottom 96-well plate, and the final concentration of CC312 was 0, 0.01193, 0.1193, 3.58, 11.93, 119.3, 1193 pM. After the antibody drug was mixed evenly with the PBMCs, it was placed in a constant temperature incubator at 37°C, 5% CO2 for 120 hours of incubation. After 120 hours, the cells in each well were stained with FITC anti-human CD20 antibody (BD Pharmingen) and Near-IR fluorescent reactive dye (BioLegend), and the CD20 + The ratio and number of cells were calculated, and the killing efficiency was calculated. Killing efficiency (%) = (1 - sample well cell number / vehicle control well cell number) x 100%.

[0283] The results are shown in Figure 17, and CC312 can dose-dependently mediate effective depletion of autologous B cells in PBMCs from three patients, and has a better killing ability at a low concentration than Blincyto at an equimolar concentration.

[0284] Example 15

[0285] CC312 mediates the activation of T cells in MDA5+ dermatomyositis pulmonary fibrosis patient PBMCs in vitro

[0286] In this example, the activation activity of T cells mediated by CC312 in the PBMC system was detected.

[0287] PBMCs from MDA5+ dermatomyositis patients with pulmonary interstitial fibrosis (subject from Shanghai Renji Hospital) were counted and adjusted to 1.11E6 / mL with RPMI 1640 complete medium (Gibco). CC312 and Blincyto were gradient diluted with RPIM 1640 complete medium. 180 μL of PBMC cell suspension and 20 μL of antibody diluent were added to each well of a U-bottom 96-well plate, and the final molar concentration of CC312 was 0, 0.0119, 0.119, 1.19, 11.9, 119, 1190, 11900 pM. After the drug was mixed evenly with PBMC, it was placed in a constant temperature incubator at 37°C, 5% CO2 for 24 hours. After 24 hours, the cells in each well were stained with Pacific Blue anti-human CD4 antibody (BioLegend), Brilliant Violet 605 TM anti-human CD8 antibody (BioLegend), APC anti-human CD25 antibody (BioLegend), PE anti-human CD69 antibody (BioLegend) and Near-IR fluorescent reactive dye (BioLegend), and then the proportion of the following various target cell subpopulations in each well of the 96-well plate was detected with a Thermo Fisher Attune N×T flow cytometer: CD4 + CD25 + , CD4 + CD69 + , CD8 + CD25 + , CD8 + CD69 + .

[0288] The results are shown in Figure 18. Compared with Blincyto, CC312 exhibited good effect cell activation ability.

[0289] Example 16

[0290] CC312 mediates T cell proliferation in PBMCs from MDA5+ dermatomyositis patients with pulmonary interstitial fibrosis in vitro

[0291] In this example, the T cell proliferation activity mediated by CC312 in the PBMC system was detected.

[0292] PBMC from MDA5+ dermatomyositis patients with pulmonary fibrosis (donors from Shanghai Renji Hospital) were counted and labeled with CellTrace Violent at a final concentration of 5uM for 20min at 37C incubator, then washed 2-3 times with complete medium, and adjusted the PBMC cell density to 1.11E6 / ml. CC312 and Blincyto were gradient diluted with RPIM 1640 complete medium. 180ul PBMC cell suspension and 20ul antibody dilution were added to each well of a U-bottom 96-well plate, with a final molar concentration of CC312 at 0, 0.119, 0.358, 3.58, 11.93, 119.3pM. After mixing the drug with PBMC evenly, the plate was incubated in a 37C, 5% CO2 incubator for 120 hours. After 120 hours, the cells in each well were labeled with FITC anti-human CD4 antibody (BioLegend), PE anti-human CD8 antibody (BioLegend), and Near-IR fluorescent reactive dye (BioLegend), then the number of CD4+ and CD8+ T cells in each well of the 96-well plate was detected by Thermo Fisher Attune N×T flow cytometer, and the T cell proliferation ratio was calculated. Proliferation ratio (T cell Proliferation Ratio) = T cell number in sample well / T cell number in vehicle control well.

[0293] The results are shown in Figure 19, CC312 significantly promoted the proliferation of CD4 + T cells and CD8 + T cells in PBMC at 3.58, 11.93pM, far exceeding the proliferation of Blincyto at the same molar concentration.

[0294] Example 17

[0295] CC312 mediates autologous B cell depletion in PBMC from MDA5+ dermatomyositis patients with pulmonary fibrosis in vitro

[0296] In this example, PBMC from MDA5+ dermatomyositis patients with pulmonary fibrosis (donors from Shanghai Renji Hospital) were used to evaluate CC312-mediated autologous B cell depletion.

[0297] PBMCs from MDA5+ PM / IF patients were counted and the cell density was adjusted to 1.11E6 / mL with RPMI 1640 complete medium (Gibco). CC312 and Blincyto were gradient diluted with RPMI 1640 complete medium. 180 μL of PBMC cell suspension and 20 μL of antibody diluent were added to each well of a U-bottom 96-well plate, and the final concentration of CC312 was 0, 0.01193, 0.1193, 3.58, 11.93, 119.3, 1193 pM. After the antibody drug was mixed evenly with the PBMCs, it was placed in a constant temperature incubator at 37°C, 5% CO2 for 120 hours of incubation. After 120 hours, the cells in each well were labeled with FITC anti-human CD20 antibody (BD Pharmingen) and Near-IR fluorescent reactive dye (BioLegend), and the CD20 + The ratio and number of cells were calculated, and the killing efficiency was calculated. Killing efficiency (%) = (1 - sample well cell number / vehicle control well cell number) x 100%.

[0298] The results are shown in Figure 20, and CC312 can mediate the effective clearance of autologous B cells in PBMCs from patients in a dose-dependent manner, and the highest killing capacity is better than that of Blincyto at the same molar concentration.

[0299] Example 18

[0300] CC312 mediates the activation of T cells in PBMCs from patients with systemic sclerosis (SSc) in vitro

[0301] In this example, the activation of T cells mediated by CC312 in the PBMC system was detected.

[0302] The PBMCs used in the test were from SSc patients (the test subject was from Shanghai Yayu Biotechnology Co., Ltd).

[0303] PBMCs from SSc patients were counted and the density was adjusted to 1.11E6 / mL with RPMI 1640 complete medium (Gibco). CC312 and Blincyto were gradient diluted with RPIM 1640 complete medium. 180 μL of PBMC cell suspension and 20 μL of antibody diluent were added to each well of a U-bottom 96-well plate, and the final molar concentration of CC312 was 0, 0.01193, 0.1193, 0.358, 3.58, 11.93, 119.3, 1193 pM. After the drug was mixed evenly with the PBMCs, it was placed in a constant temperature incubator at 37°C, 5% CO2 for 24 hours of incubation.

[0304] 24 hours later, the cells in each well were labeled with FITC anti-human CD4 antibody (BioLegend), BV421 anti-human CD8 antibody (BioLegend), APC anti-human CD25 antibody (BioLegend), PE anti-human CD69 antibody (BioLegend) (and Near-IR fluorescent reactive dye (BioLegend), and then detected with a Thermo Fisher Attune NxT flow cytometer to detect the proportion of the following various target cell subpopulations in each well of the 96-well plate: CD4 + CD25 + , CD4 + CD69 + , CD8 + CD25 + , CD8 + CD69 + .

[0305] The results are shown in Figure 21. CC312 exhibited good effector cell activation ability compared with Blincyto.

[0306] Example 19

[0307] CC312 mediates T cell proliferation in PBMCs of systemic sclerosis (SSc) patients in vitro

[0308] In this example, the T cell proliferation activity mediated by CC312 in the PBMC system was detected.

[0309] The PBMCs used in the test were from SSc patients (the test subjects were from Shanghai Yayu Biotechnology Co., Ltd).

[0310] After counting the PBMC of SSc patients, the PBMC were labeled with CellTrace Violent at a final concentration of 5uM in a 37℃ incubator for 20min, then washed 2-3 times with complete medium, and the cell density of PBMC was adjusted to 1.11E6 / ml. CC312 and Blincyto were gradient diluted with RPIM 1640 complete medium. In each well of a U-bottom 96-well plate, 180uL of PBMC cell suspension and 20uL of antibody dilution were added, and the final molar concentration of CC312 was 0, 0.01193, 0.1193, 0.358, 3.58, 11.93, 119.3, 1193pM. After mixing the drug with PBMC evenly, the plate was placed in a 37℃, 5% CO2 incubator for incubation for 120 hours.

[0311] After 120 hours, the cells in each well were labeled with APC-cy7 anti-human CD4 antibody (BioLegend), FITC anti-human CD8 antibody (BioLegend) and 7-AAD (BioLegend), and then the number of CD4+ and CD8+ T cells in each well of the 96-well plate was detected by Thermo Fisher Attune N×T flow cytometer, and the T cell proliferation ratio was calculated. Proliferation ratio (T cell Proliferation Ratio) = T cell number in sample well / T cell number in vehicle control well.

[0312] The results are shown in Figure 22, and CC312 had the maximum proliferation ratio of CD4 + T cells and CD8 + T cells in PBMC, which was >10, far exceeding the Blincyto at the same molar concentration.

[0313] Example 20

[0314] CC312 mediates the clearance of autologous B cells by PBMC in patients with systemic sclerosis (SSc) in vitro

[0315] In this example, the functional activity of CC312 in mediating the clearance of autologous B cells by PBMC was detected.

[0316] The PBMC used in the test were from SSc patients (the test subjects were from Shanghai Yayu Biotechnology Co., Ltd).

[0317] After counting the PBMC of SSc patients, the density was adjusted to 1.11E6 / mL with RPMI 1640 complete medium (Gibco). CC312 and Blincyto were gradient diluted with RPIM 1640 complete medium. 180 μL of PBMC cell suspension and 20 μL of antibody diluent were added to each well of a U-bottom 96-well plate, and the final molar concentration of CC312 was 0, 0.01193, 0.1193, 0.358, 3.58, 11.93, 119.3, 1193 pM or 0, 0.01193, 0.1193, 11.93, 119.3, 1193 pM. After mixing the drug with PBMC uniformly, it was placed in a constant temperature incubator at 37°C, 5% CO2 for 72 hours.

[0318] After 72 hours, the cells in each well were labeled with FITC anti-human CD20 antibody (BD Pharmingen) and Near-IR fluorescent reactive dye (BioLegend), and the CD20 was detected by flow cytometry. + The proportion and number of cells were calculated, and the killing efficiency was calculated. Killing efficiency (%) = (1 - sample well CD20+% / vehicle control well CD20+%) x 100%.

[0319] The results are shown in Figure 23, and CC312 can mediate the removal of autologous B cells by PBMC of different donor patients in a concentration-dependent manner.

[0320] Example 21

[0321] CC312 mediates the activation of T cells in PBMC of IgG4-related disease (IgG4 RD) patients in vitro

[0322] In this example, the activity of CC312 in mediating the activation of T cells in PBMC system was detected.

[0323] The PBMC used in the test were from IgG4 RD patients (the test subjects were from Shanghai Yayu Biotechnology Co., Ltd).

[0324] After counting the PBMC of SSc patients, the density was adjusted to 1.11E6 / mL with RPMI 1640 complete medium (Gibco). CC312 and Blincyto were gradient diluted with RPIM 1640 complete medium. 180μL PBMC cell suspension and 20μL antibody diluent were added to each well of a U-bottom 96-well plate, and the final molar concentration of CC312 was 0, 0.01193, 0.1193, 0.358, 3.58, 11.93, 119.3, 1193pM. After mixing the drug with PBMC uniformly, it was placed in a constant temperature incubator at 37℃, 5% CO2 for 24 hours.

[0325] After 24 hours, the cells in each well were labeled with FITC anti-human CD4 antibody (BioLegend), BV421 anti-human CD8 antibody (BioLegend), APC anti-human CD25 antibody (BioLegend), PE anti-human CD69 antibody (BioLegend) (and Near-IR fluorescent reactive dye (BioLegend), and then the proportion of the following various target cell subpopulations in each well of the 96-well plate was detected with a Thermo Fisher Attune N×T flow cytometer: CD4 + CD25 + , CD4 + CD69 + , CD8 + CD25 + , CD8 + CD69 + .

[0326] The results are shown in Figure 24. Compared with Blincyto, CC312 exhibited good effect cell activation ability.

[0327] Example 22

[0328] CC312 mediates the proliferation of T cells in PBMC of IgG4-related disease (IgG4 RD) patients in vitro

[0329] In this example, the proliferation activity of CC312 in mediating T cells in PBMC system was detected.

[0330] The PBMC used in the test was from an IgG4 patient (the subject was from Shanghai Yayu Biotechnology Co., Ltd).

[0331] After counting the PBMC of SSc patients, the PBMC were labeled with CellTrace Violent at a final concentration of 5 uM in a 37 °C incubator for 20 min, and then washed 2-3 times with complete medium, and the cell density of PBMC was adjusted to 1.11E6 / ml. CC312 and Blincyto were gradient diluted with RPIM 1640 complete medium. In each well of a U-bottom 96-well plate, 180 μL of PBMC cell suspension and 20 μL of antibody diluent were added, and the final molar concentration of CC312 was 0, 0.01193, 0.1193, 0.358, 3.58, 11.93, 119.3, 1193 pM. After the drug was mixed evenly with PBMC, it was placed in a constant temperature incubator at 37 °C, 5% CO2 for incubation for 120 hours.

[0332] After 120 hours, the cells in each well were labeled with APC-cy7 anti-human CD4 antibody (BioLegend), FITC anti-human CD8 antibody (BioLegend) and 7-AAD (BioLegend), and then the number of CD4+ and CD8+ T cells in each well of the 96-well plate was detected with a Thermo Fisher Attune N×T flow cytometer, and the T cell proliferation ratio was calculated. Proliferation ratio (T cell Proliferation Ratio) = T cell number in sample well / T cell number in vehicle control well.

[0333] The results are shown in Figure 25, and CC312 has the maximum proliferation ratio of CD8 + T cells in PBMC >25, and the maximum proliferation ratio of CD4 + T cells >10, which are significantly higher than that of Blincyto at the same molar concentration.

[0334] Example 23

[0335] CC312 mediates the clearance of autologous B cells by PBMC in patients with IgG4-related disease (IgG4 RD) in vitro

[0336] In this example, the functional activity of CC312 in mediating the clearance of autologous B cells by PBMC was detected.

[0337] The PBMC used in the test were from an IgG4 patient (the subject was from Shanghai Yayu Biotechnology Co., Ltd).

[0338] After counting the PBMC of SSc patients, the density was adjusted to 1.11E6 / mL with RPMI 1640 complete medium (Gibco). CC312 and Blincyto were gradient diluted with RPIM 1640 complete medium. 180 μL PBMC cell suspension and 20 μL antibody dilution were added to each well of U-bottom 96-well plate, the final molar concentration of CC312 was all 0, 0.01193, 0.1193, 0.358, 3.58, 11.93, 119.3, 1193 pM or 0, 0.01193, 0.1193, 11.93, 119.3, 1193 pM. After mixing the drug with PBMC evenly, the mixture was incubated in a constant temperature incubator at 37°C, 5% CO2 for 72 hours.

[0339] After 72 hours, the cells in each well were labeled with FITC anti-human CD20 antibody (BD Pharmingen) and Near-IR fluorescent reactive dye (BioLegend), and the CD20 was detected by flow cytometry. + The proportion and number of cells were calculated, and the killing efficiency was calculated. Killing efficiency (%) = (1 - sample well CD20+% / vehicle control well CD20+%) x 100%.

[0340] The results are shown in Figure 26. CC312 can mediate the removal of autologous B cells by PBMC of different donor patients in a concentration-dependent manner.

[0341] Example 24

[0342] CC312 mediates the removal of autologous B cells by PBMC of patients with autoimmune hemolytic anemia (AIHA) in vitro

[0343] In this example, the functional activity of CC312 in mediating the removal of autologous B cells by PBMC was detected.

[0344] The PBMC used in the test were from AIHA patients (the test subjects were from the Hematology Hospital of Chinese Academy of Medical Sciences).

[0345] After counting the PBMC of AIHA patients, the density was adjusted to 1.11E6 / mL with RPMI 1640 complete medium (Gibco). CC312 and Blinatumomab were gradient diluted with RPIM 1640 complete medium. 180μL PBMC cell suspension and 20μL antibody diluent were added to each well of a U-bottom 96-well plate, and the final molar concentration of CC312 was 0, 0.01193, 0.1193, 0.358, 3.58, 11.93, 119.3, 1193pM. After mixing the drugs with PBMC evenly, they were placed in a constant temperature incubator at 37℃, 5% CO2 for 72 hours of incubation.

[0346] After 72 hours, the cells in each well were labeled with FITC anti-human CD20 antibody (BD Pharmingen) and Near-IR fluorescent reactive dye (BioLegend), and CD20 was detected by flow cytometry. + The proportion and number of cells were calculated, and the killing efficiency was calculated. Killing efficiency (%) = (1 - sample well CD20+% / vehicle control well CD20+%) x 100%.

[0347] The results are shown in Figure 27, and CC312 can mediate the concentration-dependent elimination of autologous B cells by PBMC of different donor patients.

[0348] Example 25

[0349] CC312 mediates the activation of T cells in PBMC of myasthenia gravis (MG) patients in vitro

[0350] In this example, the activity of CC312 in mediating the activation of T cells in PBMC systems was detected.

[0351] The PBMC used in the test were from MG patients (the test subjects were from Miaoshun (Shanghai) Biotechnology Co., Ltd.).

[0352] After counting the PBMC of MG patients, the density was adjusted to 1.11E6 / mL with RPMI 1640 complete medium (Gibco). CC312 and Blincyto were gradient diluted with RPIM 1640 complete medium. 180μL PBMC cell suspension and 20μL antibody diluent were added to each well of a U-bottom 96-well plate, and the final molar concentration of CC312 was 0, 0.01193, 0.1193, 0.358, 3.58, 11.93, 119.3, 1193pM. After mixing the drugs with PBMC evenly, they were placed in a constant temperature incubator at 37℃, 5% CO2 for 24 hours of incubation.

[0353] 24 hours later, the cells in each well were labeled with FITC anti-human CD4 antibody (BioLegend), BV421 anti-human CD8 antibody (BioLegend), APC anti-human CD25 antibody (BioLegend), PE anti-human CD69 antibody (BioLegend) (and Near-IR fluorescent reactive dye (BioLegend), and then detected with a Thermo Fisher Attune NxT flow cytometer to determine the proportion of each of the following target cell subpopulations in each well of the 96-well plate: CD4 + CD25 + CD4 + CD69 + CD8 + CD25 + CD8 + CD69 + .

[0354] The results are shown in Figure 28. CC312 exhibited good effector cell activation ability compared with Blincyto.

[0355] Example 26

[0356] CC312 mediates T cell proliferation in PBMCs of myasthenia gravis (MG) patients in vitro

[0357] In this example, the T cell proliferation activity mediated by CC312 in PBMC systems was detected.

[0358] The PBMCs used in the test were from MG patients (the test subject was from Miaoshun (Shanghai) Biotechnology Co., Ltd.).

[0359] After counting the PBMCs of the MG patients, the PBMCs were labeled with the tracer dye CellTrace Violent at a final concentration of 5 uM in a 37°C incubator for 20 min, and then washed 2-3 times with complete culture medium, and the PBMC cell density was adjusted to 1.11E6 / ml. CC312 and Blincyto were gradient-diluted with RPIM 1640 complete culture medium. In each well of a U-bottom 96-well plate, 180 uL of the PBMC cell suspension and 20 uL of the antibody dilution were added, and the final molar concentration of CC312 was 0, 0.01193, 0.1193, 0.358, 3.58, and 11.93. After the drug and PBMCs were mixed uniformly, they were incubated in a 37°C, 5% CO2 incubator for 120 hours.

[0360] After 120 hours, the cells in each well were stained with APC-cy7 anti-human CD4 antibody (BioLegend), FITC anti-human CD8 antibody (BioLegend) and 7-AAD (BioLegend), and then the number of CD4+ and CD8+ T cells in each well of the 96-well plate was detected by Thermo Fisher Attune NxT flow cytometer, and the T cell proliferation ratio was calculated. Proliferation ratio (T cell Proliferation Ratio) = T cell number in sample well / T cell number in vehicle control well.

[0361] The results are shown in Figure 29. For MG Donor 1, the maximum proliferation ratio of CD4+ T cells and CD8+ T cells in PBMC mediated by CC312 was >10, far exceeding that of Blincyto at the same molar concentration. + For MG Donor 2, the maximum proliferation ratio of CD4+ T cells and CD8+ T cells in PBMC mediated by CC312 was >7, far exceeding that of Blincyto at the same molar concentration. + For MG Donor 2, the maximum proliferation ratio of CD4+ T cells and CD8+ T cells in PBMC mediated by CC312 was >7, far exceeding that of Blincyto at the same molar concentration. + For MG Donor 2, the maximum proliferation ratio of CD4+ T cells and CD8+ T cells in PBMC mediated by CC312 was >7, far exceeding that of Blincyto at the same molar concentration. + For MG Donor 2, the maximum proliferation ratio of CD4+ T cells and CD8+ T cells in PBMC mediated by CC312 was >7, far exceeding that of Blincyto at the same molar concentration.

[0362] Example 27

[0363] CC312 mediates the clearance of autologous B cells from PBMC of myasthenia gravis (MG) patients in vitro

[0364] In this example, the functional activity of CC312 in mediating the clearance of autologous B cells from PBMC was detected.

[0365] The PBMC used in the experiment were from MG patients (donor from Miaoshun (Shanghai) Biotechnology Co., Ltd.).

[0366] After counting the PBMC of MG patients, the density was adjusted to 1.11E6 / mL with RPMI 1640 complete medium (Gibco). CC312 was gradiently diluted with RPIM 1640 complete medium. In each well of a U-bottom 96-well plate, 180 μL of PBMC cell suspension and 20 μL of antibody diluent were added, and the final molar concentration of CC312 was 0, 0.01193, 0.1193, 0.358, 3.58, 11.93, 119.3, 1193 pM. After the drug was mixed uniformly with the PBMC, it was placed in a constant temperature incubator at 37°C, 5% CO2 for incubation for 72 hours.

[0367] 72 hours later, cells in each well were labeled with Pacific Blue anti-human CD20 antibody (BioLegend) and Near-IR fluorescent reactive dye (BioLegend), and then detected by flow cytometry for CD20 + The proportion and number of cells were calculated, and the killing efficiency was calculated. Killing efficiency (%) = (1 - sample well CD20+% / solvent control well CD20+%) x 100%.

[0368] The results are shown in Figure 30. CC312 can mediate the clearance of autologous B cells from PBMCs of different donor patients in a concentration-dependent manner.

[0369] Example 28

[0370] CC312 mediates the activation of T cells in PBMCs of patients with immune thrombocytopenia (ITP) in vitro

[0371] In this example, the activity of CC312 in mediating the activation of T cells in PBMCs was detected.

[0372] The PBMCs used in the test were from patients with ITP (the test subjects were from Miaoshun (Shanghai) Biotechnology Co., Ltd.).

[0373] After counting the PBMCs of patients with ITP, the density was adjusted to 1.11E6 / mL with RPMI 1640 complete medium (Gibco). CC312 and Blincyto were gradient diluted with RPIM 1640 complete medium. 180 μL of PBMC cell suspension and 20 μL of antibody diluent were added to each well of a U-bottom 96-well plate, and the final molar concentration of CC312 was 0, 0.01193, 0.1193, 0.358, 3.58, 11.93, 119.3, 1193 pM. After the drug was mixed evenly with the PBMCs, it was placed in a constant temperature incubator at 37°C, 5% CO2 for 24 hours.

[0374] After 24 hours, the cells in each well were labeled with FITC anti-human CD4 antibody (BioLegend), BV421 anti-human CD8 antibody (BioLegend), APC anti-human CD25 antibody (BioLegend), PE anti-human CD69 antibody (BioLegend) (and Near-IR fluorescent reactive dye (BioLegend), and then detected with a Thermo Fisher Attune NxT flow cytometer to determine the proportion of each of the following target cell subpopulations in each well of the 96-well plate: CD4 + CD25 + CD4 + CD69 + CD8 + CD25 + CD8 + CD69 + .

[0375] The results are shown in FIG. 31. CC312 exhibited good effector cell activation ability compared to Blincyto.

[0376] Example 29

[0377] CC312 mediates proliferation of T cells in PBMCs of patients with immune thrombocytopenia (ITP) in vitro

[0378] In this example, the proliferation activity of CC312 in mediating T cells in PBMC systems was detected.

[0379] The PBMCs used in the test were from patients with ITP (the test subject was from Miaoshun (Shanghai) Biotechnology Co., Ltd.).

[0380] After counting the PBMCs of the ITP patient, the PBMCs were labeled with the tracer dye CellTrace Violent at a final concentration of 5 uM in a 37°C incubator for 20 minutes, then washed 2-3 times with complete culture medium, and the PBMC cell density was adjusted to 1.11E6 / ml. CC312 and Blincyto were gradient-diluted with RPIM 1640 complete culture medium. In each well of a U-bottom 96-well plate, 180 uL of the PBMC cell suspension and 20 uL of the antibody dilution were added, and the final molar concentration of CC312 was 0, 0.01193, 0.1193, 0.358, 3.58, 11.93, 119.3. After the drug and PBMCs were mixed evenly, they were placed in a constant-temperature incubator at 37°C and 5% CO2 for incubation for 120 hours.

[0381] After 120 hours, the cells in each well were stained with APC-cy7 anti-human CD4 antibody (BioLegend), FITC anti-human CD8 antibody (BioLegend) and 7-AAD (BioLegend), and then the number of CD4+ and CD8+ T cells in each well of the 96-well plate was detected by Thermo Fisher Attune NxT flow cytometer, and the T cell proliferation ratio was calculated. Proliferation ratio (T cell Proliferation Ratio) = T cell number in sample well / T cell number in vehicle control well.

[0382] The results are shown in Figure 32. For MG Donor 1, the maximum proliferation ratio of CD4 + T cells and CD8 + T cells was >7, far exceeding the equimolar concentration of Blincyto. For MG Donor 2, the maximum proliferation ratio of CD4 + T cells and CD8 + T cells was >5, far exceeding the equimolar concentration of Blincyto.

[0383] Example 30

[0384] CC312 mediates the clearance of autologous B cells by PBMC in patients with immune thrombocytopenia (ITP) in vitro

[0385] In this example, the functional activity of CC312 in mediating the clearance of autologous B cells by PBMC was detected.

[0386] The PBMC used in the experiment were from ITP patients (the subject was from Miaoshun (Shanghai) Biotechnology Co., Ltd.).

[0387] After counting the PBMC of ITP patients, the density was adjusted to 1.11E6 / mL with RPMI 1640 complete medium (Gibco). CC312 was gradient diluted with RPIM 1640 complete medium. In each well of a U-bottom 96-well plate, 180 μL of PBMC cell suspension and 20 μL of antibody dilution were added, and the final molar concentration of CC312 was 0, 0.01193, 0.1193, 0.358, 3.58, 11.93, 119.3, 1193 pM. After the drug was mixed evenly with the PBMC, it was placed in a constant temperature incubator at 37°C, 5% CO2 for incubation for 72 hours.

[0388] 72 hours later, cells in each well were stained with Pacific Blue anti-human CD20 antibody (BioLegend) and Near-IR fluorescent reactive dye (BioLegend) and detected by flow cytometry for CD20 + The ratio and number of cells were counted and the killing efficiency was calculated. Killing efficiency (%) = (1 - sample well CD20+% / vehicle control well CD20+%) x 100%.

[0389] The results are shown in Figure 33, CC312 was able to mediate the depletion of autologous B cells from different donor patient PBMCs in a concentration dependent manner.

[0390] Example 31

[0391] Pharmacodynamic study of CC312 in NCG mouse model of SLE patient PBMC transplantation

[0392] This example aims to establish a SLE patient PBMC transplantation model in NCG mice with severe immunodeficiency to test the pharmacodynamic activity of CC312 in vivo in the disease model. An equimolar concentration of bispecific CD19xCD3 antibody (Blinatumomab) was used as a control in the study.

[0393] Female NCG mice were intraperitoneally inoculated with SLE patient-derived PBMCs at a dose of 1 x 10 7 cells / 200 μL / mouse, and the inoculation day was defined as D0. On D7, the mice were randomly divided into 3 groups (vehicle control group, 0.1 mg / kg Blinatumomab group and 0.15 mg / kg CC312 group) according to body weight, with 8 mice in each group, and dosing was performed on the grouping day by tail vein injection, with a biweekly (BIW) dosing frequency for a total of three weeks. The mice were observed twice a week, and body weight changes and GVHD scores were recorded. At the end of the study (D28), the mice were euthanized, and peripheral blood was collected for anti-dsDNA antibody and human IgG detection, and lymphocytes were isolated for flow cytometry detection of B cell (hCD20 + ) and plasma cell (hCD138 + hCD38 + ) percentages; the right kidney was fresh tissue embedded for pathological immunofluorescence (to detect human IgG levels in kidney tissue).

[0394] As shown in Figure 34, there was no significant change in mouse body weight during the study compared with the vehicle control group. As shown in Figure 35, the GVHD score was normal, indicating good tolerance of the drug.

[0395] As shown in Figure 36, at the end of the study (D28), both Blinatumomab and CC312 dosing groups were observed to significantly reduce anti-dsDNA antibodies and human IgG in peripheral blood. Meanwhile, Figure 37A shows that Blinatumomab and CC312 were also able to efficiently deplete B cells. In addition, the results of Figure 37B indicate that CC312 was significantly more effective than Blinatumomab in depleting plasma cells (CD38 + CD138 + ) in peripheral blood.

[0396] As shown in Figure 38, CC312 significantly reduced the level of human IgG deposited in kidney tissue compared to the vehicle control group, and the reduction was greater than that of Blinatumomab at equimolar concentration.

[0397] Example 32

[0398] Clinical study of CC312 in treating patients with relapsed / refractory autoimmune diseases

[0399] This study is a 3+3 design, dose escalation study, aiming to determine the adverse events and maximum tolerated dose (MTD) of intravenous infusion of CC312 in patients with relapsed / refractory autoimmune diseases. Patients with moderate to severe systemic lupus erythematosus (SLE), idiopathic inflammatory myopathy (IIM), rheumatoid arthritis (RA) or systemic sclerosis (SSc) who have failed standard therapy are included. CC312 is administered at three different dose levels (5-20 μg) twice a week for four consecutive weeks. After four weeks of administration, the researchers will decide whether to continue treatment based on the efficacy of the patients and B cell depletion data. Safety, primary efficacy, pharmacodynamic (PD) parameters, including peripheral blood B cell counts, autoantibodies and other disease-related biomarkers, are observed during the administration period and the follow-up period after drug withdrawal, with a maximum follow-up time of 48 weeks. The efficacy of SLE will be evaluated by the SLE Response Index-4 (SRI-4) response rate, which includes the Systemic Lupus Erythematosus Disease Activity Score (SLEDAI-2000) score, the British Isles Lupus Assessment Group Index (BILAG-2004), and the Investigator's Global Assessment (PGA); IIM is evaluated by the American College of Rheumatology-European League Against Rheumatism (ACR-EULAR) major clinical response; RA is evaluated by ACR 20 / 50 score and Disease Activity Score in Rheumatoid Arthritis (DAS28-CRP) score; SSc is evaluated by the European Systemic Sclerosis Trials and Research Group (EUSTAR) score.

[0400] Results: A total of 5 patients with relapsed / refractory systemic lupus erythematosus were enrolled, 3 were given a dose of 5 μg, and 2 were given a dose of 10 μg, with a drug administration period of 4-12 weeks.

[0401] Baseline and dosing information of the 5 SLE patients enrolled were shown in Table 2.

[0402] Table 2. Information of patients enrolled

[0403] Results:

[0404] Safety: All 5 subjects were safe and tolerable, no DLT events occurred, all CRS were grade 1, the main clinical manifestation was fever, which lasted for a short time and recovered after symptomatic treatment, no SAE related to CC312 occurred, the adverse events related to CC312 of grade 3 or above were shown in Table 3.

[0405] Table 3. Adverse events related to CC312 of grade 3 or above

[0406] Efficacy: The clinical symptoms of 5 patients were significantly improved after treatment, the BILAG score index was significantly improved (Table 4), the SLEDAI-2K and PDA scores were significantly decreased (Figures 39 and 40), the proteinuria symptoms of 4 patients were greatly improved after administration (such as Figure 41), the anti-double-stranded DNA of 2 patients in the 10 ug dose group was observed to decrease to different degrees (one of which decreased to normal) (such as Figure 42). All patients achieved SRI-4 response.

[0407] Table 4. BILAG score index

[0408] In addition, the change of peripheral blood B cell level before and after administration was monitored in the trial, and the results were shown in Figure 43, which showed that the number of CD19+ B cells of each patient was sharply reduced or even completely cleared after administration, and for each B cell subpopulation, the 10 ug dose group showed higher clearance rate than the 5 ug dose group (Figure 44).

[0409] The results of cytokine monitoring after administration were shown in Figure 45, which showed that the overall release level of IL10, IL6, IFN-gamma, TNF-alpha factors was low, which was consistent with the good tolerability of the product.

Claims

1. Use of a trispecific antibody specifically binding to CD19, CD3 and CD28 in the manufacture of a medicament for treating an autoimmune disease, the trispecific antibody comprising: (1) a first binding domain specifically binding to CD19, comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising a heavy chain complementarity determining region 1 (HCDR1) as depicted in SEQ ID NO: 1, a heavy chain complementarity determining region 2 (HCDR2) as depicted in SEQ ID NO: 2 and a heavy chain complementarity determining region 3 (HCDR3) as depicted in SEQ ID NO: 3, the VL comprising a light chain complementarity determining region 1 (LCDR1) as depicted in SEQ ID NO: 4, a light chain complementarity determining region 2 (LCDR2) as depicted in SEQ ID NO: 5 and a light chain complementarity determining region 3 (LCDR3) as depicted in SEQ ID NO: 6; (2) a second binding domain specifically binding to CD3, comprising a VH and a VL, the VH comprising a HCDR1 as depicted in SEQ ID NO: 12, a HCDR2 as depicted in SEQ ID NO: 13 and a HCDR3 as depicted in SEQ ID NO: 14, the VL comprising a LCDR1 as depicted in SEQ ID NO: 15, a LCDR2 as depicted in SEQ ID NO: 16 and a LCDR3 as depicted in SEQ ID NO: 17; (3) a third binding domain specifically binding to CD28, comprising a VH and a VL, the VH comprising a HCDR1 as depicted in SEQ ID NO: 23, a HCDR2 as depicted in SEQ ID NO: 24 and a HCDR3 as depicted in SEQ ID NO: 25, the VL comprising a LCDR1 as depicted in SEQ ID NO: 26, a LCDR2 as depicted in SEQ ID NO: 27 and a LCDR3 as depicted in SEQ ID NO:

28.

2. The use according to claim 1, the trispecific antibody comprising: (1) a first binding domain specifically binding to CD19, comprising a VH comprising an amino acid sequence as depicted in SEQ ID NO: 7, or an amino acid sequence comprising at least 90% sequence identity to the amino acid sequence as depicted in SEQ ID NO: 7, and a VL comprising an amino acid sequence as depicted in SEQ ID NO: 8, or an amino acid sequence comprising at least 90% sequence identity to the amino acid sequence as depicted in SEQ ID NO: 8; (2) a first linker sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35 or SEQ ID NO: 36; (3) a second linker sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 or SEQ ID NO: 39; and (4) a third linker sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO:

42. (3) a second binding domain that specifically binds to CD3, comprising a VH comprising an amino acid sequence as set forth in SEQ ID NO: 18, or an amino acid sequence that comprises at least 90% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 18, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 19, or an amino acid sequence that comprises at least 90% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 19; (4) a second linker sequence comprising an amino acid sequence as set forth in SEQ ID NO: 37; and (5) a third binding domain that specifically binds to CD28, comprising a VH comprising an amino acid sequence as set forth in SEQ ID NO: 29, or an amino acid sequence that comprises at least 90% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 29, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 30, or an amino acid sequence that comprises at least 90% sequence identity to the amino acid sequence as set forth in SEQ ID NO:

30.

3. The use according to claim 1 or 2, wherein the trispecific antibody is a single chain fusion protein comprising a scFv that specifically binds to CD19 as a first binding domain, a scFv that specifically binds to CD3 as a second binding domain, and a scFv that specifically binds to CD28 as a third binding domain.

4. The use according to any one of claims 1 to 3, wherein in the trispecific antibody, the first binding domain comprises an amino acid sequence as set forth in SEQ ID NO: 11, or an amino acid sequence that comprises at least 90% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 11; the second binding domain comprises an amino acid sequence as set forth in SEQ ID NO: 22, or an amino acid sequence that comprises at least 90% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 22; and / or the third binding domain comprises an amino acid sequence as set forth in SEQ ID NO: 33, or an amino acid sequence that comprises at least 90% sequence identity to the amino acid sequence as set forth in SEQ ID NO:

33.

5. The use according to any one of claims 1 to 4, wherein the trispecific antibody comprises an amino acid sequence as set forth in SEQ ID NO: 38, or an amino acid sequence that comprises at least 90% sequence identity to the amino acid sequence as set forth in SEQ ID NO:

38.

6. The use according to any one of claims 1 to 5, wherein the autoimmune disease is a B cell-based autoimmune disease.

7. The use according to claim 6, wherein the autoimmune disease is an autoimmune disease based on autoreactive B cells.

8. The use according to claim 7, wherein the autoimmune disease based on self-reactive B cells is selected from the group consisting of systemic lupus erythematosus, lupus nephritis, type 1 diabetes, systemic sclerosis, idiopathic inflammatory myopathy, Sjogren syndrome, systemic vasculitis, rheumatoid arthritis, immune thrombocytopenia, anti-synthetase syndrome, MDA5+ dermatomyositis, MDA5+ dermatomyositis interstitial lung fibrosis, primary membranous nephropathy, limbic encephalitis, scleromyositis, anti-phospholipid syndrome, autoimmune hemolytic anemia, IgG4-related disease, Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, polyarteritis nodosa, chronic autoimmune hepatitis, primary biliary cirrhosis, myasthenia gravis, neuromyelitis optica spectrum disorder, multiple sclerosis, Lambert-Eaton syndrome, Hashimoto's thyroiditis, Graves' disease and paraneoplastic cerebellar syndrome.

9. The use according to claim 7, wherein the autoimmune disease based on self-reactive B cells is selected from the group consisting of systemic lupus erythematosus, systemic sclerosis, IgG4-related disease, anti-synthetase syndrome, autoimmune hemolytic anemia, myasthenia gravis, immune thrombocytopenia, rheumatoid arthritis, idiopathic inflammatory myopathy and MDA5+ dermatomyositis interstitial lung fibrosis.

10. The use according to any one of claims 7 to 9, wherein the autoimmune disease based on self-reactive B cells is systemic lupus erythematosus, systemic sclerosis, rheumatoid arthritis, idiopathic inflammatory myopathy, anti-synthetase syndrome, IgG4-related disease, autoimmune hemolytic anemia or MDA5+ dermatomyositis interstitial lung fibrosis.

11. A pharmaceutical composition for treating an autoimmune disease, comprising a trispecific antibody specifically binding to CD19, CD3 and CD28, and / or a pharmaceutically acceptable carrier; the trispecific antibody comprises an amino acid sequence as set forth in SEQ ID NO: 38, or an amino acid sequence having at least 90% sequence homology to the amino acid sequence as set forth in SEQ ID NO: 38.

Images