Therapy using recombinant fusion protein targeting CD47 and CD20

Abstract

Provided is a use of a recombinant fusion protein in the preparation of a drug for treating an autoimmune disease that can benefit from the reduction or elimination of CD20+B cells (for example, CD20+ B cell depletion therapy). The recombinant fusion protein comprises i) a CD47-binding peptide, and ii) an anti-CD20 antibody or an antigen-binding portion thereof, wherein the CD47-binding peptide comprises a first extracellular Ig-like domain (SIRPαD1) of signal regulatory protein α (SIRPα), and the anti-CD20 antibody or the antigen-binding portion thereof comprises a heavy chain variable region, a heavy chain constant region, a light chain variable region, and a light chain constant region. The heavy chain variable region comprises VH-CDR1, VH-CDR2, and VH-CDR3, and the light chain variable region comprises VL-CDR1, VL-CDR2, and VL-CDR3, wherein the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 respectively comprise amino acid sequences as shown in GYTFTSYN (SEQ ID NO: 1), IYPGNGDT (SEQ ID NO: 2), ARSTYYGGDWYFNV (SEQ ID NO: 3), SSVSY (SEQ ID NO: 4), ATS, and QQWTSNPPT (SEQ ID NO: 5). The heavy chain constant region has the binding capacity for an FcR or a complement system protein. The CD47-binding peptide is linked to the N-terminus of the heavy chain variable region or light chain variable region of the anti-CD20 antibody or the antigen-binding portion thereof.

Description

Therapies using recombinant fusion proteins targeting CD47 and CD20 Invention Field

[0001] This application relates to the application of a recombinant fusion protein capable of simultaneously targeting CD47 and CD20 in disease treatment. Specifically, this recombinant fusion protein can be used to treat patients who benefit from CD20. + Autoimmune diseases with reduced or eliminated B cells, such as systemic lupus erythematosus (SLE), neuromyelitis optica spectrum disorder (NMOSD), IgG4-related disease (IgG4-RD), membranous nephropathy, Sjögren's syndrome, polymyositis, dermatomyositis, multiple sclerosis, scleroderma, or vasculitis. Alternatively, this recombinant fusion protein can be used to kill B cells, particularly CD20. + B cells, such as CD20 + CD47 + B cells, or those used to implement B cell depletion therapy (BCDT). Background Technology

[0002] B cells, like T cells, are an important component of the immune system. They secrete antigen-specific antibodies and play a role in antigen presentation. B cells are generated in the bone marrow and express unique B cell receptors (BCRs). After tolerance checkpoint testing, B cells containing non-autoreactive BCRs migrate through the bloodstream to the spleen for further development, becoming naïve B cells (NBCs). NBCs circulate in the blood and secondary lymphoid organs until stimulated by antigens. The immune response to antigens occurs in secondary lymphoid organs such as lymph nodes, tonsils, and spleen. In the germinal centers (GCs) within follicles, activation of cytidine deaminase (AID) activity leads to the accumulation of mutations in the antigen-binding domain of the gene encoding the BCR, resulting in high-frequency somatic mutations. B cells with the highest antigen affinity become the main force in the immune response, and these B cells undergo antibody type switching and recombination to generate B cells that produce other types of antibodies, effectively resisting pathogens carrying foreign antigens. Some of these B cells become memory B cells (MBCs) or plasma cells (PCs). NBC cells, in conditions such as infection, can also be activated in the extrafollicular environment, such as T-cell regions or myelin cords, and rapidly transform into PCs. B cells can also express T-bet in the presence of IFNγ, and some T-bet... + B cells remain in the spleen and become MBC (Lee, DSWet al., (2021) B cell depletion therapies in autoimmune disease: advances and mechanistic insights. Nat Rev Drug Discov 20:179–199).

[0003] During secondary BCR diversification in GC using AID, BCRs may develop an affinity for self-antigens. Furthermore, B cell mutations may occur during events such as viral infections. These mutated B cells proliferate and secrete autoantibodies, all of which can potentially trigger autoimmune diseases. Additionally, T-bet... + B cells are considered to play a particularly important role in autoimmune diseases. MBCs that highly express T-bet accumulate in autoimmune diseases such as multiple sclerosis (MS) (Claes, N. et al., (2016) Age-associated B cells with proinflammatory characteristics are expanded in a proportion of multiple sclerosis patients. J. Immunol. 197:4576-4583), and in patients with systemic lupus erythematosus (SLE), B cells that highly express T-bet can rapidly differentiate into PCs in an IL-21-dependent manner (Wang, S. et al., (2018) IL-21drives expansion and plasma cell differentiation of autoreactive CD11chiT-bet+ B cells in SLE. Nat. Commun. 9:1758).

[0004] In B-cell depletion therapy (BCDT) for autoimmune diseases such as SLE and MS, CD20 antibodies are primarily used. CD20 is mainly expressed in naïve B cells, plasmablasts, and T-cell blasts. + B cells, among others, are not expressed on precursor B cells, hematopoietic stem cells, and terminally differentiated long-lived plasma cells. Using CD20 antibodies for BCDT can preserve the stem cell pool, limit targeted toxicity, and is crucial for post-treatment B cell regeneration. BCDT can also be performed using CD19 and BAFF antibodies; CD19 is mainly expressed on naive B cells, plasma cells, and T-cells. + B cells, etc. In BCDT, the level of autoantibodies did not decrease, suggesting that cytokines secreted by B cells, such as IL-6, may play a role in the occurrence and development of autoimmune diseases.

[0005] Systemic lupus erythematosus (SLE) is a typical diffuse connective tissue disease characterized by prominent autoimmune inflammation. Its pathogenesis is complex and not yet fully understood. As a B-cell-related disease, type-switching antibodies target intracellular antigens, particularly nuclear antigens (antinuclear antibodies, ANA), causing tissue damage to multiple organs. For example, lupus nephritis (LN) in SLE damages the kidneys. The short-term goals of SLE treatment are to control disease activity, improve clinical symptoms, and achieve clinical remission or low disease activity. Long-term goals are to prevent and reduce relapses, minimize adverse drug reactions, prevent and control disease-induced organ damage, achieve long-term sustained remission, reduce disability and mortality rates, and improve patients' quality of life. The etiology of SLE is heterogeneous; currently approved BAFF antibodies, belimumab, do not respond to many patients. Rituximab, which targets CD20, failed to meet the primary endpoint in clinical trials (Merrill, J.T. et al., (2010) Efficacy and safety of rituximab in moderately-to-severely active systemic lupus erythematosus: the randomized, double-blind, phase II / III systemic lupus erythematosus evaluation of rituximab trial. Arthritis Rheum. 62:222-233), and sudden exacerbations may occur after discontinuation of the drug (Merrill, J. et al., (2011) Assessment of flares in lupus patients enrolled in a phase II / III study of rituximab (EXPLORER). Lupus 20:709-716), but it has been approved in Japan and other countries for the treatment of lupus nephritis (LN) (Masanobu Ueno et al., (2025) EFFICACY). AND safety of rituximab (RTX) in reproductive lupus nephritis (LN) from real-world lows regimen. The Journal of Rheumatology 52(Suppl 1):36). Obexelimab, which targets CD19, also failed to meet its primary endpoint in a phase II clinical trial. CAR-T cell therapy targeting CD19 is currently in clinical trials.

[0006] Neuromyelitis optica spectrum disorder (NMOSD) is a central nervous system disorder caused by NMO-IgG antibodies targeting aquaporin 4 (AQP4), affecting the spinal cord, optic nerve, and brainstem. AQP4 is mainly expressed on glial cells and is highly concentrated in the foot processes of astrocytes in the spinal cord gray matter and the periventricular region of the cerebral aqueduct. When bound by NMO-IgG, AQP4 expression on the cell surface decreases, leading to increased permeability of the blood-brain barrier and activation of the complement system, which in turn promotes glial proliferation and axonal damage (Bruscolini, A. et al., (2018) Diagnosis and management of neuromyelitis optica spectrum disorders-an update. Autoimmun. Rev. 17:195-200; Mader, S. et al., (2011) Complement activating antibodies to myelin oligodendrocyte glycoprotein in neuromyelitis optica and related disorders. J. Neuroinflammation 8:184). NMOSD is a disease with a high relapse rate and high disability rate. More than 90% of patients have a relapse course, and most patients are left with relatively severe neurological disabilities. Inbilizumab, which targets CD19, has been approved for the treatment of NMOSD, while rituximab is widely used to prevent NMOSD relapse (Barreras P, et al., (2022) Long-term Effectiveness and Safety of Rituximab in Neuromyelitis Optica Spectrum Disorder and MOG Antibody Disease. Neurology. 2022 99(22):e2504-e2516).

[0007] IgG4-related disease (IgG4-RD), also known as IgG4-related autoimmune disease, is a multi-organ, fibro-inflammatory disease characterized by tumor-like lesions. It can affect almost any part of the body, most commonly the pancreas, kidneys, ring structures of the eye, salivary glands, and retroperitoneum. Patients typically have elevated serum IgG4 levels, and pathological features include dense lymphoplasmacytic infiltration in tissues, accompanied by abundant IgG4. +Plasma cells, accompanied by striations, obliterative phlebitis, and eosinophilic infiltration. Tumor-like lesions and fibrosis caused by persistent immune inflammatory response can compress and cause irreversible damage to the affected organs and surrounding tissues, even leading to organ failure (Yamamoto M, et al., (2025) Clinical profile of IgG4-related disease in Japan based on the rare disease data registry. Immunol Med. 48(3):192-202). Most IgG4-RD cases show a significant relapse course. Glucocorticoids are widely used and effective in the initial treatment and relapse treatment of IgG4-RD; however, they have significant limitations, cannot effectively prevent disease relapse after discontinuation, and long-term use can lead to significant toxic reactions. Even with other medications for IgG4-RD, such as non-hormonal immunosuppressants, patients may still face disease relapse (Omar D, et al., (2020) Glucocorticoids and steroid sparing medications monotherapies or in combination for IgG4-RD: a systematic review and network meta-analysis. Rheumatology (Oxford). 59(4):718-726). Therefore, this patient group urgently needs more effective treatments. Clinical studies have shown that B-cell depletion therapy can rapidly improve both clinical symptoms and serological indicators. Even without concurrent glucocorticoid (GC) therapy, B-cell depletion therapy remains an effective treatment for IgG4-RD. Using B-cell depletion therapy can minimize patients' glucocorticoid exposure, potentially reducing known adverse effects of hormones on tissues and organs such as bones, skin, muscles, adrenal glands, and eyes. Simultaneously, it is expected to reduce the associated risks of hormones with weight gain, diabetes, and hypertension.

[0008] CD47 is a membrane protein belonging to the immunoglobulin superfamily. It is widely expressed in various cells as a "self" marker. It interacts with signaling proteins (SIRP) on the surface of immune cells such as macrophages to send a "don't eat me" signal. Tumor cells often use this signaling pathway to evade immune regulation, and blocking the CD47-SIRP signaling pathway has achieved good results in tumor treatment. In the central nervous system, microglia perform functions similar to macrophages. CD47 on the surface of nerve cells interacts with SIRPα on the surface of microglia, thereby escaping the phagocytosis of microglia (Hutter G, et al., (2019) Microglia are effector cells of CD47-SIRPα antiphagocytic axis disruption against glioblastoma. Proc Natl Acad Sci US A. 116(3):997-1006).

[0009] The CD47-SIRP signaling pathway is also associated with autoimmune diseases. For example, in multiple sclerosis (MS) lesions, CD47 is downregulated, thereby promoting the phagocytosis of dense myelin and spleen cells by immune cells (Koning N, et al., (2007) Downregulation of macrophage inhibitory molecules in multiple sclerosis lesions. Ann Neurol. 62(5):504-514). However, the role of CD47 in autoimmune diseases is not very clear in general, and no related studies have entered the clinical stage. In SLE, the expression level of CD47 on monocytes of patients is higher than that of healthy controls, and the expression level of CD47 is related to the degree of disease activity. In addition, the amount of autoantibodies targeting CD47 is also higher than that of healthy controls (Park JK, et al., (2021) CD47 Potentiates Inflammatory Response in Systemic Lupus Erythematosus. Cells. 10(5):1151).

[0010] Reference to any document in this application is not an admission that such document is prior art. Summary of the Invention

[0011] The inventors of this application discovered in in vitro experiments that when the blood of SLE patients was treated with the recombinant fusion protein of this application, which simultaneously targets CD47 and CD20, a better B-cell depletion effect was achieved compared to rituximab. This is particularly relevant in relapsed or refractory CD20-positive SLE patients. + In clinical trials of patients with B-cell non-Hodgkin lymphoma, the recombinant fusion protein of this application was found to effectively eliminate B cells, and the number of B cells in peripheral blood remained at a low level after drug withdrawal. Combined with CD47 targeting, the recombinant fusion protein of this application also targets CD20... + B cell binding may be more robust, thereby enhancing CD20 binding. + B cell killing.

[0012] Furthermore, when the inventors applied this recombinant fusion protein to patients with SLE, NMOSD, and IgG4-RD, they found it achieved excellent B-cell depletion effects, inhibiting disease progression. This included, but was not limited to, causing a decrease in systemic IgG levels (such as AHA, AQP4-IgG, and IgG4), a decrease in proteinuria, renal function improvement, improved SLEDAI-2000 scores, achieving the Systemic Lupus Reactivity Index (SRI-4), and inhibiting or delaying clinical relapse of NMOSD, without causing serious side effects. Most importantly, B-cell remodeling was observed in patients; the remodeled B cells were predominantly naive B cells, while memory cells were at a lower level, indicating that the current application may be able to reconstruct a healthy B-cell system, fundamentally eliminating the cause of the disease.

[0013] Based on existing clinical data, the inventors of this application believe that the recombinant fusion protein of this application can also treat membranous nephropathy, Sjögren's syndrome, polymyositis, dermatomyositis, multiple sclerosis, scleroderma, and vasculitis, and clinical trials for these conditions are underway. Membranous nephropathy is a group of diseases characterized by the deposition of immune complexes in the lateral glomerular basement membrane (GBM) and subepithelial cells, accompanied by diffuse thickening of the GBM. It may be caused by autoantibodies against PLA2R, etc. CD20 antibody is the first-line treatment for intermediate- to high-risk primary membranous nephropathy (Zhao Cui, et al., (2023)#3776THE TYPEⅡANTI-CD20 MONOCLONAL ANTIBODY MIL62 OR CYCLOSPORINE IN CHINESE PRIMARY MEMBRANOUS NEPHROPATHY:PRELIMINARY RESULTS OF A PHASEⅠB / ⅡTRIAL, Nephrology Dialysis Transplantation, 38(Supplement_1):gfad063c_3776). Sjogren's syndrome is a common chronic autoimmune disease caused by the immune system mistakenly attacking its own glands, leading to inflammation and dysfunction. It primarily affects exocrine glands, especially the salivary and lacrimal glands, resulting in symptoms such as dry mouth and dry eyes. CD20 antibody therapy is one of the treatment methods for primary Sjogren's syndrome (Chen S, et al., (2014) Anti-CD20 antibody in primary Sjogren's syndrome). syndrome management. Curr Pharm Biotechnol. 15(6):535-41). Dermatomyositis and polymyositis are both idiopathic inflammatory myopathies caused by autoimmune reactions, characterized by muscle weakness. In severe cases, they can cause damage to various internal organs. Rituximab is effective for both dermatomyositis and polymyositis (Oddis CV, et al., (2013) Rituximab in the treatment of refractory adult and juvenile dermatomyositis and adult polymyositis: a randomized, placebo-phase trial. Arthritis Rheum. 65(2):314-24). The British Society for Rheumatology (BSR) guidelines for the management of myositis state that rituximab can be considered as a treatment option for refractory myositis, particularly for adolescent-onset myositis, patients with a positive myositis autoantibody profile, and patients with low disease severity (Oldroyd AGS et al., (2022) British Society for Rheumatology guideline on management of paediatric, adolescent and adult patients with idiopathic inflammatory myopathy. Rheumatology, Volume 61, Issue 5, May 2022, Pages). 1760–1768). Multiple sclerosis (MS) is a chronic autoimmune demyelinating disease affecting the central nervous system (CNS). Clinical symptoms include decreased vision, limb weakness, sensory abnormalities (such as tingling, numbness, cold limbs, etc.), ataxia, etc. As the disease progresses, patients may experience autonomic dysfunction (such as constipation, urinary frequency, etc.), mental symptoms (such as depression, irritability) and cognitive impairment (such as memory loss). The phase III trial of CD20 antibody ofatumumab for the treatment of MS is still underway (Merino-Vico A, et al., (2023) Targeting B cells and plasma cells in autoimmune diseases: From established treatments to novel therapeutic approaches. Eur J Immunol. 53(1):e2149675).Scleroderma is a chronic connective tissue disease, mainly characterized by vasculitis and fibrosis of the whole organs, leading to hardening of the skin, muscles and internal organs. It is one of the connective tissue diseases with the highest mortality rate and is divided into systemic sclerosis (SSc) and localized sclerosis. Patients have highly activated B cells, secreting autoantibodies (such as anti-Scl-70) and pro-fibrotic cytokines (such as IL-6 and TGF-β). Restoring B cell homeostasis may help control the disease. Rituximab, although not officially approved, has been used in clinical practice for the control of SSC (Bosello SL, et al., (2023) More to B: the growing evidence to inform targeting B cells in scleroderma. Rheumatology (Oxford). 62(5):1730-1732). For patients with early diffuse SSc (rapid skin progression), CD20 antibodies are the preferred second- or third-line treatment (Ebata S et al., (2022) Safety and efficacy of rituximab in systemic sclerosis (DESIRES): open-label extension of a double-blind, investigator-initiated, randomized, placebo-controlled trial. Lancet Rheumatol. 4(8): e546-e555). For patients with interstitial lung disease (ILD), CD20 antibodies are a powerful alternative to the traditional therapy cyclophosphamide, especially for patients who cannot tolerate the side effects of chemotherapy (Maher TM, et al., (2023) Rituximab versus intravenous cyclophosphamide in patients with connective tissue disease-associated interstitial lung disease in the UK (RECITAL): a double-blind, double-dummy, randomized, controlled, phase 2b trial. Lancet Respir Med. 11(1): 45-54). Antineutrophil cytoplasmic antibodies (ANCA) are autoantibodies that target cytoplasmic components of neutrophils and monocytes, such as MPO and PR3.ANCA-associated vasculitis is a rare autoimmune disease characterized by inflammation of small blood vessels. ANCA antibodies cause inflammation of the blood vessel walls and tissue damage by activating neutrophils and monocytes, often endangering organs and even life. Rituximab has been used to treat B-cell exhaustion and has caused symptom relief in patients in clinical trials (Trivioli G, et al., (2025) Advances in the treatment of ANCA-associated vasculitis. Nat Rev Rheumatol. 21(7):396-413).

[0014] Therefore, in the first aspect, this application provides a treatment in subjects in need that can benefit from CD20. + B cells are reduced or eliminated (e.g., CD20) + A method for treating autoimmune diseases using B-cell depletion therapy includes administering a recombinant fusion protein to a subject, wherein the recombinant fusion protein may comprise i) a CD47-binding peptide and ii) a CD20 antibody or its antigen-binding moiety, wherein the CD47-binding peptide may comprise a first extracellular Ig-like domain (SIRPαD1) of signal regulatory protein α (SIRPα), wherein the CD20 antibody or its antigen-binding moiety may comprise a heavy chain variable region, a heavy chain constant region, a light chain variable region, and optionally a light chain constant region, wherein the heavy chain variable region comprises VH-CDR1, VH-CDR2, and VH-CDR3, and the light chain variable region comprises VL-CDR1, VL-CDR2, and VL-CDR3, wherein VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 may each comprise GYTFTSYN (SEQ ID NO:1), IYPGNGDT (SEQ ID NO:1), and VL-CDR3, respectively. The amino acid sequences of (SEQ ID NO:2), ARSTYYGGDWYFNV (SEQ ID NO:3), SSVSY (SEQ ID NO:4), ATS, and QQWTSNPPT (SEQ ID NO:5), wherein the heavy chain constant region may contain binding forces for Fc receptors (especially FcγR) or complement system proteins, wherein the CD47 binding peptide is linked to the CD20 antibody or its antigen-binding moiety.

[0015] able to benefit from CD20 + B cells are reduced or eliminated (e.g., CD20) + B-cell depletion therapy is used to treat autoimmune diseases, including, for example, systemic lupus erythematosus (SLE), neuromyelitis optica spectrum disorder (NMOSD), IgG4-related disease (IgG4-RD), membranous nephropathy, Sjögren's syndrome, polymyositis, dermatomyositis, multiple sclerosis, scleroderma, and vasculitis.

[0016] able to benefit from CD20 + B cells are reduced or eliminated (e.g., CD20) + Autoimmune diseases treated with B-cell depletion therapy can be primary or secondary.

[0017] Methods may include administering the recombinant fusion protein to the subject at a dose of 0.8–2.0 mg / kg body weight (e.g., 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mg / kg) once a week, once every two weeks, or once every three weeks.

[0018] In some implementations, the method may include administering the recombinant fusion protein at a dose of 1.6 or 2.0 mg / kg body weight to the subject once a week, once every two weeks, or once every three weeks.

[0019] In some implementations, the method may include administering 0.8-2.0 mg / kg body weight of the recombinant fusion protein to the subject once a week.

[0020] The method can be administered in 4 applications as one course of treatment.

[0021] Methods may include an interval of 4-6 months between two treatment courses.

[0022] In some implementations, the method may include administering a recombinant fusion protein to a subject at a dose of 0.8–2.0 mg / kg body weight (e.g., 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mg / kg) once a week, once every two weeks, or once every three weeks for four consecutive weeks, and then, after 4–6 months, administering the recombinant fusion protein to the subject at a dose of 0.8–2.0 mg / kg body weight (e.g., 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mg / kg) once a week, once every two weeks, or once every three weeks for four consecutive weeks, and so on, until the desired outcome is achieved. In some implementations, the method may include administering the recombinant fusion protein at a dose of 0.8-2.0 mg / kg body weight to the subject once a week for four weeks, and then administering the recombinant fusion protein at a dose of 0.8-2.0 mg / kg body weight to the subject once a week for four weeks after four to six months, and so on, until the desired result is achieved.

[0023] In the methods of this application, systemic lupus erythematosus may include lupus nephropathy (LN). In some embodiments, the methods of this application may include treating lupus nephropathy in subjects of need.

[0024] In the methods of this application, vasculitis may include ANCA vasculitis. In some embodiments, the methods of this application may include treating ANCA vasculitis in subjects in need.

[0025] In the methods of this application, scleroderma may include systemic sclerosis. In some embodiments, the methods of this application may include treating systemic sclerosis in subjects in need.

[0026] The method of this application may include administering a composition comprising a recombinant fusion protein to a subject. The composition may be a pharmaceutical composition and may also include a pharmaceutically acceptable carrier. The method of this application may include administering the pharmaceutical composition comprising the recombinant fusion protein to a subject via intravenous or subcutaneous administration, including, for example, intravenous injection, intravenous infusion, or subcutaneous injection. In some embodiments, the method includes administering an intravenous formulation of the recombinant fusion protein or a subcutaneous formulation of the recombinant fusion protein to a subject.

[0027] In the recombinant fusion protein of this application, the heavy chain variable region of the CD20 antibody or its antigen-binding moiety may contain an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:6, and the light chain variable region may contain an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:7. The heavy chain constant region may contain Fc receptor (e.g., FcγR) and / or complement system protein binding capacity, particularly high FcR (e.g., FcγR) and / or complement system protein binding capacity. In some embodiments, the heavy chain constant region may contain binding capacity for FcR, such as FcγRIIa / IIIa, particularly high binding capacity. The heavy chain constant region can be a natural or engineered heavy chain constant region with FcR (e.g., FcγR) and / or complement system protein binding capacity, particularly a heavy chain constant region with high FcR (e.g., FcγR) and / or complement system protein binding capacity, or a functional fragment thereof, such as a hinge region, CH2, or CH3 fragment containing the heavy chain constant region, like the Fc region. The heavy chain constant region may contain mutations capable of inducing stronger antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). The heavy chain constant region can be an IgG1 constant region, such as the human IgG1 constant region, or a functional fragment thereof. In some embodiments, the heavy chain constant region may contain the amino acid sequence shown in SEQ ID NO:8. The light chain constant region can be a κ or λ light chain constant region, such as the human κ or λ light chain constant region. In some embodiments, the light chain constant region may contain the amino acid sequence shown in SEQ ID NO:9. In some embodiments, a CD20 antibody or its antigen-binding portion may comprise two heavy chains and two light chains, or be composed of two heavy chains and two light chains. In some embodiments, the CD20 antibody or its antigen-binding portion may be a single-chain antibody (scFv) or composed of antibody fragments, such as Fab or F(ab')2 fragments.

[0028] In the recombinant fusion protein of this application, the CD47-binding peptide may contain a first extracellular Ig-like domain (SIRPαD1) of signal regulatory protein α (SIRPα) and contain Ala(A) at position 80 corresponding to SEQ ID NO:10. In some embodiments, the CD47-binding peptide may contain an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:10. In some embodiments, the CD47-binding peptide may contain the amino acid sequence shown in SEQ ID NO:10.

[0029] In the recombinant fusion protein of this application, the CD47-binding peptide is linked to the N-terminus of the heavy chain variable region or light chain variable region of the CD20 antibody or its antigen-binding moiety. In some embodiments, the CD47-binding peptide is linked to the N-terminus of the heavy chain variable region of the CD20 antibody or its antigen-binding moiety. The CD47-binding peptide can be linked to the N-terminus of the heavy chain variable region or light chain variable region of the CD20 antibody or its antigen-binding moiety via a linker or directly. In some embodiments, the CD47-binding peptide can be linked to the N-terminus of the heavy chain variable region or light chain variable region of the CD20 antibody or its antigen-binding moiety via a linker. The linker can be a peptide of 5-30 amino acids in length, particularly a peptide of 10-30 amino acids in length or a peptide of 10-20 amino acids in length. The linker can be a GS linker, such as a GS linker containing the amino acid sequence shown in SEQ ID NO:11.

[0030] The recombinant fusion protein of this application may comprise i) a CD47-binding peptide-adaptor-CD20 antibody heavy chain variable region-heavy chain constant region chain, and ii) a CD20 antibody light chain variable region-light chain constant region chain; or i) a CD20 antibody heavy chain variable region-heavy chain constant region chain, and ii) a CD47-binding peptide-adaptor-CD20 antibody light chain variable region-light chain constant region chain. In some embodiments, the recombinant fusion protein of this application may comprise i) a CD47-binding peptide-adaptor-CD20 antibody heavy chain variable region-heavy chain constant region chain, and ii) a CD20 antibody light chain variable region-light chain constant region chain, wherein i) and ii) respectively comprise amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 12 and 13.

[0031] In some embodiments, the recombinant fusion protein of this application may comprise:

[0032] i) The first polypeptide chain, from the N-terminus to the C-terminus, includes a CD47-binding peptide, a linker, a heavy chain variable region capable of specifically binding to CD20, and a heavy chain constant region.

[0033] ii) The second polypeptide chain, from the N-terminus to the C-terminus, includes a light chain variable region capable of specifically binding CD20 and an optional light chain constant region.

[0034] iii) The third polypeptide chain, from the N-terminus to the C-terminus, includes a CD47-binding peptide, a linker, a heavy chain variable region capable of specifically binding to CD20, and a heavy chain constant region.

[0035] iv) The fourth polypeptide chain, from the N-terminus to the C-terminus, includes a light chain variable region capable of specifically binding CD20 and an optional light chain constant region.

[0036] The heavy chain variable region in the first polypeptide chain that specifically binds to CD20 and the light chain variable region in the second polypeptide chain that specifically binds to CD20 can form a CD20 binding domain. The heavy chain variable region in the third polypeptide chain that specifically binds to CD20 and the light chain variable region in the fourth polypeptide chain that specifically binds to CD20 can form a CD20 binding domain. The heavy chain constant region in the first polypeptide chain and the heavy chain constant region in the third polypeptide chain can be linked together by, for example, a pestle-mortar structure or a disulfide bond.

[0037] In some embodiments, the first, second, third and fourth polypeptide chains may contain the amino acid sequences shown in SEQ ID NO:12, 13, 12 and 13, respectively.

[0038] Accordingly, this application also provides the recombinant fusion protein of this application in the preparation of a treatment that can benefit CD20. + B cells are reduced or eliminated (e.g., CD20) + Use in medications for autoimmune diseases (B-cell depletion therapy). Able to benefit from CD20. + B cells are reduced or eliminated (e.g., CD20) + B-cell depletion therapy is used for autoimmune diseases, including, for example, systemic lupus erythematosus (SLE), neuromyelitis optica spectrum disorder (NMOSD), IgG4-related disease (IgG4-RD), membranous nephropathy, Sjögren's syndrome, polymyositis, dermatomyositis, multiple sclerosis, scleroderma, and vasculitis. Patients can benefit from CD20. + B cells are reduced or eliminated (e.g., CD20) + Autoimmune diseases treated with B-cell depletion therapy can be primary or secondary. Systemic lupus erythematosus can include lupus nephropathy (LN). Vasculitis can include ANCA vasculitis. Scleroderma can include systemic sclerosis.

[0039] In a second aspect, this application provides a composition configured in a single-dose form, wherein the single-dose form comprises 40 mg to 200 mg of the recombinant fusion protein of this application. The recombinant fusion protein of this application is the recombinant fusion protein defined in the first aspect of this application.

[0040] In some embodiments, the single-dose form comprises 40 mg to 80 mg of the recombinant fusion protein of this application.

[0041] In some embodiments, the single-dose form comprises 60 mg to 120 mg of the recombinant fusion protein of this application.

[0042] In some embodiments, the single-dose form comprises 80 mg to 160 mg of the recombinant fusion protein of this application.

[0043] In some embodiments, the single-dose form comprises 100 mg to 200 mg of the recombinant fusion protein of this application.

[0044] The recombinant fusion protein of this application may be in the form of lyophilized powder, and the composition may also contain water for injection.

[0045] In a third aspect, this application provides a method for reducing or eliminating B cells in a subject in need, or for administering B cell depletion therapy to a subject in need, comprising administering the recombinant fusion protein of this application to the subject. The recombinant fusion protein of this application is the recombinant fusion protein defined in the first aspect of this application.

[0046] B cells can be CD20 + B cells, especially CD20 + CD47 + B cells.

[0047] Methods may include administering the recombinant fusion protein to the subject at a dose of 0.8–2.0 mg / kg body weight (e.g., 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mg / kg) once a week, once every two weeks, or once every three weeks.

[0048] In some implementations, the method may include administering the recombinant fusion protein at a dose of 1.6 or 2.0 mg / kg body weight to the subject once a week, once every two weeks, or once every three weeks.

[0049] In some implementations, the method may include administering 0.8-2.0 mg / kg body weight of the recombinant fusion protein to the subject once a week.

[0050] The method can be administered in 4 applications as one course of treatment.

[0051] Methods may include an interval of 4-6 months between two treatment courses.

[0052] In some implementations, the method may include administering a recombinant fusion protein to a subject at a dose of 0.8–2.0 mg / kg body weight (e.g., 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mg / kg) once a week, once every two weeks, or once every three weeks for four consecutive weeks, and then, after 4–6 months, administering the recombinant fusion protein to the subject at a dose of 0.8–2.0 mg / kg body weight (e.g., 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mg / kg) once a week, once every two weeks, or once every three weeks for four consecutive weeks, and so on, until the desired outcome is achieved. In some implementations, the method may include administering the recombinant fusion protein at a dose of 0.8-2.0 mg / kg body weight to the subject once a week for four weeks, and then administering the recombinant fusion protein at a dose of 0.8-2.0 mg / kg body weight to the subject once a week for four weeks after four to six months, and so on, until the desired result is achieved.

[0053] The method of this application may include administering a composition comprising a recombinant fusion protein to a subject. The composition may be a pharmaceutical composition and may also include a pharmaceutically acceptable carrier. The method of this application may include administering the pharmaceutical composition comprising the recombinant fusion protein to a subject via intravenous or subcutaneous administration, including, for example, intravenous injection, intravenous infusion, or subcutaneous injection. In some embodiments, the method includes administering an intravenous formulation of the recombinant fusion protein or a subcutaneous formulation of the recombinant fusion protein to a subject.

[0054] Subjects may be able to benefit from CD20 + Patients with autoimmune diseases characterized by B-cell depletion or elimination, including, but not limited to, patients with SLE (e.g., LN), NMOSD, IgG4-RD, membranous nephropathy, Sjögren's syndrome, polymyositis, dermatomyositis, multiple sclerosis, scleroderma, and vasculitis. Systemic lupus erythematosus can include lupus nephropathy (LN). Vasculitis can include ANCA vasculitis. Scleroderma can include systemic sclerosis.

[0055] Accordingly, this application also provides the use of the recombinant fusion protein of this application in the preparation of a medicament for reducing or eliminating B cells. This application also provides the use of the recombinant fusion protein of this application in the preparation of a medicament for B cell depletion therapy. B cells may be CD20... + B cells.

[0056] Other features and advantages disclosed herein will become readily apparent from the following detailed description and embodiments, which should not be construed as limiting. All references, Genbank registration numbers, patents, and published patent applications cited in this specification are incorporated herein by reference. Attached Figure Description

[0057] The following detailed description, given by way of example but not intended to limit the invention to the specific embodiments described, can be better understood in conjunction with the accompanying drawings.

[0058] Figure 1 shows the expression of CD47 in lymphocytes and B cells in the blood of SLE patients. * and *** indicate p<0.05.

[0059] Figure 2 shows the B-cell depletion in the blood of SLE patients after treatment with the recombinant fusion protein of this application, rituximab, and negative control antibody (A), including the B-cell depletion in the blood of patients with mild / inactive SLE (B) and moderate-to-severe SLE (C), and the comparison of B-cell depletion between moderate-to-severe patients and mild / inactive patients (D). *** indicates p<0.05, and ns indicates no statistical difference.

[0060] Figure 3 shows the changes in tumor volume (A) and body weight (B) in tumor-bearing mice after intravenous and subcutaneous administration of the recombinant fusion protein of this application.

[0061] Figure 4 shows the changes in blood concentrations of the recombinant fusion protein of this application administered intravenously and subcutaneously in mice.

[0062] Figure 5 shows the changes in the number of B cells in the peripheral blood of lymphoma patients treated with the recombinant fusion protein of this application. The numbers after each icon represent "patient number - dosage".

[0063] Figure 6 shows the percentage of patients who achieved a reduction of ≥4 in their SLEDAI-2000 score at different time points.

[0064] Figure 7 shows the time it took for symptoms (including arthritis, rash, hair loss, and vasculitis) to disappear based on the SLEDAI-2000 score for each patient.

[0065] Figure 8 shows the changes in 24-hour proteinuria, dsDNA antibodies, and complement components C3 and C4 at baseline (BL) and after the most recent administration (LV) for each patient. Detailed Implementation

[0066] Unless otherwise specified, the terms used herein have their common meanings as found in dictionaries, textbooks, and technical reference books, or as commonly understood by those skilled in the art. The following descriptions of some terms are for the purpose of understanding this application only and are not intended to impose any particular limitations on these terms, unless otherwise specified.

[0067] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include the plural form of the object referred to, unless the context clearly specifies otherwise.

[0068] The term "or" refers to a single element among the listed selectable elements, unless the context explicitly indicates otherwise.

[0069] The terms "comprising" or "including" mean that the stated elements, integers, or steps are included, but do not exclude the inclusion of any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise specified, they also cover combinations of the stated elements, integers, or steps. The terms "consisting of" or "comprises of" generally mean that only the stated elements, integers, or steps are included, without the addition of other elements, integers, or steps.

[0070] The terms “optional” or “optionally” in this document mean that a component or step is not mandatory or necessary to include, that is, a component or step may be included in some cases and may not be included in others.

[0071] In this article, "antibody" is intended to include full-length antibodies of IgG, IgA, IgD, IgE, and IgM, as well as any antigen-binding fragments (i.e., antigen-binding portions). Full-length antibodies are typically glycoproteins containing at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain is separated by a heavy chain variable region (V). H It consists of (or VH) and the heavy chain constant region. The heavy chain constant region consists of three structural domains, namely C H1 C H2 and C H3 Each light chain consists of a light chain variable region (V for short). L It consists of a light chain constant region (or VL) and a light chain constant region. The light chain constant region consists of a structural domain C. L Composition. V H and V L The region can also be divided into highly variable regions called complementarity-determining regions (CDRs), which are separated by more conservative framework regions (FRs). Each V H and V LThe antibody heavy chain constant region consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The antibody heavy chain constant region mediates the binding of immunoglobulins to host tissues or factors, including binding to various immune system cells (e.g., effector cells) and the first component (C1q) of the conventional complement system. The "functional fragment" of the antibody heavy chain constant region refers to a segment within the constant region that retains certain desired functions; for example, a fragment in the heavy chain constant region that retains FcR / complement system component binding activity, such as an Fc fragment. The antibody light chain constant region is typically used to stabilize the antibody structure.

[0072] As used herein, the “antigen-binding portion” (or simply antigen-binding fragment) of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., the CD20 protein). It has been demonstrated that the antigen-binding function of an antibody can be performed using fragments of the full-length antibody. Examples of binding fragments contained in the “antigen-binding portion” of an antibody include (i) the Fab fragment, which is composed of V… L V H C L and C H1 (ii) F(ab')2 segment, a divalent segment containing two Fab segments connected by a disulfide bridge in the hinge region; (iii) composed of V H and C H1 (iv) Fd fragments composed of antibody single-arm V L and V H The Fv segment is composed of (v) and V. H The dAb fragment composed of H (Ward et al., (1989) Nature 341:544-546); (vi) separated complementarity-determining regions (CDRs); and (vii) dAb-V L A fragment containing a single variable structural domain and a heavy-chain constant structural domain. Furthermore, although the two structural domains V of the Fv fragment... L and V H Encoded by different genes, they can be linked via a synthetic linker that makes them single-protein chains through recombination, where V L and V H Regions pair to form monovalent molecules. These single-chain antibodies are also intended to be included in the terminology. These antibody fragments can be obtained using common techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as intact antibodies.

[0073] The crystallizable region (Fc region) is the tail region of an antibody and is the domain that determines the effector function of the antibody (i.e., how the antibody interacts with specific cellular receptors or other defense proteins). Fc receptors (FcRs) are proteins expressed on the surface of some cells, such as B lymphocytes, natural killer cells, and macrophages. They can be bound by the Fc portion of an antibody, triggering phagocytosis and cytotoxicity against target cells, playing a crucial role in the immune system. FcRs include Fcα receptors, Fcε receptors, and Fcγ receptors. Among them, Fcγ receptors belong to the immunoglobulin superfamily and are the most important FcRs in initiating phagocytosis of microorganisms, including FcγRI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), and FcγRIIIA (CD16A).

[0074] "Antibody-dependent cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" refers to cell-mediated immune defense in which immune system effector cells (such as NK cells) actively bind cell membrane surface antigens (such as CD20 and CD47) to the recombinant fusion protein of this application, resulting in the lysis of target cells.

[0075] "Antibody-dependent phagocytosis" or "ADCP" refers to an immune elimination mechanism in which the recombinant fusion protein of this application binds to target cells and binds to the FcγRIIa of immune effector cells, such as phagocytes (e.g., macrophages), via a constant region such as Fc, thereby recruiting immune effector cells and facilitating the phagocytosis of target cells by immune effector cells.

[0076] "Effective cells" or "immune effector cells" generally refer to immune cells that participate in clearing foreign antigens and performing effector functions in the immune response, such as plasma cells, cytotoxic T cells, NK cells, APSC pluripotent cells, mast cells, etc.

[0077] "Complement-dependent cytotoxicity" or "CDC" refers to the effector function of the recombinant fusion protein of this application, which, upon binding to a surface antigen, triggers a typical complement pathway, including the formation of a membrane attack complex and target cell lysis. The recombinant fusion protein of this application, upon binding to a target cell, triggers CDC on the target cell.

[0078] "Drug concentration in blood" refers to the total concentration of a drug in plasma, including drugs bound to plasma proteins or free in the plasma. It can also sometimes refer to the concentration of a drug in whole blood. The potency of a drug's effect is generally proportional to its concentration in plasma, and the concentration of a drug in the body changes over time. Drug concentrations in blood samples can be measured using methods such as high-performance liquid chromatography (HPLC).

[0079] "Specific recognition" or "specific binding" to a target, such as CD20, refers to a binding molecule, such as an antibody or its antigen-binding fragment, or a recombinant fusion protein, that can distinguish this target biomolecule from one or more reference molecules, and whose binding affinity or binding activity to the target biomolecule is, for example, 1, 5, or 10 times higher than that of other reference molecules. Specific assay methods include, but are not limited to, Western blotting, ELISA, RIA, ECL, IRMA assays, and peptide scanning.

[0080] The term "identity" or "sequence identity" as used herein refers to the percentage of nucleotides / amino acids in a sequence that are identical to those in a reference sequence after sequence alignment. If necessary, spaces are introduced in the sequence alignment to achieve the maximum percentage of sequence similarity between the two sequences. Those skilled in the art can use various methods, such as computer software, to perform pairwise or multiple sequence alignments to determine the percentage of sequence similarity between two or more nucleic acid or amino acid sequences. Such computer software includes, for example, ClustalOmega, T-coffee, Kalign, and MAFFT.

[0081] The term “subject” includes any human or non-human animal. The term “non-human animal” includes all vertebrates, such as mammals and non-mammalians, such as non-human primates, sheep, dogs, cats, cattle, horses, chickens, amphibians, and reptiles, although mammals, such as non-human primates, sheep, dogs, cats, cattle, and horses, are preferred.

[0082] Autoimmune diseases are diseases in which the immune system abnormally attacks its own normal tissues, causing organ or systemic damage. B cells participate directly or indirectly in autoimmune diseases by presenting antigens to T cells and producing pro-inflammatory cytokines or autoantibodies. "Being able to benefit from CD20..." + "Reduction or elimination of B cells" or "Ability to benefit from CD20" + "B-cell exhaustion therapy" for autoimmune diseases refers to the use of CD20 in the treatment of this autoimmune disease. + B cell reduction or elimination, or CD20 + B-cell depletion therapy, compared to no treatment, can reduce the severity of symptoms in this autoimmune disease, or increase the frequency and duration of asymptomatic periods. CD20 + B-cell elimination refers to CD20 + Complete or near-complete elimination of B cells, i.e., depletion. For example, CD20 administration is used in patients with systemic lupus erythematosus (SLE), neuromyelitis optica spectrum disorder (NMOSD), IgG4-related disease (IgG4-RD), membranous nephropathy, Sjögren's syndrome, polymyositis, dermatomyositis, multiple sclerosis, scleroderma, and vasculitis.+ B cell reduction or elimination or CD20 + B-cell depletion therapy, compared with control subjects, reduces the severity of symptoms by at least about 20%, at least about 40%, more preferably at least about 60%, or increases the frequency and duration of asymptomatic periods by at least about 20%, at least about 40%, more preferably at least about 60%, or even achieves complete cure or no recurrence.

[0083] "Treatment" refers to preventing or slowing the progression of a disease, and / or preventing or slowing the symptoms associated with the disease or condition.

[0084] B cells, like T cells, are an important component of the immune system, secreting antigen-specific antibodies and playing a role in antigen presentation. However, during the secondary BCR diversification process in the GC with the help of AID, BCRs may develop an affinity for self-antigens. Furthermore, B cell mutations may occur during events such as viral infections. These mutated B cells proliferate and secrete autoantibodies, all of which can potentially trigger autoimmune diseases. Killing pathogenic B cells with CD20 antibodies, CD19 antibodies, or BAFF antibodies has become one approach to treating autoimmune diseases, and clinically approved therapies already exist. However, not all patients respond to these therapies, or develop tolerance during treatment, necessitating new treatments to address these issues.

[0085] CD47 is a self-identified biomarker widely expressed in various cells. It interacts with signaling proteins (SIRPs) on the surface of immune cells, such as macrophages, sending a "don't eat me" signal to avoid attack by the immune system. It is well known that tumor cells frequently utilize this signaling pathway to evade immune surveillance, and blocking the CD47-SIRP signaling pathway has achieved good results in tumor treatment. In the central nervous system, microglia function similarly to macrophages. CD47 on the surface of nerve cells interacts with SIRPα on the surface of microglia, thereby escaping phagocytosis by microglia.

[0086] Pathological attenuation of the CD47-SIRP signaling pathway may be associated with autoimmune diseases. For example, in multiple sclerosis (MS) lesions, CD47 is downregulated, thereby promoting the phagocytosis of dense myelin sheaths and spleen cells by immune cells. Furthermore, it has been found that in some patients with autoimmune diseases, the expression level of certain immune cells, such as monocytes, is higher than in healthy volunteers. However, overall, the role of CD47 in autoimmune diseases is not fully understood, and no related research has entered the clinical stage.

[0087] The inventors of this application attempted to use a recombinant fusion protein capable of simultaneously targeting CD47 and CD20, through CD47 or CD20 pathway blockade, and / or ADCC, ADCP, or CDC action, to target CD20. + Treatment of B-cell-related autoimmune diseases, such as SLE, NMOSD, IgG4-RD, membranous nephropathy, Sjögren's syndrome, polymyositis, dermatomyositis, multiple sclerosis, scleroderma, or vasculitis. ADCP is mainly induced by the interaction of the Fc on the recombinant fusion protein with FcγRIIα on immune cells such as macrophages, while ADCC is mainly induced by the interaction of the Fc on the recombinant fusion protein with FcγRIIIα on immune cells such as NK cells. Macrophages are found only in tissues, while NK cells are found in both tissues and peripheral blood. The recombinant fusion protein of this application, by enhancing ADCC and ADCP function, may be able to better eliminate B cells.

[0088] Unexpectedly, the recombinant fusion protein of this application is located at CD20. + Good therapeutic effects have been achieved in patients with B-cell-related autoimmune diseases such as SLE, NMOSD, and IgG4-RD, including, but not limited to, a decrease in systemic IgG levels, a decrease in proteinuria, renal function improvement, an improvement in the SLEDAI-2000 score, achievement of the Systemic Lupus Reactivity Index (SRI-4), and inhibition or delay of clinical relapse in NMOSD. Furthermore, the recombinant fusion protein of this application is specific for B-cell killing because CD45 levels are high in blood samples treated with it. + Lymphocytes were not killed, nor did they cause erythrocytotoxicity or T-lymphocyte toxicity. Furthermore, B cell remodeling was observed in the patient, with the remodeled B cells being predominantly naive B cells and memory cells at a lower level, suggesting that the current application may be able to reconstruct a healthy B cell system, fundamentally eliminating the cause of the disease.

[0089] Specifically, this application provides a method for treating CD20 in subjects in need. + Methods for treating B-cell-related autoimmune diseases include administering the recombinant fusion protein of this application to a subject.

[0090] CD20 + B-cell-related autoimmune diseases may benefit from CD20. + B cells are reduced or eliminated (e.g., CD20) +B-cell depletion therapy is indicated for autoimmune diseases, including, for example, systemic lupus erythematosus (SLE), neuromyelitis optica spectrum disorder (NMOSD), IgG4-related disease (IgG4-RD), membranous nephropathy, Sjögren's syndrome, polymyositis, dermatomyositis, multiple sclerosis, scleroderma, and vasculitis. The method may include administering the recombinant fusion protein at doses of 0.8–2.0 mg / kg body weight (e.g., 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mg / kg) to the subject once weekly, once every two weeks, or once every three weeks. Four doses constitute one treatment cycle. After one cycle, the medication may be discontinued for a period of observation, such as 4–6 months (e.g., 4, 5, or 6 months), followed by continued administration once weekly, once every two weeks, or once every three weeks for a total of four cycles.

[0091] The recombinant fusion protein of this application can be administered to subjects via intravenous or subcutaneous routes. When administered intravenously or subcutaneously to tumor-bearing mice, the blood concentration of the recombinant fusion protein remained essentially consistent after two days of administration. Intravenous administration achieved higher blood concentrations one to two days after administration. However, despite the differences in blood concentrations in the first two days, the overall tumor-suppressive effect was comparable. Therefore, intravenous and subcutaneous administration should achieve similar therapeutic effects in the treatment of SLE, NMOSD, or IgG4-RD. Subcutaneous administration may reach the lesion faster than intravenous administration and has a lower chance of causing systemic adverse reactions. However, intravenous infusion can be used if the drug needs to be administered to organs far from the epidermis.

[0092] The recombinant fusion protein applicable to the therapeutic methods described in this application comprises two parts: a CD47-binding peptide and a CD20 antibody or its antigen-binding portion. Those skilled in the art will recognize that many design options exist for these two components. Preferably, human or humanized sequences can be used in human treatment because the strong immunogenicity of non-human animal proteins or peptides may cause allergic reactions and other adverse reactions. However, other animal proteins or peptides may also be used in this application depending on the specific application.

[0093] Any extracellular Ig-like domain of any SIRP (SIRPα, SIRPβ, and SIRPγ) capable of binding to CD47 can be selected as a CD47-binding peptide. In one embodiment, the signal regulatory protein in the recombinant fusion protein is SIRPα, specifically the first extracellular Ig-like domain of SIRPα (SIRPαD1). In one embodiment, SIRPαD1 is a mutant SIRPαD1, which, compared to wild-type SIRPαD1, has an N80A mutation at position 80 corresponding to SEQ ID NO:10. This mutation at this site achieves a deglycosylation effect. SIRPα proteins with this specific structure bind weakly to human T, B, and NK cells and almost not to human erythrocytes, reducing blood toxicity and improving safety. Amino acid residues other than this site can be mutated while retaining their desired properties.

[0094] The CD20 antibody or its antigen-binding moiety comprises heavy and light chain variable regions, each variable region containing three CDRs. The heavy chain variable region CDRs and light chain variable region CDRs of the CD20 antibody or its antigen-binding moiety used in the recombinant fusion protein of this application are determined using the IMGT numbering system. It is known in the art that the heavy chain variable region CDRs and light chain variable region CDRs can also be determined based on the full-length sequence of the variable region using numbering systems such as Chothia, Kabat, AbM, or Contact.

[0095] The CD47-binding peptide can be directly or via a linker to the N-terminus of the heavy chain variable region or light chain variable region of the CD20 antibody or its antigen-binding moiety. The linker primarily functions as a spacer between the CD47-binding peptide and the N-terminus of the heavy chain or light chain of the CD24 antibody. The linker can be composed of peptide-linked amino acids, for example, 5-30, 10-30, 10-20, or 15 amino acids linked by peptide bonds, wherein the amino acids are selected from 20 naturally occurring amino acids. One or more of these amino acids can be glycosylated or deglycosylated, as understood by those skilled in the art. In one embodiment, the 5-30, 10-30, 10-20, or 15 amino acids can be selected from glycine, alanine, proline, asparagine, glutamine, serine, and lysine. In one embodiment, the linker is composed mostly of sterically hindered amino acids with empty bonds, such as glycine and alanine. Exemplary linkers can be polyglycine (especially Gly, poly(Gly-Ala)), and polyalanine, such as the GS linker. The linker can also be a non-peptide linker. For example, alkyl linkers can be used, such as -NH-, -(CH2)sC(O)-, where s = 2-20. These alkyl linkers can also be connected to any non-sterically hindered group, such as lower alkyl groups (e.g., C). 1-4 Substitution can be achieved by lower acyl groups, halogens (e.g., Cl, Br), CN, NH2, phenyl, etc.

[0096] The CD47-binding peptide, CD20 antibody, or its antigen-binding moiety or linker of this application may contain one or more modifications (mutations), particularly conserved modifications. It is known in the art that some conserved sequence modifications do not alter the properties of a protein or peptide, particularly the primary or properties of interest, such as the antigen-binding property of an antibody or the CD47-binding property of a CD47-binding peptide. "Conserved sequence modification" refers to an amino acid modification that does not significantly affect or alter the properties of a protein or peptide, such as the antigen-binding property of an antibody or the CD47-binding property of a CD47-binding peptide. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into peptides and proteins using standard techniques known in the art, such as point mutations and PCR-mediated mutations, and their modified function and effects can be tested using detection methods known in the art. Conserved amino acid substitution involves replacing an amino acid residue with an amino acid residue having a similar side chain. Groups of amino acid residues with similar side chains are known in the art. These amino acid residue groups include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), nonpolar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0097] The pharmaceutical compositions containing recombinant fusion proteins of this application may include pharmaceutically acceptable carriers, such as excipients. Excipients that may be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersants or suspending agents, stabilizers, colorants, flavoring agents, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof.

[0098] The primary medium or carrier in a pharmaceutical composition can be inherently aqueous or non-aqueous. For example, suitable mediums or carriers may be water for injection, physiological saline, or artificial cerebrospinal fluid, supplemented with other materials commonly used in injection. For example, a medium or carrier may be a neutral buffered saline solution or a saline solution mixed with serum albumin. Other exemplary pharmaceutical compositions contain Tris buffer or acetate buffer, which may also contain sorbitol or suitable alternatives thereof. In one embodiment of this application, the composition can be prepared for storage by mixing selected components of desired purity with any formulation, in lyophilized or aqueous form. Furthermore, the pharmaceutical composition can be formulated as a lyophilized form using suitable excipients such as sucrose. Pharmaceutical compositions can be in the form of sterile aqueous solutions or suspensions. They can also be formulated as microemulsions, liposomes, or other ordered structures suitable for high concentrations of the drug.

[0099] Specifically, the pharmaceutical compositions are suitable for intravenous, intramuscular, subcutaneous, enteric, spinal, or epidermal administration (e.g., by injection or bolus). Depending on the route of administration, the active molecules may be encapsulated in a material to protect them from acids and other natural conditions that could inactivate them. In particular, the pharmaceutical compositions of this application can be administered intravenously or subcutaneously.

[0100] The amount of active ingredient that can be combined with a carrier material to prepare a single-dose formulation varies depending on the subject to be treated and the specific route of administration, and is generally the amount of the composition that produces therapeutic efficacy. Typically, this amount, in percentage terms, is about 0.01% to about 99% of the active ingredient combined with a pharmaceutically acceptable carrier.

[0101] Dosing regimens can be adjusted to achieve the optimal desired response (e.g., therapeutic response). For example, multiple fractions can be administered over time, or the dose can be reduced or increased proportionally according to the severity of the treatment condition. Particularly advantageous are parenteral compositions formulated in dose units for ease of administration and to promote dose uniformity. The dose unit type used herein refers to a physically separated unit suitable for a single dose to the treated subject; each unit contains a pre-calculated amount of active compound, together with the drug carrier, to produce the desired therapeutic effect. Alternatively, recombinant fusion proteins can be administered in a sustained-release formulation, in which case the frequency of administration is reduced.

[0102] In some embodiments, the recombinant fusion protein of this application can be formulated to ensure suitable in vivo distribution. For example, to ensure that the recombinant fusion protein of this application crosses the blood-brain barrier, the fusion protein is formulated in liposomes and may additionally contain targeting groups to enhance selective delivery to specific cells or organs.

[0103] The pharmaceutical compositions of this application can be used in combination with other therapeutic agents, vaccines, etc. The combinations of therapeutic agents discussed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or as separate compositions, wherein each agent is contained in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents can be administered sequentially.

[0104] Furthermore, if multiple combination therapies are administered and the drugs are administered sequentially, the order of administration at each time point can be reversed or kept the same, and sequential administration can be combined with simultaneous administration or any combination thereof.

[0105] This application also provides a composition configured as a single-dose formulation, wherein the single-dose formulation comprises 40 mg to 200 mg of the recombinant fusion protein of this application. A single dose refers to the amount of drug provided to a subject for a single administration. The composition configured as a single-dose formulation can be used for a single administration to a subject.

[0106] Based on the doses of 0.8, 1.2, 1.6 and 2.0 mg / kg body weight in this application, and the body weight of the subject being 50-100 kg, the composition configured as a single dose may contain 40 mg-200 mg of the recombinant fusion protein of this application.

[0107] When the dose is 0.8 mg / kg, based on the subject's body weight of 50-100 kg, this single-dose form contains 40 mg-80 mg of the recombinant fusion protein of this application.

[0108] When the dose is 1.2 mg / kg, based on the subject's body weight of 50-100 kg, this single-dose form contains 60 mg-120 mg of the recombinant fusion protein of this application.

[0109] When the dose is 1.6 mg / kg, based on the subject's body weight of 50-100 kg, this single-dose form contains 80 mg-160 mg of the recombinant fusion protein of this application.

[0110] When the dose is 2.0 mg / kg, based on the subject's body weight of 50-100 kg, this single-dose form contains 100 mg-200 mg of the recombinant fusion protein of this application.

[0111] The recombinant fusion protein in the composition can be in the form of a lyophilized powder, and the composition may also contain water for injection. The recombinant fusion protein can be dissolved in water for injection, and an appropriate volume is taken according to the concentration of the recombinant fusion protein and the specific weight of the subject. Therefore, the composition of this application, which is formulated as a single dose, is also suitable for subjects weighing less than 50 kg.

[0112] In particular, this application provides a method for treating systemic lupus erythematosus (SLE) in subjects in need.

[0113] For the treatment of SLE, the applicant of this application recruited several dose groups in Phase Ib clinical trials, within a dose range of 0.8-1.6 mg / kg, such as 0.8 mg / kg, 1.2 mg / kg, and 1.6 mg / kg, with at least 6 subjects in each group; in Phase II clinical trials, they recruited an experimental group and a placebo group, with approximately 100 subjects in each group. Dosage was administered once weekly, once every two weeks, or once every three weeks for four consecutive weeks. After an interval of 4-6 months, a continuity study was initiated, continuing the administration of once weekly, once every two weeks, or once every three weeks for four consecutive weeks. In the Phase II placebo group, dosing was administered once weekly, once every two weeks, or once every three weeks for four consecutive weeks, followed by a 4-6 month break before resuming the administration of once weekly, once every two weeks, or once every three weeks for four consecutive weeks. Phase Ib includes a screening period (maximum 4 weeks), a treatment period (approximately 24 weeks), a continuation study (approximately 24 weeks), and a follow-up period (4 weeks). Subjects who only complete the first treatment cycle have a 7-day follow-up period. Phase II includes a screening period (maximum 4 weeks), a treatment period (approximately 48 weeks), and a follow-up period (4 weeks). In clinical trials, the dosage, frequency, and intervals between treatment cycles in the continuation study can be adjusted based on the obtained clinical data. The dosing frequency in Phase II can be adjusted based on the PK / PD data from Phase Ib.

[0114] During the Phase Ib and Phase II studies, subjects continued to receive standard treatment, including glucocorticoids, antimalarial drugs, and immunosuppressants (immunosuppressants were not allowed to be used in combination with two drugs and did not include cyclophosphamide, cyclosporine, or tacrolimus).

[0115] However, if the subject's condition permits, the medication should be used according to the following requirements during the study.

[0116] - Oral corticosteroids - As standard treatment, oral corticosteroids can be continued at a stable dose until SLE disease improves. After SLE disease improves, the dose can be gradually tapered. The recommended tapering rate is as follows: Baseline prednisone (or equivalent dose) 40-20 mg / day: reduce by 5 mg / day every 1-2 weeks; 19-10 mg / day: reduce by 2.5 mg / day every 1-2 weeks; <10 mg / day: reduce by 2.5 mg / day every 2 weeks.

[0117] -Antimalarial drugs-- From the screening period until the end of week 52, the dosage regimen should be kept as stable as possible. If toxic reactions or related adverse events occur, the dosage can be reduced or discontinued. If the toxic reactions or adverse events disappear, the dosage should be restored to baseline.

[0118] -Immunosuppressants-- From the screening period until 52 weeks, the dosage regimen should be kept as stable as possible. Dosage can be reduced or discontinued if toxic reactions or related adverse events (such as infection) occur. If the toxic reaction or adverse event disappears, the dosage should be restored to baseline. Permissible doses during the study period are: azathioprine ≤2.5 mg / kg / day; methotrexate ≤25 mg / week; leflunomide ≤20 mg / day; mycophenolate mofetil ≤1.5 g / day.

[0119] The medication is administered intravenously or subcutaneously. The infusion time for the first cycle (i.e., the first four doses) is 180 min ± 20 min. The second cycle can be adjusted based on safety data from the first cycle and individual circumstances. Pretreatment can be performed 0.5–1.0 hours before infusion to prevent infusion reactions. For example, corticosteroids such as dexamethasone 5 mg–10 mg can be administered intramuscularly or intravenously; antihistamines such as diphenhydramine 50 mg or promethazine 25 mg–50 mg can be administered intramuscularly or intravenously; or antipyretic analgesics such as acetaminophen 0.5 g–0.65 g or indomethacin 50 mg can be administered orally or rectally.

[0120] Subjects must meet all of the following criteria to be enrolled in the current clinical trial:

[0121] - Patients aged 18-75 years (inclusive), male or female;

[0122] - ≥12 weeks prior to screening, diagnosed with systemic lupus erythematosus (SLE) according to the 2019 European League Against Rheumatism (EULAR) / American College of Rheumatology (ACR) diagnostic classification criteria;

[0123] - Screening visit: Antinuclear antibody (ANA) titer ≥1:80 (HEp-2 cell immunofluorescence assay) / ANA test positive (equivalent solid-phase immunoassay) or anti-double-stranded DNA antibody positive;

[0124] - During screening visits, BILAG-2004: one or more organ systems have Category A symptoms and / or two or more organ systems have Category B symptoms; SLEDAI-2000 score ≥6;

[0125] - The patient is receiving stable standard medical therapy prior to the first dose, including single-agent oral corticosteroid therapy (≤prednisone 40 mg / day or equivalent dose) or combination therapy with a stable dose of an antimalarial drug within 8 weeks prior to the first dose, or stable immunosuppressant therapy within 8 weeks prior to the first dose (immunosuppressants cannot be used in combination with two drugs and do not include cyclophosphamide, cyclosporine, or tacrolimus; permitted immunosuppressants include methotrexate, azathioprine, leflunomide, and mycophenolate mofetil). Immunosuppressants should meet the following dosage requirements: azathioprine: ≤2.5 mg / kg / day; methotrexate: ≤25 mg / week; leflunomide: ≤20 mg / day; mycophenolate mofetil: ≤1.5 g / day;

[0126] - Voluntarily sign the informed consent form.

[0127] Subjects who meet any of the following criteria are ineligible for the current clinical trial:

[0128] - Under the premise of no blood transfusion within 7 days, abnormal laboratory indicators include, but are not limited to: neutrophils <1.5×10 9 / L, hemoglobin <100g / L, platelet count ≤100×10 9 / L, total bilirubin > 1.5 × ULN, international normalized ratio (INR) > 2 × ULN or activated partial thromboplastin time (APTT) > 2 × ULN, aspartate aminotransferase (AST) > 2 × ULN and alanine aminotransferase (ALT) > 2 × ULN, glomerular filtration rate (eGFR) < 30 mL / min / 1.73m 2；

[0129] - If you have received rituximab or any B-cell clearance drug within 12 months (for Phase Ib only) or 9 months (for Phase II only) prior to the first dose, or if you have received B-cell stimulating factor inhibition, such as belimumab or teltascept, within 12 weeks or 5 times the half-life (whichever is longer) prior to the first dose;

[0130] -CD4-positive T lymphocyte count <200 cells / μL;

[0131] - Received cyclophosphamide within 12 weeks or 5 times the half-life (whichever is longer) before the first dose; received calcineurin inhibitors (cyclosporine, tacrolimus, etc., excluding topical medications) or plasma exchange therapy within 12 weeks or 5 times the half-life (whichever is longer) before the first dose;

[0132] - Received TNF inhibitors, interleukin monoclonal antibodies, JAK inhibitors, BTK inhibitors, TYK2 inhibitors, or thalidomide within 12 weeks or 5 times the half-life (whichever is longer) prior to the first dose;

[0133] - Administer a live or attenuated vaccine within 28 days prior to the first dose. Inactivated vaccines may be administered.

[0134] - Have received any clinical trial drug within 28 days prior to the first dose or within 5 times the half-life of the investigational drug;

[0135] - Comorbidities: a. History of other malignant tumors within the past 5 years, excluding basal or squamous cell carcinoma of the skin, superficial bladder cancer, or cervical or breast carcinoma that has received radical treatment; b. History of other chronic diseases requiring oral or injectable corticosteroids within the past year (e.g., asthma, Crohn's disease); c. Active infections, including lung infections, infections requiring systemic antibiotic treatment within 4 weeks prior to the first dose, and untreated EBV, cytomegalovirus, herpes simplex virus, or herpes zoster virus infections; d. Severe active central nervous system lupus currently requiring intervention (including epilepsy, psychosis, organic brain syndrome, cerebrovascular accident, encephalitis, or central nervous system vasculitis, and others); e. Known hereditary or acquired bleeding disorders; uncontrolled active bleeding or coagulation. f. Patients with blood disorders or requiring therapeutic use of anticoagulants; g. Patients with deep vein thrombosis or pulmonary embolism within 6 months prior to screening; h. Patients with acute lung disease, interstitial lung disease, such as interstitial pneumonia (excluding local interstitial pneumonia induced by radiotherapy and chemotherapy), pulmonary fibrosis, etc.; i. Systemic diseases that are not stably controlled by treatment, such as diabetes or severe organic cardiovascular and cerebrovascular diseases; j. Patients with a clear history of neurological or psychiatric disorders, such as epilepsy or dementia, or poor compliance; k. Patients with a history of inflammatory bowel disease, gastritis, gastrointestinal ulcers, rectal bleeding, persistent nausea, or other clinically significant gastrointestinal abnormalities; k. Patients with myocardial infarction, unstable angina, stroke, or New York Heart Association class III or IV heart failure that led to hospitalization within 6 months; l. Patients with active tuberculosis currently receiving anti-tuberculosis treatment or with a positive tuberculin skin test or a positive T-SPOT test.

[0136] - Positive for hepatitis B surface antigen (HBsAg) and / or hepatitis B core antibody (HBcAb), and HBV DNA titer exceeding the normal range (patients who are HBsAg positive and / or HBcAb positive need regular HBV DNA testing and receive antiviral prophylaxis); positive for hepatitis C virus (HCV) antibody; positive for human immunodeficiency virus (HIV) serological reaction. Positive for Treponema pallidum antibody (TP-Ab) (if the Treponema pallidum serological test is positive, further non-Treponema pallidum serological tests are performed; patients who are negative for the latter and are judged by the researcher to have been previously infected with syphilis but have been cured meet the inclusion criteria);

[0137] - A history of severe allergic reaction to humanized monoclonal antibodies, or a known allergy to any component of the recombinant fusion protein drug of this application;

[0138] -Pregnant and lactating women; For women of childbearing age who have not undergone sterilization: We do not agree to use appropriate methods of contraception, such as oral contraceptives, intrauterine devices, or barrier contraception combined with spermicides, from the start of the screening period until 6 months after the end of treatment;

[0139] - For men who have not undergone sterilization: We do not agree to the use of barrier contraception from the start of the screening period to 6 months after the end of treatment, and we do not agree to request their partners to use other methods of contraception, such as oral contraceptives, intrauterine devices, barrier methods, or spermicides;

[0140] - Other situations that the researchers deem unsuitable for participation in this study.

[0141] In accordance with GCP and ICH guidelines, all participants have the right to withdraw from the trial at any time during the trial without needing to give any reason. Participants may withdraw from the trial in the following circumstances: the occurrence of intolerable toxicity; the withdrawal of informed consent by the participant; or withdrawal decided by other investigators.

[0142] The primary objective of the Phase Ib clinical trial was to evaluate the safety of recombinant fusion protein at different doses and frequencies in the treatment of active SLE, and to determine the recommended dose (RP2D) and dosing frequency for Phase II. Secondary objectives included evaluating the pharmacokinetic / pharmacodynamic (PK / PD) characteristics, efficacy, and immunogenicity of the recombinant fusion protein in patients with active SLE. Exploratory objectives included evaluating the correlation between relevant biomarkers and the efficacy of the recombinant fusion protein in treating patients with active SLE, and observing the safety and efficacy of long-term treatment.

[0143] The primary objective of the Phase II clinical trial was to evaluate the efficacy of the recombinant fusion protein compared to placebo in treating patients with active SLE. Secondary objectives included evaluating the safety, population pharmacokinetic / proliferative characteristics, and immunogenicity of the recombinant fusion protein in treating patients with active SLE. An exploratory objective was to evaluate the correlation between relevant biomarkers and the efficacy of the recombinant fusion protein in treating patients with active SLE.

[0144] The primary endpoint for Phase Ib is safety and tolerability (occurrence and frequency of adverse events (AEs) and sarcastic anemia (SAEs) according to NCI CTCAE 5.0).

[0145] Secondary endpoints include (1) pharmacokinetics, peak concentration at first dose (C0). max Peak time (T) max The area under the blood drug concentration-time curve (AUC) from 0 to the last quantifiable time point 0-tIf data permits, the area under the blood drug concentration-time curve (AUC) from time 0 to infinity will also be calculated. 0-∞ ), elimination phase half-life (t) 1 / 2 ), volume of distribution (V), clearance rate (CL), etc.; steady-state trough concentration (C) during multiple administrations. min ,ss), steady-state peak concentration (C max ,ss), steady-state mean plasma concentration (C av ss), the area under the curve (AUC) of drug-time during the dosing interval. 0-tau Peak time (T) max C max Accumulation ratio (Rac_C) max ), AUC 0- tau Accumulation ratio (Rac_AUC) 0-tau If data permits, the steady-state distributed volume (V) will also be calculated. ss Steady-state clearance rate (CL) ss and elimination phase half-life (t) 1 / 2 (2) Efficacy, a. Efficacy of treatment in SLE patients over 24 weeks (proportion of patients achieving SRI-4); b. Changes in 24-hour urinary protein in subjects with elevated baseline 24-hour urinary protein (24-hour urinary protein ≥0.5g) over 24 weeks; c. Changes in quality of life in SLE patients (EQ-5D scale score); (3) Immunogenicity: Positive rate and titer of anti-drug antibodies (ADA); Neutralizing antibodies (Nab) may be further evaluated.

[0146] The exploratory endpoints were (1) changes in TBNK (including but not limited to CD3, CD4, CD56, CD16, Treg, etc.), B cell subsets (including but not limited to CD19, CD20, CD27, CD38, CD24, etc.), cytokines (CXCL-10, TNFα, IL-6, IFNg), peripheral CD47RO, serum IgG, IgA, IgM, complement (C3, C4), antinuclear antibody (ANA), anti-dsDNA antibody, Th1, Th2, etc. before and after administration, and their relationship with efficacy; (2) the safety and efficacy of long-term treatment, the occurrence of adverse events; and the efficacy of treatment in SLE patients for 52 weeks (the proportion of patients reaching SRI-4).

[0147] The primary endpoint for Phase II was the percentage of patients with active SLE who achieved the Systemic Lupus Response Index (SRI-4) at week 52 after medication. SRI-4 criteria were: a decrease of ≥4 points in SLEDAI-2000 from baseline; no new ≥1 Grade A score or ≥2 Grade B score from baseline in BILAG-2004; and no or <0.3 points increase in PGA score from baseline.

[0148] Secondary endpoints were: (1) efficacy, a. the percentage of subjects achieving the Systemic Lupus Response Index (SRI-4) at week 24; b. changes in 24-hour urinary protein levels at week 24 and week 52 in patients with elevated baseline 24-hour urinary protein (≥0.5 g); c. the percentage of subjects achieving or maintaining prednisone ≤7.5 mg / day (or equivalent dose) between weeks 40 and 52; d. changes in EQ-5D scale scores from baseline to week 52; e. changes in serological markers (IgG, IgA, IgM, anti-dsDNA antibody, complement C3, C4) from baseline. (2) Safety: Evaluation of the safety of recombinant fusion protein or placebo in combination with standard therapy in SLE subjects. All adverse events (AEs) will be classified according to the system organ classification (SOC) and preferred term (PT) of the International Medical Dictionary (MedDRA) version 27.0 or above, and the incidence of AEs will be calculated according to the Common Adverse Event Evaluation Criteria (CTCAE) v5.0. (2) Pharmacokinetics: trough and peak concentrations of the drug in the blood. (3) Immunogenicity: positive rate and titer of anti-drug antibodies (ADA); neutralizing antibodies (Nab) may be further evaluated.

[0149] The exploratory endpoints are changes in TBNK (including but not limited to CD3, CD4, CD56, CD16, Treg, etc.), B cell subsets (including but not limited to CD19, CD20, CD27, CD38, CD24, etc.), cytokines (CXCL-10, TNFα, IL-6, IFNg), peripheral CD47RO, serum IgG, IgA, IgM, complement (C3, C4), antinuclear antibody (ANA), anti-dsDNA antibody, Th1, Th2, etc. before and after drug administration, and their relationship with efficacy.

[0150] During the study, if the standard treatment regimen was adjusted due to poor control of SLE activity in the subjects, the following conditions will be considered as failure to achieve SRI-4 in the efficacy assessment of this and subsequent visits: The average daily dose of prednisone (or equivalent dose) of glucocorticoids increased by >5 mg / day or >30 mg / day from baseline in the 7 days prior to each visit during weeks 24-40 (W25D1-W41D1); the average daily dose of prednisone (or equivalent dose) of glucocorticoids increased by >5 mg / day or >10 mg / day from baseline in the 7 days prior to each visit during weeks 44-52 (W45D1-W53D1); the dose of antimalarial drugs was added or increased after week 16 (W17D1 visit); the dose of immunosuppressants needed to be added during the study or increased after week 16 (W17D1 visit).

[0151] Statistical analysis of the data is performed in SAS 9.4 or later.

[0152] The population to be analyzed will include:

[0153] The All Participants Set (ENR) includes all participants who provided informed consent for this study. It is used for analysis of the distribution of all participants.

[0154] The full analysis set (FAS) includes all enrolled subjects who have received the study drug at least once. The FAS will be used for demographic and baseline data analysis.

[0155] The safety analysis set (SS) includes subjects who have received the study drug at least once and have undergone at least one post-dosage safety assessment. The SS is used for safety analysis.

[0156] The Protocol Set (PPS) includes participants who received at least one dose of the study drug and completed the week 52 efficacy evaluation, or who withdrew early due to severe SLE relapse or treatment failure, deemed unsuitable for continued participation, and whose protocol deviations did not significantly affect the efficacy evaluation. This is used for efficacy analysis.

[0157] The PK analysis set (PKAS) includes all subjects who received the study drug, had no protocol deviations that would seriously affect PK evaluation, and had evaluable PK data.

[0158] The Pharmacodynamic Analysis Set (PDAS) includes all subjects who received the study drug, had no protocol deviations that would seriously affect pharmacodynamic evaluation, and had evaluable pharmacodynamic data.

[0159] The Immunogenicity Analysis Set (IMS) includes all subjects who received the study drug, had no protocol deviations that would seriously affect immunogenicity assessment, and had evaluable immunogenicity data.

[0160] The DLT evaluable analysis set (DES) will include all subjects who, in the designated dose group during Phase Ib, achieved a relative dose intensity ≥80% and completed a safety assessment during the DLT observation period, or who withdrew from the trial early due to DLT during the DLT observation period.

[0161] Descriptive statistics should be compiled or listed for all variables obtained at each observation time point, unless the protocol or statistical analysis plan (SAP) determines that statistical description is not required at a specific time point. Generally, continuous variables (such as age) will be described using the number of cases, mean, standard deviation, median, minimum, and maximum; categorical variables will be described using the frequency and percentage of each category.

[0162] All adverse events (AEs) will be classified according to SOC and PT according to MedDRA version 27.0 or later, and graded according to CTCAE V5.0. All treatment-related adverse events (TEAEs), grade 3 or higher TEAEs, SAEs, TEAEs related to the study drug, grade 3 or higher TEAEs related to the study drug, SAEs leading to discontinuation of study drug, and adverse events of particular concern (AESIs) will be summarized by SOC and PT in terms of number and percentage. Furthermore, the severity of TEAEs will also be summarized by SOC and PT.

[0163] Laboratory test results, vital signs, and 12-lead ECG values ​​will be analyzed using descriptive statistics to examine their observed values ​​and changes relative to baseline. Where applicable, baseline clinical significance and clinical significance at each planned time point after baseline will be presented in cross-tabulation format. Descriptive statistics will be performed on the frequencies and percentages of physical examination categorization results at each planned time point.

[0164] Descriptive statistical analysis was performed on all pharmacokinetic parameters, and the number of cases, mean, standard deviation, median, minimum, maximum, coefficient of variation, geometric mean, and coefficient of variation of the geometric mean were reported. Individual and mean (mean ± SD) pharmacokinetic curves were also plotted.

[0165] Effectiveness analysis was conducted based on the full analysis set and the set of compliant protocols. The main effectiveness assessment parameters included SLEDAI-2000, BILAG-2004, PGA, and EQ-5D scale scores. Effectiveness assessments were performed at 24 and 52 weeks.

[0166] The report summarizes the overall number and percentage of ADA cases at each planned time point. A list presents the changes in ADA and Nab for each subject (ADA-positive subjects only). Based on the safety and tolerability data obtained in Phase Ib, including the occurrence and frequency of adverse events (AEs), the recommended dose for Phase II (RP2D) is derived.

[0167] In particular, this application also provides a method for treating neuromyelitis optica spectrum disorder (NMOSD) in subjects in need.

[0168] For the treatment of NMOSD, the applicant of this application recruited several dose groups in Phase Ib clinical trials, within a dose range of 0.8-1.6 mg / kg, such as 0.8 mg / kg, 1.2 mg / kg, and 1.6 mg / kg, with at least 8 subjects in each group, including 6 in the treatment group and 2 in the positive control group. After subjects in the low-dose group completed the DLT observation period, a decision was made on whether to escalate to the next dose group. In Phase III clinical trials, the applicant recruited experimental and positive control groups, planning for approximately 50 subjects in each group. Dosage was administered once weekly, once every two weeks, or once every three weeks for four consecutive weeks. After an interval of 4-6 months, a continuity study was initiated, continuing the administration of once weekly, once every two weeks, or once every three weeks for four consecutive weeks. Subjects enrolled in the positive control group received mycophenolate mofetil (MMF) orally, 1.0 g twice daily.

[0169] The Phase Ib study period is approximately 24 weeks. Dosing after week 24 is not included in the Phase Ib safety and efficacy assessment; it serves only as an extension of the long-term efficacy and safety observation of Phase Ib, extending to week 52. Efficacy and safety evaluations are conducted at least every 3 months according to standard clinical practice. After week 52, subjects who have not experienced a relapse are followed up by telephone until their disease recurs. During telephone follow-ups, if the patient is not experiencing a relapse, their medication information (drug name and time of administration) is recorded; if a relapse occurs, the time of the relapse is recorded, and the telephone follow-up ends. During the Phase Ib clinical trial, subject safety will be closely monitored, with a comprehensive assessment every month. The decision to initiate the next phase, Phase III, will be based on safety data from all subjects 60 days after injection.

[0170] The Phase III study lasts 52 weeks and evaluates the efficacy and safety of the recombinant fusion protein in the treatment of NMOSD. After completing the prescribed 52-week Phase III clinical trial, all subjects have the opportunity to continue treatment with the recombinant fusion protein during the study extension period (subjects meeting the onset diagnosis are considered to have completed the treatment period and entered the study extension period) for no more than 2 years. During the study extension period, subjects receive repeated recombinant fusion protein treatment every 6 months, with efficacy and safety evaluations performed at least once every 3 months at the clinical trial center, following standard clinical practice. The dosing frequency and intervals between treatments in Phase III may be adjusted based on the results of the Phase Ib PK / PD data.

[0171] The drug is administered via intravenous injection or subcutaneous injection. The infusion time for the first cycle (i.e., the first 4 doses) is 180 min ± 20 min. The second cycle can be adjusted based on the safety data from the first cycle and individual circumstances.

[0172] Subjects must meet all of the following criteria to be enrolled in the current clinical trial.

[0173] - Male or female patients, aged 18 to 70 years (inclusive);

[0174] - Patients who meet the 2015 international consensus diagnostic criteria for neuromyelitis optica spectrum disorder (NMOSD) (the presence of optic neuritis and / or myelitis in clinical symptoms) and are positive for AQP4-IgG;

[0175] - Extended Disability Status Scale (EDSS) score ≤ 7.0;

[0176] Stage Ib: At least one NMOSD seizure requiring salvage therapy within the two years prior to screening; Stage III: At least one NMOSD seizure requiring salvage therapy within the year prior to screening; or at least two NMOSD seizures requiring salvage therapy within the two years prior to screening. This includes the first seizure.

[0177] - Patients who have received glucocorticoid therapy prior to screening, and whose dose of prednisone or its equivalent dose has been ≤20 mg / day within 14 days prior to the first administration;

[0178] - Voluntarily sign the informed consent form.

[0179] Subjects who meet any of the following criteria are ineligible for enrollment in the current clinical trial:

[0180] - Abnormal laboratory indicators that need to be excluded from the subject's examination include, but are not limited to, the following: neutrophils <1.5×10⁻⁶. 9 / L, hemoglobin <90g / L, platelet count <100×10 9 Serum creatinine > 1.5 × ULN, total bilirubin > 1.5 × ULN, aspartate aminotransferase (AST) > 1.5 × ULN, alanine aminotransferase (ALT) > 1.5 × ULN, alkaline phosphatase > 2 × ULN; glomerular filtration rate (GFR) < 60 mL / min / 1.73 cm 2 ;

[0181] - Those who have received rituximab or any B-cell clearance drug within 6 months prior to the screening period, or who have used B-cell clearance drugs, have a CD20 level during the screening period. + B cell count below the lower limit of normal (LLN);

[0182] - Use of alkylating agents such as tocilizumab, eculizumab, mitoxantrone, or cyclophosphamide within 3 months prior to the first dose;

[0183] Phase Ib: Patients who have used immunosuppressants such as azathioprine (half-life = 6h), mycophenolate mofetil (half-life = 16h), tacrolimus (half-life = 43h), cyclosporine (half-life = 27h), methotrexate (half-life = 14h), or cyclophosphamide (half-life = 6h) before the first dose, but whose drug discontinuation interval exceeds 5 times the half-life, are eligible for enrollment. Phase III: Patients who have used immunosuppressants other than glucocorticoids within 1 month before the first dose, including but not limited to azathioprine, mycophenolate mofetil, tacrolimus, cyclosporine, and methotrexate (if continuous use is ≤7 days, exclusion is not required). Leflunomide and teriflunomide require cholestyramine washout. These medications can be discontinued and the following measures taken: Administer cholestyramine 8g three times daily for 11 days. If the 8g dose is not tolerated, it can be changed to 4g orally each time, at the same time and frequency as before.

[0184] - Those who have used plasma exchange (PE) or moderate-volume transfusion, or immunomodulatory drugs such as interferon beta, interferon gamma, or intravenous immunoglobulin (IVIG) within 28 days prior to the first dose;

[0185] - Combined with other chronic active immune system diseases that require treatment with glucocorticoids, biologics, or immunosuppressants (such as rheumatoid arthritis, scleroderma, etc.);

[0186] - Received a live or attenuated live vaccine within 4 weeks prior to screening; received an inactivated vaccine within 2 weeks prior to baseline; received a novel coronavirus vaccine within 4 weeks prior to baseline;

[0187] - Previous treatments include bone marrow transplantation, hematopoietic stem cell transplantation, whole lymph node irradiation, and T-cell vaccine therapy;

[0188] - Have received any clinical trial drug within 28 days prior to the first dose or within 5 times the half-life of the investigational drug, whichever is shorter;

[0189] -Having any of the following conditions: ①Having had another malignant tumor within the past 3 years, except for basal or squamous cell skin cancer, superficial bladder cancer, cervical or breast carcinoma in situ, or other malignant tumors with low risk of metastasis and death (5-year survival rate >90%) that have received radical treatment; ②Severe liver or kidney dysfunction; ③Active bacterial, viral, fungal, mycobacterial, parasitic, or other infections (excluding nail bed fungal infection) that require anti-infective treatment or hospitalization; ④Having a severe mental illness;

[0190] - Patients' hearts meet any of the following criteria: ① Left ventricular ejection fraction (LVEF) ≤ 55%; ② New York Heart Association (NYHA) class II or above congestive heart failure or active heart disease; ③ Severe arrhythmia requiring treatment (excluding atrial fibrillation and paroxysmal supraventricular tachycardia deemed by the investigator to have no impact on the trial); ④ QTc interval ≥ 450 ms for men and ≥ 470 ms for women (QTcB = QT / RR1 / 2); ⑤ Myocardial infarction or bypass surgery, stenting surgery within 6 months prior to drug administration, or other heart diseases deemed unsuitable for enrollment by the investigator.

[0191] - Positive for hepatitis B surface antigen (HBsAg) and / or hepatitis B core antibody (HBcAb), and HBV DNA greater than the upper limit of normal; positive for hepatitis C virus (HCV) antibody, and HCV RNA greater than the upper limit of normal; positive for human immunodeficiency virus (HIV) serological reaction; positive for Treponema pallidum antibody (TP-Ab) (if the Treponema pallidum serological test is positive, further non-Treponema pallidum serological tests are performed, and patients who are negative for the latter and are judged by the researcher to have been previously infected with syphilis but have been cured meet the inclusion criteria);

[0192] - Suffering from acute lung disease, interstitial lung disease, such as interstitial pneumonia (excluding local interstitial pneumonia induced by radiotherapy and chemotherapy), pulmonary fibrosis, active pulmonary tuberculosis (positive tuberculin test, positive T-SPOT test), etc.;

[0193] - Allergic to parenteral contrast agents or unable to undergo magnetic resonance imaging;

[0194] - History of severe allergic reaction to humanized monoclonal antibodies, or known allergy to any component of recombinant fusion protein;

[0195] -Pregnant and lactating women; For women of childbearing age who have not undergone sterilization: We do not agree to use appropriate methods of contraception, such as oral contraceptives, intrauterine devices, or barrier contraception combined with spermicides, from the start of the screening period until 6 months after the end of treatment;

[0196] - For men who have not undergone sterilization: We do not agree to the use of barrier contraception from the start of the screening period to 6 months after the end of treatment, and we do not agree to request their partners to use other methods of contraception, such as oral contraceptives, intrauterine devices, barrier methods, or spermicides;

[0197] - Other situations that the researchers deem unsuitable for participation in this study.

[0198] According to GCP and ICH guidelines, all participants have the right to withdraw from the trial at any time during the trial without any reason. Participants may withdraw from the trial in the following circumstances: loss to follow-up; occurrence of intolerable toxicity; withdrawal of informed consent; or withdrawal decided by other investigators.

[0199] The primary objective of Phase Ib was to evaluate the safety of recombinant fusion protein at different doses and dosing frequencies in treating NMOSD patients, and to determine the optimal therapeutic dose and dosing frequency of recombinant fusion protein in conjunction with Phase Ib clinical trials of recombinant fusion protein in treating SLE.

[0200] Secondary objectives were to evaluate the PK / PD characteristics, efficacy, and immunogenicity of recombinant fusion protein in the treatment of NMOSD patients, and to evaluate changes in AQP4-IgG titers.

[0201] The exploratory objective was to evaluate the correlation between relevant biomarkers and the efficacy of recombinant fusion protein in treating NMOSD patients.

[0202] The primary objective of Phase III was to evaluate the time to first clinical relapse at 52 weeks or within 52 weeks in NMOSD patients compared to the positive control.

[0203] Secondary objectives were to evaluate the efficacy, safety, population PK / PD characteristics, and immunogenicity of recombinant fusion protein in treating NMOSD patients, and to evaluate changes in AQP4-IgG titers.

[0204] The exploratory objective was to evaluate the correlation between relevant biomarkers and the efficacy of recombinant fusion protein in treating NMOSD patients.

[0205] The primary endpoint for Phase Ib is safety, specifically the occurrence and frequency of adverse events (AEs) and sarcastic anxieties (SAEs) (according to NCICTCAE 5.0).

[0206] Secondary endpoints were: (1) pharmacokinetics, first dose: peak concentration (C max Peak time (T) max The area under the blood drug concentration-time curve (AUC) from 0 to the last quantifiable time point 0-tlast If data permits, the area under the blood drug concentration-time curve (AUC) from time 0 to infinity will also be calculated. 0-inf ), elimination phase half-life (t) 1 / 2 ), volume of distribution (V), clearance rate (CL), etc.; multiple administrations: steady-state trough concentration (C) min,ss ), steady-state peak concentration (C) max,ss ), steady-state mean plasma concentration (C av,ss The area under the curve (AUC) of drug-time during the dosing interval 0-tau Peak time (T)max C max The accumulation ratio (R) ac _C max ), AUC 0-tau The accumulation ratio (R) ac _AUC 0-tau If data permits, the steady-state distributed volume (V) will also be calculated. ss Steady-state clearance rate (CL) ss and elimination phase half-life (t) 1 / 2 (2) Efficacy, time to first clinical relapse within 24 and 52 weeks, changes in Extended Disability Status Scale (EDSS) scores; number of cumulative active lesions on magnetic resonance imaging (MRI); changes in visual function; annualized relapse rate; proportion of subjects hospitalized due to NMOSD relapse; changes in 25-foot walk test, nine-hole pedicle test (9HPT), visual evoked potential (VEP) P100 latency, and changes in RNFL thickness on optical coherence tomography (OCT); (3) Immunogenicity, positivity rate and titer of anti-drug antibody (ADA); further evaluation of neutralizing antibody (Nab); (4) Evaluation of the correlation between serum AQP4-IgG titer and disease prognosis or relapse.

[0207] The exploratory endpoints were changes in TBNK (including but not limited to CD3, CD4, CD56, CD16, etc.), B cell subsets (including but not limited to CD19, CD20, CD27, CD38, CD24, etc.), cytokines (CXCL-10, TNFα, IL-6, IFNg), peripheral CD47 RO, serum IgG, IgA, IgM, complement (C3, C4, C5), etc., before and after drug administration, and their relationship with efficacy. The time from week 52 to the first clinical relapse was also considered.

[0208] The primary endpoint for Phase III is the time to first clinical relapse at or within 52 weeks.

[0209] Secondary endpoints were: (1) efficacy, time to first clinical relapse within 3 years, changes in Extended Disability Status Scale (EDSS) scores; number of cumulative active lesions on magnetic resonance imaging (MRI); changes in visual function; annualized relapse rate; proportion of subjects hospitalized due to NMOSD relapse; changes in 25-foot walk test, nine-hole pedicle test (9HPT), visual evoked potential (VEP) P100 latency, and changes in RNFL thickness on optical coherence tomography (OCT); (2) safety: occurrence and frequency of adverse events (AEs) and severe acute exacerbations (SAEs) (according to NCICTCAE 5.0); (3) pharmacokinetics, first dose: peak concentration (C max Peak time (T) maxThe area under the blood drug concentration-time curve (AUC) from 0 to the last quantifiable time point 0-tlast If data permits, the area under the blood drug concentration-time curve (AUC) from time 0 to infinity will also be calculated. 0-inf ), elimination phase half-life (t) 1 / 2 ), volume of distribution (V), clearance rate (CL), etc.; multiple administrations: steady-state trough concentration (C) min,ss ), steady-state peak concentration (C) max,ss ), steady-state mean plasma concentration (C av,ss The area under the curve (AUC) of drug-time during the dosing interval 0-tau Peak time (T) max C max The accumulation ratio (R) ac _C max ), AUC 0-tau Accumulation ratio (Rac_AUC) 0-tau If data permits, the steady-state distributed volume (V) will also be calculated. ss Steady-state clearance rate (CL) ss and elimination phase half-life (t) 1 / 2 (4) Immunogenicity, positive rate and titer of anti-drug antibodies (ADA); neutralizing antibodies (Nab) may be further evaluated; (5) Evaluation of the correlation between serum AQP4-IgG titer and disease prognosis or relapse.

[0210] The exploratory endpoints were changes in TBNK (including but not limited to CD3, CD4, CD56, CD16, etc.), B cell subsets (including but not limited to CD19, CD20, CD27, CD38, CD24, etc.), cytokines (CXCL-10, TNFα, IL-6, IFNg), peripheral CD47 RO, serum IgG, IgA, IgM, complement (C3, C4, C5) before and after drug administration, and their relationship with efficacy.

[0211] Statistical analysis will be performed in SAS 9.4 or later. This study will be grouped according to different dosage groups. Descriptive statistical methods will be used to summarize patient characteristics, treatment outcomes, and the safety, efficacy, and pharmacokinetic properties of the drugs in each group. All summaries, tables, graphs, and analyses will be presented and described according to each dosage group. Quantitative data will be described using the number of cases, missing data, mean, standard deviation, maximum, minimum, and median; pharmacokinetic parameters will provide the coefficient of variation, geometric mean, and geometric standard deviation; categorical data will be described using frequency tables, percentages, or proportions.

[0212] MedDRA was used to code all adverse events (AEs) by system organ classification and preferred terminology. AEs occurring before treatment and those occurring during treatment were listed separately, with a focus on AEs occurring during treatment. Based on SS, the safety and tolerability of the drug were assessed through the incidence and severity of AEs and severe acute exacerbations (SAEs), physical examination, vital signs, laboratory tests, and 12-lead electrocardiogram.

[0213] The study primarily employed descriptive statistical analysis, listing adverse events and reactions occurring in each dosage group of this trial (where adverse reactions were defined as: adverse events that were definitely related to, very likely related to, or possibly related to the study drug). Laboratory test results were described as follows: those that were normal before the trial but abnormal after treatment, and the relationship between these abnormal changes and the study drug.

[0214] Calculate the mean, standard deviation, median, quartiles, minimum, and maximum values ​​of vital signs and laboratory indicators before and after medication for each dosage group. Perform descriptive analysis on the changes in vital signs and laboratory indicators among the dosage groups.

[0215] Pharmacokinetic analysis was performed using the WinNonlin 8.0 or later version non-compartmental model method to calculate pharmacokinetic parameters. Descriptive statistics were compiled and tabulated for plasma drug concentration data at each time point before and after drug administration, according to dosage groups. These statistics included the number of effective cases (N), arithmetic mean, standard deviation (SD), coefficient of variation (CV%), median, minimum (Min), maximum (Max), geometric mean, geometric standard deviation, and geometric coefficient of variation.

[0216] Based on the actual blood collection time, blood drug concentration-time curves and semi-logarithmic curves were plotted for each subject. Mean blood drug concentration-time curves and semi-logarithmic curves were provided for each dose group according to the planned blood collection time points. Blood drug concentration data were tabulated and descriptively summarized according to the sampling time points specified in the protocol, and the statistical summaries were presented according to dose groups.

[0217] Efficacy analysis was conducted based on the full analysis set and the protocol set. Efficacy assessments included time to first clinical relapse, proportion of subjects without relapse, low contrast visual acuity score (LCVA), VEP P100 latency, OCT RNFL thickness, 9-hour high-frequency test (9HPT), and changes in the 25-foot walk test compared to baseline.

[0218] Immunogenicity analysis included a quantitative description of the positivity rate and titer of anti-drug antibody (ADA).

[0219] Current clinical data show that the treatment method described in this application has essentially met the primary endpoint and / or secondary endpoints, and the adverse events (AEs) are within acceptable limits.

[0220] This application will be further described with reference to the following non-limiting embodiments.

[0221] Example

[0222] In the following embodiments, an exemplary recombinant fusion protein of this application is used, which comprises a CD20 antibody and a CD47-binding peptide linked to the N-terminus of the heavy chain variable region of the CD20 antibody. Specifically, the exemplary recombinant fusion protein comprises four peptide chains, wherein two chains comprise a CD47-binding peptide, a linker, a CD20 heavy chain variable region, and a heavy chain constant region from the N-terminus to the C-terminus, and the other two chains comprise a CD20 light chain variable region and a light chain constant region from the N-terminus to the C-terminus, wherein the CD47-binding peptide-linker-CD20 heavy chain variable region-heavy chain constant region chain comprises the amino acid sequence shown in SEQ ID NO:12, and the CD20 light chain variable region-light chain constant region chain comprises the amino acid sequence shown in SEQ ID NO:13.

[0223] Example 1. CD47 expression level in B cells of blood samples from SLE patients

[0224] Peripheral blood samples were collected from 14 SLE patients (provided by the Basic Research Project of the First Affiliated Hospital of Zhengzhou University) to detect the expression of CD47 in B cells.

[0225] Invert the whole blood sample to mix thoroughly. Add 1200 μL of erythrocyte lysis buffer (Sangon Biotech (Shanghai) Co., Ltd., Cat#B541001) that has been brought to room temperature beforehand to a 1.5 mL EP centrifuge tube, then add 200 μL of whole blood sample, making the whole blood:erythrocyte lysis buffer volume ratio 1:6. After pipetting and mixing, incubate at room temperature in the dark for about 4-8 minutes, until the sample becomes clear. Centrifuge at 3000 rpm for 3 minutes, discard the supernatant, and resuspend the cells in 200 μL of 0.5% BSA-PBS. Take 30 μL of resuspended solution, add 4 μL of FITC mouse anti-human CD45 (BD, Cat#555482), 1 μL of PE mouse anti-human CD47 (Biolegend, Cat#323108), 1 μL of PE-cy5 mouse anti-human CD20 (Biolegend, Cat#302308), 1 μL of APC / Cy7 anti-human CD38 (Biolegend, Cat#303534), 1 μL of APC mouse anti-human CD24 (Biolegend, Cat#311117), and 1 μL of APC mouse anti-human CD19 (Biolegend, Cat#363005), mix well, and incubate at 4℃ for 45 min. Among them, CD45 antibody is used to label lymphocytes, CD19 antibody is used to label B cells, and CD20 antibody, CD38 antibody, and CD24 antibody are used to label different B cell subsets, respectively. Afterwards, the cells were washed once with 200 μL of 0.5% BSA-PBS, and the expression of CD47 in various B cells was measured by flow cytometry. The differences between groups were compared using the t-test.

[0226] Figure 1 shows the cell subpopulations in the sample, including CD19. + CD24 高 CD38 低 Memory B cells, CD19 + CD38 高 CD24 高 Transitional B cells, CD19 + CD38 高 CD24 低 Plasmablasts and CD19 + CD38 中 CD24 中 The expression level of CD47 in mature naïve B cells. It can be seen that the total CD19... + CD47 expression levels in B cells and transitional B cells are higher than in non-B cells (i.e., CD45 expression levels). + (Lymphocytes), and the expression level of CD47 in other B cell subsets is slightly higher than that in non-B cells.

[0227] Example 2. Recombinant fusion protein on CD19 in blood samples from SLE patients+ B cell killing

[0228] Peripheral blood samples were taken from 14 SLE patients in Example 1 to test the efficacy of the recombinant fusion protein of this application on B cell depletion. Among these 14 SLE patients, according to the SLEDAI index, 6 had mild or inactive lupus nephropathy (LN) and the other 8 had moderate to severe LN.

[0229] Invert the whole blood sample to mix thoroughly, then dilute it 1:1 v / v with 1640 complete culture medium (Gibco, Cat#31800089) for later use.

[0230] The exemplary recombinant fusion protein (molecular weight 174 kDa), rituximab (Roche, molecular weight approximately 144.5 kDa), and a negative control antibody that does not target CD47 or CD20 (molecular weight approximately 144.5 kDa) were diluted to 0.4 μg / mL with 1640 complete medium.

[0231] Add 50 μL of the exemplary recombinant fusion protein, rituximab, and negative control antibody to each well of a 96-well plate, followed by 150 μL of whole blood diluent. Incubate at 37°C for 3 days. The final concentrations of the exemplary recombinant fusion protein, rituximab, and negative control antibody are all 0.1 μg / ml. After incubation, mix well and add 30 μL of sample from each well to a U-shaped plate containing 180 μL / well of erythrocyte lysis buffer (Sangon Biotech (Shanghai) Co., Ltd., Cat#B541001). Let stand for 8-10 minutes until the sample becomes red and transparent. Then wash twice with 0.5% BSA (Sangon Biotech, Cat#A500023-0100) and resuspend the cells in 40 μL of 0.5% BSA-PBS. Take 30 μL of the resuspended solution, add 0.5 μL of PE mouse anti-human CD45 (BD, Cat#555483) and 0.5 μL of APC mouse anti-human CD19 (Biolegend, Cat#302212), mix well, and incubate at 4°C for 45 min. CD45 antibody is used to label lymphocytes, and CD19 is used to label B cells. Afterwards, wash once with 200 μL of 0.5% BSA-PBS, and detect the number of B cells by flow cytometry.

[0232] The B-cell exhaustion rate was calculated using the following formula: B-cell exhaustion% = 100% - B-cell percentage in the treated sample / B-cell percentage in the blank control. The p-value was then calculated using a t-test to compare the differences between groups.

[0233] Figure 2 shows the B-cell depletion in the blood of SLE patients after treatment with 0.1 μg / ml of the exemplary recombinant fusion protein, rituximab, and a negative control antibody (A), including B-cell depletion in the blood of patients with mild / inactive SLE (B) and moderate-to-severe SLE (C), and a comparison of B-cell depletion between moderate-to-severe patients and mild / inactive patients (D). It can be seen that, compared to rituximab, the recombinant fusion protein of this application can kill B cells better, especially in blood samples obtained from patients with mild / inactive LN, where the specific B-cell killing effect of the recombinant fusion protein of this application is more significant (B). Furthermore, the B-cell killing effect of the recombinant fusion protein of this application is comparable in blood samples from patients with moderate-to-severe LN and patients with mild / inactive LN, with no significant difference (D).

[0234] Example 3. Comparison of pharmacodynamics and pharmacokinetics of recombinant fusion protein administered intravenously and subcutaneously.

[0235] The exemplary recombinant fusion protein of this application was administered to tumor-bearing mice via both intravenous and subcutaneous injection to compare the efficacy and pharmacokinetics of the two administration routes.

[0236] Specifically, human B-cell lymphoma Raji was cultured in RPMI-1640 medium supplemented with 10% FBS in an incubator at 37°C containing 5% CO2, and the cells were harvested before ten consecutive generations of culture.

[0237] 100 μL containing 5 × 10 6 Raji cells were mixed with PBS suspension and Matrigel (1:1 v / v) and subcutaneously injected into the right side of the mouse back near the axilla. Mice were anesthetized with 3-4% isoflurane prior to inoculation. The tumors grew to an average size of approximately 117.81 mm. 3 Around [time period missing], 48 tumor-bearing mice were randomly divided into 4 groups of 12 mice each, based on tumor volume and body weight. The day of grouping was defined as day 0, and drug administration began on that day. The administration regimen is shown in Table 1.

[0238] Table 1. Dosing Regimen

[0239] Observe the appearance and behavior of each mouse daily, including tumor nodule ulceration, animal mental state, and diet. Measure and record mouse weight and tumor volume twice a week. Tumor volume (TV) is calculated as (length × width). 2 ) / 2. When the tumor volume of the animal exceeds 3000 mm 3Euthanasia was necessary in some cases. Furthermore, during the experiment, each group of mice was divided into three subgroups, and blood samples were collected at the time points shown in Table 2 for pharmacokinetic analysis. Sampling time points included before each drug administration (0 min), 5 min, 0.5 h, 2 h, 6 h, 24 h, 48 h, and 72 h after each drug administration, and day 28. Before blood collection, mice were anesthetized with 3-4% isoflurane, and blood was collected through the orbital venous plexus, separating approximately ~50 μL of serum. The serum was stored below -80°C. After the experiment, mouse tumor tissue was harvested, weighed, and photographed.

[0240] Table 2. Blood Sample Collection Time Points

[0241] The blood drug concentration was determined according to the following steps. The self-prepared monoclonal antibody against the recombinant fusion protein was diluted to 1 μg / mL with CBS buffer and placed in a 96-well plate, 100 μL / well, and incubated overnight at 2–8°C. 200 μL / well of CBS buffer containing 3% skim milk was added to the 96-well plate, and the plate was blocked at room temperature for 2 hours. The recombinant fusion protein was then diluted to 5 μg / mL with CBS buffer containing 3% skim milk, and then serially diluted 3-fold to obtain the standard. Mouse serum obtained by centrifugation was diluted 500-fold with CBS buffer containing 3% skim milk. The recombinant fusion protein standard and serum dilution were added to 96-well plates, 100 μL / well, and incubated at 37°C for 1 hour. Afterwards, the plate was washed three times with 200 μL / well of washing buffer (PBST, PBS (pH 7.4, 0.01 M) + 0.05% Tween-20). Add 100 μL / well of peroxidase-conjugated AffiniPure F(ab')2 fragment goat anti-human IgG-Fc-γ fragment-specific (1:10000 dilution, Jackson, Cat#109-036-098) to a 96-well plate and incubate at 37°C for 1 hour. Wash 5 wells with 200 μL / well of PBST. Add 100 μL / well of TMB chromogenic buffer (KPL, Cat#51200050) to the 96-well plate and incubate in the dark for 15 minutes. Then, stop the reaction by adding 50 μL / well of 1M H2SO4 and read the absorbance at 450 nm using a microplate reader (Tecan, Sunrise 4).

[0242] Relative tumor volume (RTV): RTV = Vt / V0, where Vt is the tumor volume measured on day t and V0 is the tumor volume measured on day 0.

[0243] Relative tumor inhibition rate (TGI): TGI% = (1-T / C) × 100%, where T and C are the relative tumor volumes (RTV) of the treatment group and the solvent control group at a specific time point, respectively.

[0244] T / C% is the relative tumor proliferation rate, which is the percentage of relative tumor volume (RTV) between the treatment group and the negative control group at a certain time point. The calculation formula is as follows: T / C% = T RTV / C RTV ×100%, where T RTV C represents the mean RTV of the treatment group. RTV This represents the average RTV of the solvent control group.

[0245] Tumor weight inhibition rate (I RTW ):I RTW % = (average tumor weight in solvent control group – average tumor weight in treatment group) / average tumor weight in solvent control group × 100%.

[0246] The obtained data was first processed using IDBS E-WorkBook 10.5.0 (64-bit). In the IDBS version, the F-test is used for analysis of homogeneity of variance. If the variances are homogeneous (p>0.05), a one-way ANOVA is performed. Comparisons between two groups are analyzed using a t-test, and comparisons among multiple groups are analyzed using Dunnett's multiple comparisons. If the variances are unequal (p≤0.05), the original data are subjected to a Kruskal-Wallis nonparametric test, followed by one-sided Dunn's multiple comparisons (Kruskal-Wallis parametric test shows no significant difference, p>0.05) or pairwise comparisons are performed using the Mann-Whitney U test (Kruskal-Wallis parametric test shows a significant difference, p≤0.05). If p<0.05, the difference is considered statistically significant.

[0247] Figure 3 shows the changes in B-cell tumor volume (A) and mouse body weight (B) after treatment with the recombinant fusion protein in each group of mice. It can be seen that when the same dose is administered, intravenous injection and subcutaneous injection can achieve almost the same effect of inhibiting B-cell tumors, and there is no difference in body weight between the two groups of mice.

[0248] Figure 4 shows the blood drug concentrations in mice of each group. When the recombinant fusion protein was administered at the same dose, intravenous injection achieved higher blood drug concentrations 1-2 days after administration compared to subcutaneous injection. After 2 days of administration, the blood drug concentrations achieved by the two administration methods were comparable.

[0249] Example 4. The ability of recombinant fusion protein to clear B cells from peripheral blood of patients with B-cell lymphoma

[0250] This application relates to the treatment of relapsed or refractory CD20. + In a clinical trial of patients with B-cell non-Hodgkin lymphoma, the scavenging power of the recombinant fusion protein of this application against B cells in peripheral blood was measured.

[0251] Specifically, patients were intravenously injected with 0.04, 0.1, 0.25, 0.5, 0.8, 1.2, 1.6, and 2.0 mg / kg of the exemplary recombinant fusion protein of this application, referred to as the first administration (the administration time point is denoted as C0D1). Two weeks later, intravenous administration at the same dose was started once a week for four weeks as one course of treatment, with the administration time point denoted as CnDn (day n of the nth course of treatment). One hour before the first administration of each course, a peripheral blood sample was taken from the patient and CD45 was analyzed by FACS. + CD3 - CD19 + B cells were counted in absolute numbers.

[0252] Figure 5 shows the levels of CD19 in peripheral blood of patients receiving doses of 0.5 mg / kg or higher during treatment. + Changes in absolute B cell count (cells / μl). It can be seen that doses above 500 μg per kilogram of body weight can effectively maintain the clearance of B cells in peripheral blood. In one patient in the 1200 μg dose group, the B cell count remained at a low level without rebound for nearly 6 months from day 22 of the 12th cycle (C12D22) to the end-of-treatment visit (EOT), even in the absence of medication; a low B cell count was also observed in the subsequent safety follow-up (F / U). In another patient in the 1600 μg dose group, the B cell count remained at a low level without rebound after the end of treatment.

[0253] Example 5. Phase Ib / II clinical trial of recombinant fusion protein for the treatment of SLE

[0254] In a multicenter, dose-escalation phase Ib clinical trial, SLE patients, including those with SLE-LN, were recruited according to inclusion criteria. Patients were administered the medication intravenously or subcutaneously at doses ranging from 0.8 to 1.6 mg / kg, such as 0.8 mg / kg, 1.2 mg / kg, or 1.6 mg / kg. The medication was administered once weekly, once every two weeks, or once every three weeks, with four doses constituting one treatment cycle. After one cycle, medication was discontinued for 4–6 months before starting the next cycle.

[0255] The exemplary recombinant fusion protein used in clinical trials is a lyophilized powder, which is dissolved in water for injection before administration.

[0256] In some practical studies, doses of 0.8 mg / kg, 1.2 mg / kg, and 1.6 mg / kg were selected and administered once weekly via intravenous infusion from week 1 to week 4. Treatment was discontinued from week 5 to week 24. A follow-up study was conducted from week 25 to week 28, with weekly intravenous infusion, followed by a discontinuation until week 48. The follow-up period was from week 49 to week 52. Throughout the study, patients received the necessary standard therapy.

[0257] Blood samples were collected for pharmacokinetic analysis before administration on day 1 of week 1, immediately after administration on day 1 of week 1, and at 2, 4, 8, 24, 48, 72, and 120 hours after administration on day 1 of week 1; before administration on day 1 of week 2; before administration on day 1 of week 3; before administration on day 1 of week 4; immediately after administration on day 1 of week 4; and at 2, 4, 8, 24, 48, 72, 120, 168, and 336 hours after administration on day 1 of week 4. Blood samples were also collected at other time points for PD and biomarker studies. Immunogenicity analysis was performed before administration on day 1 of week 1, before administration on day 1 of week 3, and 336 hours after administration on week 4.

[0258] Based on the data obtained from Phase Ib, especially the safety and tolerability data, the recommended dose (RP2D), recommended dosing frequency, and interval between treatment courses were determined for Phase II.

[0259] As of October 2025, 8 patients had been enrolled in the 0.8 mg / kg dose group, 11 patients in the 1.2 mg / kg group, and 6 patients in the 1.6 mg / kg group. Table 3 shows the baseline levels of parameters for enrolled patients in the 0.8 mg / kg and 1.2 mg / kg dose groups.

[0260] Twenty-two patients were included in the pharmacokinetic analysis, including single-dose and multiple-dose administrations. Results showed that the mean elimination half-life of the recombinant fusion protein was approximately 29.0–40.0 h. Serum drug exposure levels increased with increasing dose after both single-dose and multiple-dose administrations, with no significant accumulation observed.

[0261] Table 3. Baseline parameters of enrolled patients

[0262] SLE patients tolerated the 0.8 mg / kg, 1.2 mg / kg, and 1.6 mg / kg doses well. As shown in Tables 4 and 5, treatment-related adverse events (TRAEs) were observed in a total of 12 patients: 4 in the 0.8 mg / kg dose group and 8 in the 1.2 mg / kg dose group. No treatment-related serious adverse events (SAEs) or deaths caused by TRAEs occurred. One patient in each group experienced a grade 3 or higher TRAE. In the 0.8 mg / kg dose group, a patient experienced thrombocytopenia (grade 4) and headache (grade 3) after the fourth dose; the thrombocytopenia resolved spontaneously within 4 days, and the headache was relieved by a single dose of acetaminophen within 2 days. In the 1.2 mg / kg dose group, a patient experienced thrombocytopenia (grade 3) after the fourth dose, which resolved spontaneously within 5 days. One patient in the 0.8 mg / kg group and two patients in the 1.2 mg / kg group experienced infusion reactions, leading to infusion interruptions, which were resolved and completed on the same day. One patient in the 1.2 mg / kg dose group experienced an allergic reaction during the sixth dose (week 26), recovered after an injection of pantoprazole sodium on day 2, but withdrew from the clinical trial after further investigation.

[0263] Furthermore, in the 0.8 mg / kg dose group, the IL-6 levels measured in the 7 patients remained at a low level, except for a small peak of less than 100.0 pg / ml 5 minutes after the first injection in week 1, which returned to a low level within 4 hours. No cytokine release syndrome (CRS) was observed. In the 0.8 mg / kg and 1.2 mg / kg dose groups, 16 of the 18 patients had IgG levels within the normal range throughout the study, while one patient in each group had slightly elevated IgG levels.

[0264] Table 4. Summary of TRAE

[0265] Table 5. TRAE Details

[0266] Seven patients in the 0.8 mg / kg dose group and eleven patients in the 1.2 mg / kg dose group who received at least four doses and at least one efficacy assessment were evaluated to determine the therapeutic effect of the recombinant fusion protein of this application on SLE. Table 6 lists the symptom improvement in patients in the 0.8 mg / kg and 1.2 mg / kg dose groups. Only one patient in the 0.8 mg / kg group did not show improvement in the SLEDAI-2000 score. 28.6% (2 / 7) and 63.6% (7 / 11) of patients in the 0.8 mg / kg and 1.2 mg / kg dose groups, respectively, achieved the Systemic Lupus Reactivity Index (SRI-4) at week 12, and 71.4% (5 / 7) and 80.0% (4 / 5) of patients in the 0.8 mg / kg and 1.2 mg / kg dose groups, respectively, achieved SRI-4 at week 24, demonstrating the effectiveness of the recombinant fusion protein treatment of this application.

[0267] Specifically, in the 0.8 mg / kg dose group, a decrease in glucocorticoid (GC) dosage was observed in 57.1% (4 / 7) of patients, a decrease in SLEDAI-2000 score ≥4 was observed in 57.1% (4 / 7) of patients, and no worsening of PGA score was observed in any of the patients (100%, 7 / 7). In the 1.2 mg / kg dose group, a decrease in glucocorticoid (GC) dosage was observed in 27.3% (3 / 11) of patients, a decrease in SLEDAI-2000 score ≥4 was observed in 72.7% (8 / 11) of patients, and no worsening of PGA score was observed in any of the patients (100%, 11 / 11).

[0268] Figure 6 shows the percentage of patients achieving a SLEDAI-2000 score reduction of ≥4 at different time points, and Figure 7 shows the time to disappearance of SLEDAI-2000 score symptoms (including arthritis, rash, hair loss, and vasculitis) for each patient. It can be seen that as treatment progresses, more and more patients achieve a SLEDAI-2000 score reduction of ≥4, and symptoms other than the rash gradually disappear.

[0269] Figure 8 shows the changes in 24-hour proteinuria, dsDNA antibodies, and complement components C3 and C4 levels at baseline (BL) and after the most recent drug administration (LV) for each patient. It can be seen that 24-hour proteinuria and dsDNA antibodies generally showed a decreasing trend, while complement component levels generally showed an increasing trend, all indicating that the condition of SLE patients is continuously improving.

[0270] In addition, 23 patients in the 0.8 mg / kg, 1.2 mg / kg, and 1.6 mg / kg groups underwent flow cytometry analysis to assess the absolute count and percentage of B cells and their subsets. The longest follow-up times for each dose group were 44–48 weeks, 12–32 weeks, and 4 weeks, respectively. Results showed that all patients achieved CD19 positivity after the first administration of the recombinant fusion protein. + Complete exhaustion of B cells (CD19) + B cell counts were <5 cells / μL, and other B cell subsets (including memory B cells and plasmablasts) were also depleted after the first dose. During follow-up, B cell remodeling was observed in 9 patients between weeks 12 and 24. The remodeled B cells were predominantly naive B cells, while memory cells remained at a low level, and plasmablast levels remained extremely low. Patients who showed B cell remodeling all demonstrated a significant decrease in SLEDAI-2000 efficacy and all achieved an SRI-4 response at week 24.

[0271] Based on the above data, the SRC conference researchers and sponsors have jointly decided that the dosage will be increased to 2.0 mg / kg.

[0272] In the Phase II clinical trial, SLE patients, including SLE-LN patients, were recruited according to the inclusion criteria and administered the drug to patients via intravenous or subcutaneous injection, using the RP2D, recommended dosing frequency, and interval between treatment courses.

[0273] Blood samples were collected before administration on day 1 of week 1, immediately after administration on day 1 of week 1, 4 hours after administration on day 1 of week 1, before administration on day 1 of week 2, before administration on day 1 of week 3, before administration on day 1 of week 4, immediately after administration on day 1 of week 4, 4 hours after administration on day 1 of week 4, and 336 hours after administration on day 1 of week 4; before administration on day 1 of week 25, immediately after administration on day 1 of week 25, 4 hours after administration on day 1 of week 25, before administration on day 1 of week 26, before administration on day 1 of week 27, before administration on day 1 of week 28, immediately after administration on day 1 of week 28, 4 hours after administration on day 1 of week 28, and 336 hours after administration on day 1 of week 28 for pharmacokinetic analysis. Blood samples were also collected at other time points for PD and biomarker studies. Immunogenicity analysis was performed before administration on day 1 of week 1, before administration on day 1 of week 3, 336 hours after administration on week 4, before administration on day 1 of week 25, and 336 hours after administration on week 28.

[0274] Data from the Phase II clinical trial showed that, compared with placebo administration, the treatment method described in this application resulted in a significantly higher percentage of subjects achieving the Systemic Lupus Responsive Index (SRI-4) at week 52 after treatment, with reduced urinary protein levels, reduced AHA levels, and treatment-related adverse events within acceptable limits.

[0275] Example 6. Phase Ib / III clinical trial of recombinant fusion protein for the treatment of NMOSD

[0276] In the phase Ib clinical trial, NMOSD patients were recruited according to the inclusion criteria and administered the medication intravenously or subcutaneously at doses ranging from 0.8 to 2.0 mg / kg, including 0.8 mg / kg, 1.2 mg / kg, 1.6 mg / kg, and 2.0 mg / kg. The medication was administered once weekly, once every two weeks, or once every three weeks, with four doses constituting one treatment cycle. After one cycle, the medication was discontinued for 4–6 months before starting the next cycle. Subjects in the positive control group received mycophenolate mofetil (MMF) orally, 1.0 g twice daily. After completing the DLT observation period in the low-dose group, a decision was made regarding whether to escalate to the next dose group. Use of ≤20 mg prednisone / day or its equivalent dose of glucocorticoids was permitted within 14 days prior to the first dose. A fixed dose of 20 mg prednisone / day or its equivalent dose of glucocorticoids was administered for the first week after enrollment, gradually tapering off from day 1 of week 2, with a tapering-off period not exceeding 4 weeks.

[0277] The exemplary recombinant fusion protein used in clinical trials is a lyophilized powder, which is dissolved in water for injection before administration.

[0278] In some practical applications, doses of 0.8 mg / kg, 1.2 mg / kg, 1.6 mg / kg, and 2.0 mg / kg were administered intravenously once weekly from week 1 to week 4. Subjects in the positive control group received mycophenolate mofetil (MMF) orally, 1.0 g twice daily.

[0279] Blood samples were collected for pharmacokinetic analysis before administration on day 1 of week 1, immediately after administration on day 1 of week 1, and at 2, 4, 8, 24, 48, 72, and 120 hours after administration on day 1 of week 1; before administration on day 1 of week 2; before administration on day 1 of week 3; before administration on day 1 of week 4; immediately after administration on day 1 of week 4; and at 2, 4, 8, 24, 48, 72, 120, 168, and 336 hours after administration on day 1 of week 4. Blood samples were also collected at other time points for PD and biomarker studies. Immunogenicity analysis was performed before administration on day 1 of week 1, before administration on day 1 of week 3, and 336 hours after administration on week 4.

[0280] Based on the data obtained from Phase Ib, especially the safety and tolerability data, and combined with the data from the Phase Ib clinical trial of SLE, the recommended dose, recommended dosing frequency, and interval between treatment courses for Phase III are derived.

[0281] Seven patients were included in the pharmacokinetic analysis, including single-dose and multiple-dose administrations. Results showed that the mean elimination half-life of the recombinant fusion protein was approximately 28.7–39.0 h. Serum drug exposure levels increased with increasing dose after both single and multiple doses, without significant accumulation. NMOSD patients tolerated doses of 0.8 mg / kg, 1.2 mg / kg, and 1.6 mg / kg well. As of November 10, 2025, in the recombinant fusion protein administration group, all 10 patients were included in the safety analysis set. Seven patients (70.0%) experienced treatment-related adverse events (TEAEs), all of which were treatment-related (TRAEs). The main TEAEs (incidence ≥10%) included: decreased platelet count (3 / 10, 30.0%), nausea (2 / 10, 20.0%), rash (1 / 10, 10.0%), urticaria (1 / 10, 10.0%), abdominal distension (1 / 10, 10.0%), chest discomfort (1 / 10, 10.0%), arthralgia (1 / 10, 10.0%), infusion-related reactions (1 / 10, 10.0%), urinary tract infection (1 / 10, 10.0%), anemia (1 / 10, 10.0%), hypertensive emergency (1 / 10, 10.0%), elevated gamma-glutamyl transferase (1 / 10, 10.0%), elevated alanine aminotransferase (1 / 10, 10.0%), insomnia (1 / 10, 10.0%), and asthma (1 / 10, 10.0%). Two treatment-associated adverse events (TEAEs) of grade ≥3 (CTCAE) occurred in the same patient in the 1.6 mg dose group. These events were related to the study drug and included elevated gamma-glutamyl transferase (1 / 10, 10.0%) and decreased platelet count (1 / 10, 10.0%). Both outcomes were remission without sequelae. No serious adverse events (SAEs) occurred during treatment. One patient (10.0%) experienced a TEAE leading to drug discontinuation; no TEAEs resulted in drug discontinuation, withdrawal from the trial, or death. No cytokine release syndrome (CRS) occurred during treatment. One patient (10%) experienced an infusion-related reaction (IRR) during treatment, both grade 1-2, occurring in the same patient. Specifically, a grade 1 IIR occurred during week 3 administration, and a grade 2 IIR occurred during week 4 administration; both outcomes were remission.

[0282] In the 0.8 mg / kg dose group, one patient experienced the first clinical relapse on day 160 (W23D6) after administration. No patients in the 1.2 mg / kg group (N=3), 1.6 mg / kg group (N=4), and control group (N=3) experienced the first clinical relapse within 24 weeks.

[0283] In addition, the absolute counts and percentages of B cells and their subsets were assessed by flow cytometry. The longest follow-up visits were 24 weeks, 24 weeks, and 17–24 weeks in the 0.8 mg / kg, 1.2 mg / kg, and 1.6 mg / kg groups, respectively. Results showed that all patients achieved CD19 positivity after the first administration of the recombinant fusion protein. + Complete exhaustion of B cells (CD19) + B cell count <5 cells / μL). In the 0.8 mg / kg dose group, B cell remodeling was observed in two patients between weeks 9 and 17. The remodeled B cells were mainly naive B cells, with some regression in plasmablast levels, but the overall level remained low. Peripheral B cell data for other dose groups are still pending.

[0284] Based on the above data, the dosage will be increased to 2.0 mg / kg.

[0285] In the Phase III clinical trial, NMOSD patients were recruited according to the inclusion criteria and administered the drug intravenously or subcutaneously, using the Phase III recommended dose, recommended dosing frequency, and interval between treatment courses. Subjects in the positive control group received mycophenolate mofetil (MMF) orally, 1.0 g twice daily.

[0286] Blood samples were collected before administration on day 1 of week 1, immediately after administration on day 1 of week 1, 4 hours after administration on day 1 of week 1, before administration on day 1 of week 2, before administration on day 1 of week 3, before administration on day 1 of week 4, immediately after administration on day 1 of week 4, and 4 hours after administration on day 1 of week 4, before administration on day 1 of week 25, immediately after administration on day 1 of week 25, and 4 hours after administration on day 1 of week 25, before administration on day 1 of week 26, before administration on day 1 of week 27, before administration on day 1 of week 28, immediately after administration on day 1 of week 28, and 4 hours after administration on day 1 of week 28 for pharmacokinetic analysis. Blood samples were also collected at other time points for PD and biomarker studies. Immunogenicity analysis (ADA) was performed before administration on day 1 of week 1, before administration on day 1 of week 3, 336 hours after administration on week 4, before administration on day 1 of week 25, and 336 hours after administration on week 28.

[0287] Data from the Phase III clinical trial showed that, compared with the positive control group, the treatment method of this application significantly delayed the time to first clinical relapse within 52 weeks after medication, increased the proportion of subjects without relapse, reduced patients' AQP4-IgG titers, and the treatment-related adverse events were within an acceptable range.

[0288] Example 7. Phase II / III clinical trial of recombinant fusion protein for the treatment of IgG4-related disease (IgG4-RD)

[0289] Based on the safety demonstrated by the recombinant fusion protein in Examples 5 and 6, a multicenter, randomized, double-blind, controlled phase II / III clinical study for the treatment of IgG4-RD was conducted.

[0290] In the Phase II clinical trial, IgG4-RD patients were recruited according to the inclusion criteria and administered the drug intravenously at doses ranging from 0.8 to 2.0 mg / kg, including 0.8 mg / kg, 1.2 mg / kg, 1.6 mg / kg, and 2.0 mg / kg. The drugs were administered once weekly, once every two weeks, or once every three weeks, with four doses constituting one treatment cycle. After one cycle, the drug was discontinued for 4-6 months before starting the next cycle.

[0291] The exemplary recombinant fusion protein used in clinical trials is a lyophilized powder, which is dissolved in water for injection before administration.

[0292] In some practical studies, doses of 1.6 mg / kg and 2.0 mg / kg were selected and administered once weekly via intravenous infusion from week 1 to week 4. The drug was discontinued from week 5 to week 26. A follow-up study was conducted from week 27 to week 30, during which the drug was administered once weekly via intravenous infusion. The drug was then discontinued until week 52.

[0293] Patient inclusion criteria were:

[0294] - Adult males or females aged 18 and over;

[0295] - Clinically diagnosed as IgG4-related disease;

[0296] - Based on the eligibility committee's determination, the candidate must meet the 2019 ACR / EULAR classification criteria. Specifically, the candidate must meet the inclusion criteria of the classification criteria (including involvement of one of the following organs: pancreas, bile duct / bile tree, orbit, lung, kidney, lacrimal gland, major salivary gland, retroperitoneum, aorta, dura mater, or thyroid [Riedel's thyroiditis]), and must not meet any exclusion criteria of the classification criteria, and the inclusion score of the classification criteria must be at least 20 points;

[0297] - IgG4-related disease involving at least two organs / sites at any time during the course of the disease, supported by written evidence. One organ must meet the ACR / EULAR classification criteria; the second organ may be defined by the investigator.

[0298] - At the time of informed consent, the individual is experiencing (or has recently experienced) a relapse of IgG4-related disease requiring the initiation or continuation of glucocorticoid (GC) therapy. This criterion can be met in two ways: - Currently receiving GC therapy for a recent relapse of IgG4-related disease, having received up to 4 weeks of treatment prior to informed consent at a dose not exceeding 60 mg / day of prednisone or an equivalent, and having received 20 mg / day of prednisone or an equivalent before randomization on the day of first dose; or - At the time of informed consent, the individual is experiencing active disease but is not receiving treatment, plans to initiate GC therapy at a dose not exceeding 60 mg / day of prednisone (or an equivalent) to treat the relapse, and plans to receive 20 mg / day of prednisone (or an equivalent) before randomization on the day of first dose, with a total duration of GC therapy received during the screening period of at least 3 weeks on the day of first dose. This GC therapy can be a newly initiated treatment or a dose escalation from a previous maintenance dose of ≤10 mg / day of prednisone or an equivalent. Subjects whose GC dose cannot be reduced to 20 mg / day prednisone or an equivalent drug by Visit 2 of Phase III cannot be randomized. The total duration of GC treatment prior to randomization must be ≥3 weeks and ≤8 weeks;

[0299] - IgG4-related diseases: Individuals with IgG4-related diseases voluntarily sign informed consent forms, are willing and able to comply with the research protocol, complete research evaluations, and complete the research period.

[0300] Subjects who meet any of the following criteria should not be enrolled in this study:

[0301] - A history of retroperitoneal fibrosis, sclerosing mesenteric inflammation, fibrotic mediastinitis, or other fibrotic manifestations of IgG4-RD, where fibrosis is the only clinical manifestation of this recurrence;

[0302] - Blood tests performed during screening meet any of the following criteria: - Hemoglobin < 7.5 g / dL; - Neutrophils < 1200 / mm³ 3 ;-Platelets<110×10 9 / L; - Eosinophil count >3000 / mm 3 - Prothrombin time > 1.2 × upper limit of normal (ULN), however, this does not exclude subjects receiving anticoagulation therapy for atrial fibrillation with aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤ 2 × ULN; - Total immunoglobulin < 6 g / dL; - CD19 at screening time + B cells <40 cells / μL; this exclusion value is reproducible;

[0303] - Subjects with the following abnormal liver function test results in the absence of hepatobiliary IgG4-RD activity: - Aspartate aminotransferase (AST) > 2 × ULN; - Alanine aminotransferase (ALT) > 2 × ULN; - Total bilirubin (TBil) > 2 × ULN, unless AST, ALT, and hemoglobin are within the normal range in a central laboratory and the patient has a known history of Gilbert's syndrome. Or subjects with the following abnormal liver function test results in the presence of hepatobiliary IgG4-RD activity: - AST > 10 × ULN; - ALT > 10 × ULN; - TBil > 5 × ULN. Screening liver function tests may be repeated before randomization to allow for responses to GC treatment to abnormal values ​​due to hepatobiliary IgG4-RD activity.

[0304] - Received B-cell clearance drugs (such as rituximab, inelizumab, etc.) within 6 months prior to the screening period;

[0305] - Use of alkylating agents such as mitoxantrone or cyclophosphamide within 3 months prior to the screening period; use of non-biological DMARDs or immunosuppressants (such as azathioprine, mycophenolate mofetil, methotrexate, etc.) within 4 weeks prior to the screening period.

[0306] - The patient has other chronic active immune system diseases requiring long-term treatment with glucocorticoids, biologics, or immunosuppressants (such as systemic lupus erythematosus, rheumatoid arthritis, scleroderma, etc.). Based on patient records, the patient is known to be positive for anti-neutrophil cytoplasmic antibodies (ANCA) targeting protein 3 or myeloperoxidase. The patient has received a live or attenuated live vaccine within 4 weeks prior to screening; an inactivated vaccine within 2 weeks prior to baseline; or a COVID-19 vaccine within 4 weeks prior to baseline.

[0307] - Individuals who have previously received bone marrow transplantation, hematopoietic stem cell transplantation, organ or cell transplantation, whole lymph node irradiation, or T-cell vaccine therapy; or individuals known to have primary or secondary immunodeficiency diseases;

[0308] - Have received any clinical trial medication within 28 days prior to the first dose or within 5 times the half-life of another investigational drug (whichever is shorter); or are currently participating in other clinical trials requiring drug intervention or device-type clinical trials;

[0309] - Individuals with an active malignant tumor or a history of active malignant tumors within the past 10 years. The following are exceptions: cervical carcinoma in situ cured more than 12 months prior to screening; cured basal cell carcinoma or squamous cell carcinoma of the skin; those who underwent radical prostatectomy or radiotherapy more than 3 years prior to screening without recurrence; and those with cured superficial bladder cancer, breast carcinoma in situ, or thyroid cancer without active disease.

[0310] - Exclusion criteria include: - Left ventricular ejection fraction (LVEF) ≤ 55%; - New York Heart Association (NYHA) class II or higher congestive heart failure or active heart disease; - Serious arrhythmias requiring treatment (excluding atrial fibrillation or paroxysmal supraventricular tachycardia deemed by the investigator to have no impact on the trial); - History of myocardial infarction, bypass surgery, or stent placement within 6 months prior to administration; - Other cardiac conditions deemed unsuitable for enrollment by the investigator.

[0311] - Patients who have an active bacterial, viral, fungal, mycobacterial, or parasitic infection (excluding nail bed fungal infection) before the first dose and require anti-infection or hospitalization;

[0312] - The specific inclusion and exclusion criteria for patients with evidence of TB infection are as follows: - Patients with active TB will be excluded. Active TB is defined by imaging and laboratory tests. Patients with a history of previously diagnosed TB or latent TB, but with evidence that they have completed treatment according to local guidelines, can be enrolled; - Patients without active TB, but with a positive interferon-gamma release test at screening, can be enrolled if they have completed prophylactic treatment according to local guidelines.

[0313] - Active lung disease (such as interstitial pneumonia, pulmonary fibrosis);

[0314] - Individuals with a history of ≥1 episode of herpes zoster or other opportunistic infections within the 12 months prior to screening;

[0315] - Individuals who are positive for hepatitis B surface antigen (HBsAg) or hepatitis B core antibody (HBcAb) and have HBV DNA above the upper limit of normal; individuals who are positive for hepatitis C virus (HCV) antibody and have HCV RNA above the upper limit of normal (those who have been cured for ≥24 weeks after antiviral treatment can be included); individuals who are positive for HIV antibody; individuals who are positive for Treponema pallidum antibody (TP-Ab) and have a positive non-Treponema pallidum test.

[0316] - Severe hepatic and renal insufficiency, with an estimated glomerular filtration rate (eGFR) <30 mL / min / 1.73 m³ calculated according to the National Institute for Dietary Modification of Kidney Disease (NIDDK) formula. 2 ;

[0317] - Individuals with severe neurological, psychiatric, endocrine, hematologic, gastrointestinal, respiratory, or systemic diseases who, according to researchers, may increase risk or interfere with efficacy and safety assessments;

[0318] - Individuals who are allergic to humanized monoclonal antibodies, gamma globulin, or any component of the investigational drug, or who are intolerant to medications that prevent infusion reactions (such as antipyretics, antihistamines, or glucocorticoids);

[0319] -Those who are allergic to contrast agents or unable to undergo MRI examinations;

[0320] - Individuals with a history of alcoholism or drug abuse, or those whom researchers believe may affect adherence or safety;

[0321] -Pregnant or breastfeeding women, or those planning to become pregnant within 6 months of the last dose; unsterilized women of childbearing age who do not agree to use effective contraception (oral contraceptives, intrauterine devices, or barrier methods combined with spermicides, etc.) from the time of screening until 6 months after the last dose; unsterilized men who do not agree to use barrier methods of contraception from the time of screening until 6 months after the last dose and who request their partners to use effective contraception.

[0322] - The researchers believe that there are any other medical or psychosocial conditions that would make a person unsuitable to participate in this study.

[0323] For all patients, starting from day 1, the daily dose of oral prednisone (or equivalent) should be gradually reduced until it is completely discontinued after 8 weeks.

[0324] The infusion time is 180 min ± 20 min. Pretreatment can be performed 0.5-1.0 hours before infusion to prevent infusion reactions. For example, corticosteroids such as dexamethasone 5 mg to 10 mg can be injected intramuscularly or intravenously; antihistamines such as diphenhydramine 50 mg or promethazine 25 mg to 50 mg can be injected intramuscularly or intravenously; and antipyretic analgesics such as acetaminophen 0.5 g to 0.65 g or indomethacin 50 mg can be administered orally or rectally.

[0325] Based on the data obtained from Phase II, especially the safety and tolerability data, the recommended dose (RP2D), recommended dosing frequency, and interval between treatment courses for Phase III were determined.

[0326] In the Phase III clinical trial, IgG4-RD patients were recruited according to the inclusion criteria and administered the drug intravenously or subcutaneously, using the Phase III recommended dose, recommended dosing frequency, and interval between treatment courses.

[0327] After completing the treatment prescribed in the 52-week Phase III clinical trial, all eligible subjects entered a 3-year open-label extension period (OLP) and received treatment with the recombinant fusion protein of this application (including patients who received placebo in the randomized controlled period (RCP)). The treatment was administered once a week for a total of 4 times, i.e., W1 / W2 / W3 / W4D1, and then repeated every 6 months thereafter.

[0328] The results showed that the treatment method described in this application, compared to placebo, was able to reduce IgG4 levels in patients. + The plasma cell count, symptom relief or disease progression, and treatment-related adverse events are within acceptable limits.

[0329] The sequences used in this application are as follows.

[0330] VH-CDR1

[0331] VH-CDR2

[0332] VH-CDR3

[0333] VL-CDR1

[0334] VL-CDR2

[0335] VL-CDR3

[0336] Heavy chain variable region of CD20 antibody

[0337] Light chain variable region of CD20 antibody

[0338] Heavy chain constant region

[0339] Light chain constant region

[0340] CD47-binding peptide

[0341] connector

[0342] Long chain of recombinant fusion protein

[0343] Short chain of recombinant fusion protein

Claims

1. A recombinant fusion protein for the preparation of treatments that benefit CD20 + Use in drugs for autoimmune diseases involving B-cell depletion or elimination. The recombinant fusion protein comprises i) a CD47 binding peptide and ii) a CD20 antibody or its antigen-binding moiety. The CD47-binding peptide contains the first extracellular Ig-like domain (SIRPαD1) of signal regulatory protein α (SIRPα), which contains Ala(A) at position 80 of SEQ ID NO:

10. The CD20 antibody or its antigen-binding region comprises a heavy chain variable region, a heavy chain constant region, a light chain variable region, and a light chain constant region. The heavy chain variable region includes VH-CDR1, VH-CDR2, and VH-CDR3, and the light chain variable region includes VL-CDR1, VL-CDR2, and VL-CDR3. VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 respectively contain the amino acid sequences shown in GYTFTSYN (SEQ ID NO:1), IYPGNGDT (SEQ ID NO:2), ARSTYYGGDWYFNV (SEQ ID NO:3), SVSSY (SEQ ID NO:4), ATS, and QQWTSNPPT (SEQ ID NO:5). The heavy chain constant region contains binding affinity for Fc receptors or complement system proteins. The CD47 binding peptide is linked to the N-terminus of the heavy chain variable region or the light chain variable region of the CD20 antibody or its antigen-binding moiety.

2. The use as claimed in claim 1, wherein the heavy chain variable region and the light chain variable region respectively comprise amino acid sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 6 and 7.

3. The use as claimed in claim 1, wherein the heavy chain constant region contains a mutation capable of inducing enhanced antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC).

4. The use as described in claim 3, wherein the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:

8.

5. The use as claimed in claim 1, wherein the CD47-binding peptide comprises the amino acid sequence shown in SEQ ID NO:

10.

6. The use as claimed in claim 1, wherein the CD47 binding peptide is connected via a linker to the N-terminus of the heavy chain variable region or the light chain variable region of the CD20 antibody or its antigen-binding moiety.

7. The use as described in claim 6, wherein the recombinant fusion protein comprises a CD47-binding peptide-linker-heavy chain variable region-heavy chain constant region chain and a light chain variable region-light chain constant region chain, wherein the CD47-binding peptide-linker-heavy chain variable region-heavy chain constant region chain and the light chain variable region-light chain constant region chain respectively comprise the amino acid sequences shown in SEQ ID NO: 12 and 13.

8. The use as described in claim 1, wherein CD20 can benefit from + Autoimmune diseases with reduced or eliminated B cells include systemic lupus erythematosus (SLE), neuromyelitis optica spectrum disorder (NMOSD), IgG4-related disease (IgG4-RD), membranous nephropathy, Sjögren's syndrome, polymyositis, dermatomyositis, multiple sclerosis, scleroderma, and vasculitis.

9. A treatment that can benefit CD20 in subjects in need. + Methods for treating autoimmune diseases with reduced or eliminated B cells include administering a therapeutically effective amount of recombinant fusion protein to the subject. The recombinant fusion protein comprises i) a CD47-binding peptide and ii) a CD20 antibody or its antigen-binding moiety. The CD47-binding peptide contains the first extracellular Ig-like domain (SIRPαD1) of signal regulatory protein α (SIRPα), which contains Ala(A) at position 80 of SEQ ID NO:

10. The CD20 antibody or its antigen-binding region comprises a heavy chain variable region, a heavy chain constant region, a light chain variable region, and a light chain constant region. The heavy chain variable region includes VH-CDR1, VH-CDR2, and VH-CDR3, and the light chain variable region includes VL-CDR1, VL-CDR2, and VL-CDR3. VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 respectively contain the amino acid sequences shown in GYTFTSYN (SEQ ID NO:1), IYPGNGDT (SEQ ID NO:2), ARSTYYGGDWYFNV (SEQ ID NO:3), SVSSY (SEQ ID NO:4), ATS, and QQWTSNPPT (SEQ ID NO:5). The heavy chain constant region contains binding affinity for Fc receptors or complement system proteins. The CD47 binding peptide is linked to the N-terminus of the heavy chain variable region or the light chain variable region of the CD20 antibody or its antigen-binding moiety.

10. The method of claim 9, wherein the heavy chain variable region and the light chain variable region each comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 6 and 7.

11. The method of claim 9, wherein the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:

8.

12. The method of claim 9, wherein the CD47-binding peptide comprises the amino acid sequence shown in SEQ ID NO:

10.

13. The method of claim 9, wherein the CD47 binding peptide is linked via a linker to the N-terminus of the heavy chain variable region or the light chain variable region of the CD20 antibody or its antigen-binding moiety.

14. The method of claim 9, wherein the recombinant fusion protein comprises a CD47-binding peptide-linker-heavy chain variable region-heavy chain constant region chain and a light chain variable region-light chain constant region chain, wherein the CD47-binding peptide-linker-heavy chain variable region-heavy chain constant region chain and the light chain variable region-light chain constant region chain each comprise the amino acid sequences shown in SEQ ID NO: 12 and 13, respectively.

15. The method of claim 9, comprising administering a therapeutically effective amount of the recombinant fusion protein to the subject intravenously or subcutaneously.

16. The method of claim 9, comprising administering 0.8-2.0 mg / kg of the recombinant fusion protein to the subject.

17. The method of claim 9, comprising administering the recombinant fusion protein to the subject at a frequency of once a week, once every two weeks, or once every three weeks, for a course of treatment consisting of four treatments, with an interval of four to six months between courses of treatment.

18. The method of claim 9, wherein CD20 can benefit from it. + Autoimmune diseases with reduced or eliminated B cells include systemic lupus erythematosus (SLE), neuromyelitis optica spectrum disorder (NMOSD), IgG4-related disease (IgG4-RD), membranous nephropathy, Sjögren's syndrome, polymyositis, dermatomyositis, multiple sclerosis, scleroderma, and vasculitis.

19. A composition configured in a single-dose form, wherein the single-dose form comprises 40 mg to 200 mg of a recombinant fusion protein. The recombinant fusion protein comprises i) a CD47 binding peptide and ii) a CD20 antibody or its antigen-binding moiety. The CD47-binding peptide contains the first extracellular Ig-like domain (SIRPαD1) of signal regulatory protein α (SIRPα), which contains Ala(A) at position 80 of SEQ ID NO:

10. The CD20 antibody or its antigen-binding region comprises a heavy chain variable region, a heavy chain constant region, a light chain variable region, and a light chain constant region. The heavy chain variable region includes VH-CDR1, VH-CDR2, and VH-CDR3, and the light chain variable region includes VL-CDR1, VL-CDR2, and VL-CDR3. VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 respectively contain the amino acid sequences shown in GYTFTSYN (SEQ ID NO:1), IYPGNGDT (SEQ ID NO:2), ARSTYYGGDWYFNV (SEQ ID NO:3), SVSSY (SEQ ID NO:4), ATS, and QQWTSNPPT (SEQ ID NO:5). The heavy chain constant region contains binding affinity for Fc receptors or complement system proteins. The CD47 binding peptide is linked to the N-terminus of the heavy chain variable region or the light chain variable region of the CD20 antibody or its antigen-binding moiety.

20. The composition of claim 19, wherein the heavy chain variable region and the light chain variable region each comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 6 and 7. The heavy chain constant region contains the amino acid sequence shown in SEQ ID NO:8, or The CD47-binding peptide contains the amino acid sequence shown in SEQ ID NO:

10.

21. The composition of claim 20, wherein the recombinant fusion protein comprises a CD47-binding peptide-linker-heavy chain variable region-heavy chain constant region chain and a light chain variable region-light chain constant region chain, wherein the CD47-binding peptide-linker-heavy chain variable region-heavy chain constant region chain and the light chain variable region-light chain constant region chain each comprise the amino acid sequences shown in SEQ ID NO: 12 and 13, respectively.

22. The composition of claim 19, wherein the single-dose form comprises 80 mg-160 mg of the recombinant fusion protein of the present application, or 100 mg-200 mg of the recombinant fusion protein.

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