Application of interferon receptor 1 monoclonal antibody in preparation of medicine for treating neuromyelitis optica pedigree diseases and medicine composition

Abstract

The invention relates to the field of biological medicine, and relates to application of an interferon receptor 1 monoclonal antibody in preparation of a medicine for treating neuromyelitis optica pedigree diseases and a medicine composition. The antibody regulates immune response by inhibiting abnormal activation of an I-type interferon signal channel and down-regulating interferon stimulation gene expression, so that central nervous system inflammation and demyelination injury are relieved. The humanized IFNAR1 monoclonal antibody is independently used; the compound is combined with a B cell targeting drug to synergistically inhibit B cell activation and antibody secretion; and a bispecific antibody targeting IFNAR1 and a transferrin receptor is constructed, so that the ability of the drug to pass through the blood brain barrier is enhanced, and the concentration of the drug in the brain is improved. In-vivo and in-vitro experiments prove that the treatment scheme can effectively reduce the size of the focus of the NMOSD model and improve the neurological function, and a new precise treatment strategy is provided for AQP4 antibody positive patients.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of interferon receptor 1 monoclonal antibody in the preparation of drugs for treating neuromyelitis optica spectrum disorders (NMOSD) and its pharmaceutical composition. Background Technology

[0002] Neuromyelitis Optica Spectrum Disorders (NMOSD) are a group of autoimmune diseases characterized primarily by inflammation and demyelinating damage of the central nervous system. Clinically, they often manifest as optic neuritis, long-segment transverse myelitis, and other symptoms. Studies have shown that most NMOSD patients have autoantibodies against aquaporin-4 (AQP4), which play an important role in the occurrence and development of the disease.

[0003] Currently, clinical treatment for NMOSD primarily relies on broad-spectrum immunosuppression or immunomodulatory strategies, such as glucocorticoids, immunosuppressants, and biologics targeting B cells or cytokines. While these treatments can alleviate disease activity to some extent, they generally suffer from insufficient target specificity, limited efficacy in some patients, and high safety risks with long-term use. Therefore, developing more precise treatment strategies targeting the immunopathological mechanisms of NMOSD remains an important research direction in this field.

[0004] In recent years, the role of the type I interferon signaling pathway in autoimmune diseases has attracted attention. Interferon receptor 1 (IFNAR1) is a key component of the type I interferon signaling pathway, and its mediated signal transduction can induce changes in the expression of various interferon-stimulated genes, thereby regulating the activation, migration, and effector functions of immune cells.

[0005] Zhang et al. reported the role of the type I interferon signaling pathway in neuromyelitis optica spectrum disorders (NMOSD) in Advanced Science (2025). They revealed that enhanced IFNAR1-related signaling can promote autoimmune responses associated with NMOSD, and through animal models and mechanistic studies, demonstrated that blocking this signaling pathway helps alleviate inflammatory damage to the central nervous system. This paper elucidates the pathogenic role of IFNAR1 signaling in NMOSD from a disease pathogenesis perspective, providing important theoretical basis for understanding the immunopathology of NMOSD.

[0006] However, the aforementioned studies primarily focus on elucidating the biological role and mechanisms of the type I interferon signaling pathway in NMOSD, and have not yet addressed the application of specific humanized IFNAR1 monoclonal antibodies in the preparation of drugs for treating neuromyelitis optica spectrum disorders, nor have they provided technical solutions for direct clinical translation, such as related drug compositions, dosing combinations, or drug kits. Therefore, it remains necessary to further develop technical solutions with clearly defined drug forms and application methods based on existing research to meet the practical needs of neuromyelitis optica spectrum disorder treatment. Summary of the Invention

[0007] The purpose of this invention is to provide a new drug use and related product form, which can be used to alleviate the central nervous system inflammatory response associated with neuromyelitis optica spectrum disorders by regulating immune signaling pathways associated with these disorders.

[0008] To achieve the above objectives, the present invention proposes the following technical solution:

[0009] This invention provides the use of a humanized interferon receptor 1 (IFNAR1) monoclonal antibody in the preparation of a medicament for treating neuromyelitis optica spectrum disorders. The humanized IFNAR1 monoclonal antibody regulates the immune response associated with neuromyelitis optica spectrum disorders by inhibiting the activation of the type I interferon signaling pathway and reducing the abnormal expression of interferon-stimulated genes.

[0010] In some embodiments, the humanized IFNAR1 monoclonal antibody may be selected from anifrolumab or an antibody fragment thereof having IFNAR1 antigen-binding activity. In other embodiments, the drug is indicated for patients with aquaporin 4 antibody-positive neuromyelitis optica spectrum disorder.

[0011] Furthermore, the technical solution of the present invention also relates to the application of the humanized IFNAR1 monoclonal antibody in regulating immune cell function, including inhibiting the migration of pathogenic T cells to the central nervous system and inhibiting the activation and antigen presentation function of B cells. The pathogenic T cells may include Th17 cells and / or Th1 cells, which may express chemokine receptors CCR5, CCR6 and / or CXCR3 on their surface.

[0012] Furthermore, the present invention also provides pharmaceutical compositions, dosing combinations, and pharmaceutical kits comprising the humanized IFNAR1 monoclonal antibody, wherein the product form is configured for the treatment of neuromyelitis optica spectrum disorders, thereby improving the applicability and flexibility of the technical solutions of the present invention.

[0013] This invention provides the use of a humanized interferon receptor 1 (IFNAR1) monoclonal antibody in the preparation of a medicament for treating neuromyelitis optica spectrum disorders.

[0014] The drug is used to regulate the abnormal activation of type I interferon signaling associated with neuromyelitis optica spectrum disorders.

[0015] In some embodiments, the abnormal activation state of type I interferon signaling is manifested as an increased expression level of at least one interferon-stimulated gene.

[0016] The interferon-stimulated gene is selected from at least one of ISG15, ISG20, IFIT1, IFIT2, IFIT3, IFI44, IFI44L, IFITM2, IFITM3, MX1, and OAS1.

[0017] In some embodiments, the abnormal activation state of type I interferon signaling is further manifested as an increased proportion of chemokine receptor-positive T cells.

[0018] The chemokine receptor is selected from at least one of CCR5, CCR6 and CXCR3.

[0019] In some embodiments, the neuromyelitis optica spectrum disorder is an aquaporin 4 antibody-positive neuromyelitis optica spectrum disorder.

[0020] In some embodiments, the drug is used to selectively inhibit the migration of pathogenic T cells associated with neuromyelitis optica spectrum disorders to the central nervous system.

[0021] In some embodiments, the pathogenic T cells include Th17 cells and / or Th1 cells.

[0022] Furthermore, the migration inhibition is associated with decreased expression of CCR5, CCR6, and / or CXCR3 in the T cells.

[0023] In some embodiments, the humanized IFNAR1 monoclonal antibody is anifrolumab or an antibody fragment thereof having IFNAR1 antigen-binding activity.

[0024] Another aspect of the present invention provides a pharmaceutical composition comprising a humanized interferon receptor 1 (IFNAR1) monoclonal antibody and a pharmaceutically acceptable carrier or excipient.

[0025] The pharmaceutical composition is configured to regulate the abnormal activation state of type I interferon signaling associated with neuromyelitis optica spectrum disorders.

[0026] Another aspect of the present invention provides a dosage combination comprising the aforementioned pharmaceutical composition and at least one immunomodulatory drug for neuromyelitis optica spectrum disorders.

[0027] Another aspect of the present invention provides a drug kit comprising:

[0028] (a) A first container containing a humanized interferon receptor 1 (IFNAR1) monoclonal antibody; and

[0029] (b) Instruction manual,

[0030] The instruction manual indicates that the humanized IFNAR1 monoclonal antibody is used to regulate the abnormal activation state of type I interferon signaling associated with neuromyelitis optica spectrum disorders.

[0031] Another aspect of the present invention provides a pharmaceutical composition for preparing a treatment of neuromyelitis optica spectrum disorders, the pharmaceutical composition comprising:

[0032] (1) Interferon receptor 1 (IFNAR1) monoclonal antibody; and

[0033] (2) Immunomodulatory drugs targeting B cells.

[0034] In some embodiments, the IFNAR1 monoclonal antibody is anifrolumab or a biosimilar thereof; the B-cell-targeting immunomodulatory drug is selected from one or more of the following: anti-CD20 monoclonal antibody, anti-CD19 monoclonal antibody, anti-CD22 monoclonal antibody or antigen-binding fragment thereof.

[0035] In some embodiments, the anti-CD20 monoclonal antibody is rituximab or a biosimilar thereof. Alternatively, the anti-CD20 monoclonal antibody is rituximab, offatumumab, or ofcrelizumab, or one of these.

[0036] In some embodiments, the interferon receptor 1 monoclonal antibody and the B cell-targeting immunomodulatory drug have a synergistic effect in inhibiting B cell activation, inducing B cell apoptosis, and / or reducing the secretion of pathogenic antibodies.

[0037] In some embodiments, the neuromyelitis optica spectrum disorder is an aquaporin 4 antibody-positive neuromyelitis optica spectrum disorder.

[0038] In some embodiments, the pharmaceutical composition is an injection, a lyophilized formulation, or a pharmaceutically acceptable combination thereof, wherein the weight ratio of the IFNAR1 monoclonal antibody to the anti-CD20 monoclonal antibody is 10:1 to 1:10.

[0039] The present invention further provides a targeted drug for preparing a treatment of neuromyelitis optica spectrum disorders, the targeted drug comprising an antibody or antigen-binding fragment thereof capable of specifically binding to interferon receptor 1 and having central nervous system targeted delivery capability.

[0040] In some embodiments, the central nervous system targeted delivery capability is achieved by binding to transferrin receptors on the surface of brain endothelial cells.

[0041] In some embodiments, the targeted drug is a bispecific antibody, the bispecific antibody comprising:

[0042] (a) The antibody domain that binds to interferon receptor 1; and

[0043] (b) Antibody domain that binds to transferrin receptor.

[0044] Alternatively, the targeted drug is a bispecific antibody whose first binding domain specifically binds to interferon-α / β receptor 1 (IFNAR1) and its second binding domain specifically binds to transferrin receptor (TfR). The first binding domain contains the antigen-binding fragment of anifrolumab.

[0045] In some embodiments, the bispecific antibody employs an IgG-like asymmetric structure, with the transferrin receptor-binding domain linked to the Fc region or the C-terminus of the heavy chain via a flexible linker peptide. Alternatively, the bispecific antibody may be in the form of an asymmetric IgG-like structure, wherein the second binding domain is in the form of a single-chain antibody fragment (scFv) linked to the Fc region or the C-terminus of the heavy chain of the first binding domain antibody via a linker peptide.

[0046] In some implementations, the second binding domain is capable of cross-binding human and mouse TfR. The bispecific antibody has an equilibrium dissociation constant (KD) of less than 2 nM for IFNAR1 and a KD of less than 20 nM for TfR.

[0047] In some embodiments, in an in vitro blood-brain barrier (BBB) ​​model, the apparent permeability (Papp) of the bispecific antibody is more than five times that of the parental monoclonal antibody containing only the first binding domain. Its exposure (AUC) in brain tissue, measured in molar doses, is more than five times that of the parental monoclonal antibody containing only the first binding domain.

[0048] In some embodiments, the targeted drug is exposed at higher levels in brain tissue than interferon receptor 1 monoclonal antibodies that do not have the ability to target and deliver to the central nervous system.

[0049] In some embodiments, the neuromyelitis optica spectrum disorder is an aquaporin 4 antibody-mediated neuromyelitis optica spectrum disorder.

[0050] Compared with the prior art, the present invention has at least the following beneficial effects:

[0051] This invention targets interferon receptor 1 (IFNAR1) to regulate the type I interferon signaling pathway, intervening in the inflammatory response associated with neuromyelitis optica spectrum disorders (NMOSD) at the immune signaling level. Compared to broad-spectrum immunosuppressive strategies, this invention has a more specific target. The technical solution of this invention not only involves the regulation of humoral immunity-related B cell function but also the influence on the migration behavior of pathogenic T cells, thereby synergistically regulating NMOSD-related abnormal immune responses at multiple immune cell levels.

[0052] The use of the humanized IFNAR1 monoclonal antibody in drug preparation, along with the corresponding pharmaceutical compositions, dosing combinations, and drug kits, provides this technical solution with good feasibility and promising industrial application prospects. This technical solution is applicable to patients with aquaporin 4 antibody-positive neuromyelitis optica spectrum disorders, offering a new treatment option for this specific patient population. By functionally regulating the type I interferon signaling pathway, it provides a novel approach to the treatment of neuromyelitis optica spectrum disorders, different from existing technologies, and has potential clinical application value.

[0053] The pharmaceutical composition provided by this invention, comprising an interferon receptor 1 (IFNAR1) monoclonal antibody and an anti-CD20 monoclonal antibody, exhibits a significantly higher actual apoptosis rate in inducing apoptosis in B cells derived from NMOSD patients compared to the theoretical superposition value calculated based on the Bliss independence model. In the NMO-IgG mouse model, the combination therapy reduced the volume of lesions in the central nervous system, demonstrating significantly superior efficacy compared to monotherapy with either IFNAR1 or anti-CD20 monoclonal antibody.

[0054] This composition targets pathogenic B cells through a dual mechanism: the IFNAR1 monoclonal antibody blocks the survival and activation signals of type I interferon, promoting B cell apoptosis and inhibiting their differentiation into plasma cells; the anti-CD20 monoclonal antibody directly mediates B cell clearance. The combined use of these two agents achieves a deep, end-to-end intervention on the entire B cell cycle of activation, differentiation, survival, and clearance.

[0055] This invention provides a bispecific antibody targeting interferon receptor 1 (IFNAR1) and transferrin receptor (TfR), which can be used as a targeted drug for the treatment of neuromyelitis optica spectrum disorder (NMOSD). The bispecific antibody innovatively utilizes TfR-mediated transcytosis to achieve efficient crossing of the blood-brain barrier (BBB), successfully overcoming the core technical bottleneck of the difficulty for large molecule drugs to enter the brain. Attached Figure Description

[0056] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0057] Appendix Figure 1 This is a diagram showing the overall process and experimental results of the in vitro experiment in Embodiment 2 of the present invention;

[0058] Appendix Figure 2 Cross-sectional MRI images of the mouse brain and statistical diagram of lesion volume;

[0059] Appendix Figure 3 A comparison chart showing the ratio of IBA1+ microglia to macrophages around the lesions in the MAR1-5A3 treatment group;

[0060] Appendix Figure 4 The curves showing the changes in clinical scores of neurological function over time in the Th17 transport model;

[0061] Appendix Figure 5 The results of H&E staining on a cross-section of the spinal cord;

[0062] Appendix Figure 6 A diagram showing the immune cell gating strategy and measurement results;

[0063] Appendix Figure 7 This is a graph showing the results of flow cytometry analysis;

[0064] Appendix Figure 8 This is a graph showing the expression intensity of B cell activation markers under different stimuli. Detailed Implementation

[0065] Example 1:

[0066] This invention provides the use of a humanized interferon receptor 1 (IFNAR1) monoclonal antibody in the preparation of a medicament for treating neuromyelitis optica spectrum disorders. The humanized IFNAR1 monoclonal antibody regulates the immune response associated with neuromyelitis optica spectrum disorders by inhibiting the activation of the type I interferon signaling pathway and reducing the abnormal expression of interferon-stimulated genes.

[0067] In some embodiments, the humanized IFNAR1 monoclonal antibody may be selected from anifrolumab or an antibody fragment thereof having IFNAR1 antigen-binding activity. In other embodiments, the drug is indicated for patients with aquaporin 4 antibody-positive neuromyelitis optica spectrum disorder.

[0068] Furthermore, the technical solution of the present invention also relates to the application of the humanized IFNAR1 monoclonal antibody in regulating immune cell function, including inhibiting the migration of pathogenic T cells to the central nervous system and inhibiting the activation and antigen presentation function of B cells. The pathogenic T cells may include Th17 cells and / or Th1 cells, which may express chemokine receptors CCR5, CCR6 and / or CXCR3 on their surface.

[0069] Furthermore, the present invention also provides pharmaceutical compositions, dosing combinations, and pharmaceutical kits comprising the humanized IFNAR1 monoclonal antibody, wherein the product form is configured for the treatment of neuromyelitis optica spectrum disorders, thereby improving the applicability and flexibility of the technical solutions of the present invention.

[0070] This invention provides the use of a humanized interferon receptor 1 (IFNAR1) monoclonal antibody in the preparation of a medicament for treating neuromyelitis optica spectrum disorders.

[0071] The drug is used to regulate the abnormal activation of type I interferon signaling associated with neuromyelitis optica spectrum disorders.

[0072] In some embodiments, the abnormal activation state of type I interferon signaling is manifested as an increased expression level of at least one interferon-stimulated gene.

[0073] The interferon-stimulated gene is selected from at least one of ISG15, ISG20, IFIT1, IFIT2, IFIT3, IFI44, IFI44L, IFITM2, IFITM3, MX1, and OAS1.

[0074] In some embodiments, the abnormal activation state of type I interferon signaling is further manifested as an increased proportion of chemokine receptor-positive T cells.

[0075] The chemokine receptor is selected from at least one of CCR5, CCR6 and CXCR3.

[0076] In some embodiments, the neuromyelitis optica spectrum disorder is an aquaporin 4 antibody-positive neuromyelitis optica spectrum disorder.

[0077] In some embodiments, the drug is used to selectively inhibit the migration of pathogenic T cells associated with neuromyelitis optica spectrum disorders to the central nervous system.

[0078] In some embodiments, the pathogenic T cells include Th17 cells and / or Th1 cells.

[0079] Furthermore, the migration inhibition is associated with decreased expression of CCR5, CCR6, and / or CXCR3 in the T cells.

[0080] In some embodiments, the humanized IFNAR1 monoclonal antibody is anifrolumab or an antibody fragment thereof having IFNAR1 antigen-binding activity.

[0081] Another aspect of the present invention provides a pharmaceutical composition comprising a humanized interferon receptor 1 (IFNAR1) monoclonal antibody and a pharmaceutically acceptable carrier or excipient.

[0082] The pharmaceutical composition is configured to regulate the abnormal activation state of type I interferon signaling associated with neuromyelitis optica spectrum disorders.

[0083] Another aspect of the present invention provides a dosage combination comprising the aforementioned pharmaceutical composition and at least one immunomodulatory drug for neuromyelitis optica spectrum disorders.

[0084] Another aspect of the present invention provides a drug kit comprising:

[0085] (a) A first container containing a humanized interferon receptor 1 (IFNAR1) monoclonal antibody; and

[0086] (b) Instruction manual,

[0087] The instruction manual indicates that the humanized IFNAR1 monoclonal antibody is used to regulate the abnormal activation state of type I interferon signaling associated with neuromyelitis optica spectrum disorders.

[0088] Another aspect of the present invention provides a pharmaceutical composition for preparing a treatment of neuromyelitis optica spectrum disorders, the pharmaceutical composition comprising:

[0089] (1) Interferon receptor 1 (IFNAR1) monoclonal antibody; and

[0090] (2) Immunomodulatory drugs targeting B cells.

[0091] In some embodiments, the IFNAR1 monoclonal antibody is anifrolumab or a biosimilar thereof; the B-cell-targeting immunomodulatory drug is selected from one or more of the following: anti-CD20 monoclonal antibody, anti-CD19 monoclonal antibody, anti-CD22 monoclonal antibody or antigen-binding fragment thereof.

[0092] In some embodiments, the anti-CD20 monoclonal antibody is rituximab or a biosimilar thereof. Alternatively, the anti-CD20 monoclonal antibody is rituximab, offatumumab, or ofcrelizumab, or one of these.

[0093] In some embodiments, the interferon receptor 1 monoclonal antibody and the B cell-targeting immunomodulatory drug have a synergistic effect in inhibiting B cell activation, inducing B cell apoptosis, and / or reducing the secretion of pathogenic antibodies.

[0094] In some embodiments, the neuromyelitis optica spectrum disorder is an aquaporin 4 antibody-positive neuromyelitis optica spectrum disorder.

[0095] In some embodiments, the pharmaceutical composition is an injection, a lyophilized formulation, or a pharmaceutically acceptable combination thereof, wherein the weight ratio of the IFNAR1 monoclonal antibody to the anti-CD20 monoclonal antibody is 10:1 to 1:10.

[0096] The present invention further provides a targeted drug for preparing a treatment of neuromyelitis optica spectrum disorders, the targeted drug comprising an antibody or antigen-binding fragment thereof capable of specifically binding to interferon receptor 1 and having central nervous system targeted delivery capability.

[0097] In some embodiments, the central nervous system targeted delivery capability is achieved by binding to transferrin receptors on the surface of brain endothelial cells.

[0098] In some embodiments, the targeted drug is a bispecific antibody, the bispecific antibody comprising:

[0099] (a) The antibody domain that binds to interferon receptor 1; and

[0100] (b) Antibody domain that binds to transferrin receptor.

[0101] Alternatively, the targeted drug is a bispecific antibody whose first binding domain specifically binds to interferon-α / β receptor 1 (IFNAR1) and its second binding domain specifically binds to transferrin receptor (TfR). The first binding domain contains the antigen-binding fragment of anifrolumab.

[0102] In some embodiments, the bispecific antibody employs an IgG-like asymmetric structure, with the transferrin receptor-binding domain linked to the Fc region or the C-terminus of the heavy chain via a flexible linker peptide. Alternatively, the bispecific antibody may be in the form of an asymmetric IgG-like structure, wherein the second binding domain is in the form of a single-chain antibody fragment (scFv) linked to the Fc region or the C-terminus of the heavy chain of the first binding domain antibody via a linker peptide.

[0103] In some implementations, the second binding domain is capable of cross-binding human and mouse TfR. The bispecific antibody has an equilibrium dissociation constant (KD) of less than 2 nM for IFNAR1 and a KD of less than 20 nM for TfR.

[0104] In some embodiments, in an in vitro blood-brain barrier (BBB) ​​model, the apparent permeability (Papp) of the bispecific antibody is more than five times that of the parental monoclonal antibody containing only the first binding domain. Its exposure (AUC) in brain tissue, measured in molar doses, is more than five times that of the parental monoclonal antibody containing only the first binding domain.

[0105] In some embodiments, the targeted drug is exposed at higher levels in brain tissue than interferon receptor 1 monoclonal antibodies that do not have the ability to target and deliver to the central nervous system.

[0106] In some embodiments, the neuromyelitis optica spectrum disorder is an aquaporin 4 antibody-mediated neuromyelitis optica spectrum disorder.

[0107] Example 2:

[0108] The type I interferon (IFN-I) pathway plays a crucial role in autoimmunity. IFN-β treatment in NMOSD patients exacerbates the condition, and serum IFN-α levels are positively correlated with disease activity. Recent studies have shown aberrant activation of the IFN-I signaling pathway in NMOSD patients, which may participate in the pathogenesis of NMOSD through multiple mechanisms, including activating plasmablasts, promoting inflammatory cell infiltration, and synergistic effects with the complement system. Therefore, targeting the key node of this pathway (IFNAR1) has become a promising new therapeutic strategy. Anifrolumab, a fully human monoclonal antibody, specifically antagonizes the type I interferon receptor (IFNAR1), blocking all IFN-I biological activities, and is currently approved for the treatment of systemic lupus erythematosus. However, to date, there is no research or evidence to suggest that anifrolumab can be used to treat NMOSD. This invention, based on a new understanding of the crucial role of the IFN-I pathway in NMOSD, proposes and validates for the first time a novel application of IFNAR1 antagonists in the treatment of NMOSD.

[0109] The purpose of this invention is to provide new medical uses for IFNAR1 monoclonal antibodies (including its murine form MAR1-5A3 and its humanized form Anifrolumab), specifically, said antibodies can be used to prepare medicaments for the prevention and / or treatment of neuromyelitis optica spectrum disorders (NMOSD).

[0110] This invention demonstrates through in vitro and in vivo experiments that the antibody can significantly alleviate pathological damage to the central nervous system and improve neurological deficits in NMOSD model animals. Its mechanism of action is related to inhibiting the type I interferon signaling pathway, thereby regulating the differentiation and function of pathogenic T cells and B cells.

[0111] Combined with appendix Figure 1 To be continued Figure 8 To fully verify the technical effects of this invention, we conducted validation experiments using in vitro cell experiments, mouse in vivo disease models, and human patient cells. In the mouse experiments, the IFNAR1 antagonist used was the murine monoclonal antibody MAR1-5A3, which is the subsequent humanized antibody anifrolumab. Both have the same target binding specificity and functional activity. MAR1-5A3 is the prodrug of anifrolumab, which is a humanized version of MAR1-5A3. MAR1-5A3 is a murine monoclonal antibody targeting IFNAR1, possessing the same target specificity and functional blocking activity as the humanized antibody anifrolumab, and is commonly used for preclinical functional validation of anifrolumab.

[0112] 1. Experimental Methods

[0113] 1.1 Effects of in vitro IFN-I stimulation on AQP4-specific T cell differentiation and the intervention effect of IFNAR1 inhibitors: Experimental methods: Cell preparation: Lymph nodes of Aqp4- / - mice immunized with AQP4 peptide (p201-220) for 10 days were collected to prepare lymphocyte suspension. Experimental grouping and culture: Lymphocytes (3×10^5 / well) were seeded in 96-well plates coated with anti-CD3 / CD28 antibodies (5 μg / mL each) and cultured in three groups: Control group: cultured only under anti-CD3 / CD28 antibody stimulation. Th17+ IFN-I stimulation group: in addition to anti-CD3 / CD28 antibody stimulation, AQP4 peptide (10 μg / mL), IL-23 (20 ng / mL), IL-6 (20 ng / mL), IFN-α1 (100 U / mL) and IFN-β (100 U / mL) were added. Anti-IFNAR monoclonal antibody treatment group: Based on the exact same culture conditions as the "Th17 + IFN-I stimulation group," anti-IFNAR monoclonal antibody (10 μg / mL) was added. Detection indicators: After 72 hours of culture, the proportion of IFN-γ+ cells and IL-17A+ cells in CD4+ T cells, as well as the expression levels of key chemokine receptors CCR5, CCR6, and CXCR3 on CD4+ and CD8+ T cells were detected by flow cytometry.

[0114] 1.2. NMO-IgG Intraventricular Injection Mouse Model: Antibody Purification: AQP4-IgG was purified from the plasma of AQP4-IgG-positive NMOSD patients. Stereotactic Injection: The purified AQP4-IgG was mixed with normal human complement at a ratio of 6:4. After anesthetizing mice with isoflurane, they were fixed in a stereotaxic apparatus. A hole was drilled at the anterior fontanelle (anterior to posterior +0.5 mm, right side of the sagittal suture +2.5 mm), and the needle was inserted vertically to a depth of 3 mm. 10 μL of the mixture was injected at a rate of 0.8 μL / min. The needle was left in place for 10 minutes after injection and then slowly withdrawn in stages. Grouping and Administration: Treatment Group: Anti-IFNAR (250 μg per mouse, once daily) was injected intraperitoneally immediately after modeling. Model Control Group: Isotype control antibody was injected. Evaluation Indicators: Brain Magnetic Resonance Imaging (MRI) on day 7 after modeling: T2-weighted image (T2WI) lesion volume was analyzed.

[0115] Histopathology: Immunofluorescence staining was performed after brain perfusion (to detect GFAP, C3, IBA1, etc.).

[0116] 1.3. Th17 Cell Passive Transfer Mouse Model: Sensitization and Cell Induction: Aqp4- / - mice were immunized with AQP4 peptide (P201-220) / CFA emulsion and injected with PT. Lymphocytes were obtained from lymph nodes and spleen after 10-12 days. Th17 Polarization: AQP4-specific Th17 cell polarization was induced by culturing in a medium containing AQP4 peptide, IL-23, and IL-6 for 72 hours. Adoptive Transfer: 15-20 × 10^6 lymphocytes were intraperitoneally injected into wild-type recipient mice, followed by PT injection. Grouping and Administration: Treatment Group: Anti-IFNAR was injected intraperitoneally after cell transfer (same protocol as above). Model Control Group: Isotype control antibody was injected. Assessment Indicators: Clinical Score: Neurological function scores (0-5 points) were recorded daily. Histopathology: Spinal cord was harvested for H&E and LFB staining and scoring. Flow cytometry was used to isolate tissues from the central nervous system and analyze the proportion of infiltrating immune cells.

[0117] 1.4. Immunomodulatory Effect of Anti-IFNAR on Peripheral Blood PBMCs of NMOSD Patients: Cell Source: PBMCs were isolated from NMOSD patients diagnosed with AQP4 antibody positivity. Experimental Groups: Control Group: PBMCs were cultured in the presence of a stimulator (anti-CD3 / CD28). Anifrolumab Treatment Group: Anifrolumab (10 μg / mL) was added under the same conditions. Detection Indicators: After 24 hours of culture, flow cytometry was used to detect the expression of IFN-γ and CCR5 in CD4+ and CD8+ T cells. Simultaneously, we used magnetic bead sorting to obtain the patients' initial B cells for in vitro culture. After 24 hours of culture, the expression levels of B cell activation markers CD86 and HLA-DR were detected by flow cytometry.

[0118] 2. Experimental Results

[0119] 2.1. In vitro mouse experiments: Effects of IFN-I stimulation on AQP4-specific T cell differentiation and the intervention effect of IFNAR1 inhibitors.

[0120] We prepared lymphocyte suspensions using lymph nodes from Aqp4- / - mice immunized 10 days after immunization with AQP4 peptide (p201-220). Experimental grouping and culture: Lymphocytes (3 × 10^5 / well) were seeded in 96-well plates coated with anti-CD3 / CD28 antibodies (5 μg / mL each) and cultured in three groups: Control group: cultured only under anti-CD3 / CD28 antibody stimulation. Th17 + IFN-I stimulation group: in addition to anti-CD3 / CD28 antibody stimulation, AQP4 peptide (10 μg / mL), IL-23 (20 ng / mL), IL-6 (20 ng / mL), IFN-α1 (100 U / mL), and IFN-β (100 U / mL) were added. Anti-IFNAR monoclonal antibody treatment group: Based on the exact same culture conditions as the "Th17 + IFN-I stimulation group," anti-IFNAR monoclonal antibody (10 μg / mL) was added. Detection indicators: After 72 hours of culture, the proportion of IFN-γ+ cells and IL-17A+ cells in CD4+ T cells, as well as the expression levels of key chemokine receptors CCR5, CCR6, and CXCR3 on CD4+ and CD8+ T cells were detected by flow cytometry.

[0121] Appendix Figure 1 Figure A shows the overall procedure of the in vitro experiment. Figure B illustrates the typical gating strategy for flow cytometry analysis of CCR5, CCR6, and CXCR3. Figure C shows that compared with the control group, the proportion of CD4+ and CD8+ T cells expressing CCR5, CCR6, and CXCR3 was significantly increased in the Th17+IFN-I stimulation group. The anti-IFNAR monoclonal antibody treatment group significantly reversed this effect, restoring the proportion of chemokine receptor-positive cells to near the control group level. Figure D shows the flow cytometry peaks of IFN-γ and IL-17A expression in CD4+ T cells. Compared with the control group (green curve), the positive cell population in the Th17+IFN-I stimulation group (red curve) significantly increased and shifted to the right; the peak shape in the anti-IFNAR monoclonal antibody treatment group (dark blue curve) shifted to the left, indicating a weakened signal. Figure E shows the statistical results of the proportion of IFN-γ+ and IL-17A+ cells in CD4+ and CD8+ T cells. Data showed that the proportion of pathogenic cytokine-positive cells in the Th17+IFN-I stimulation group was significantly higher than that in the control group, while the anti-IFNAR monoclonal antibody treatment group significantly reduced the expression levels of these cytokines.

[0122] 2. NMO-IgG model:

[0123] 2.2.1 Magnetic resonance imaging (MRI) results showed that IFNAR1 monoclonal antibody significantly alleviated demyelinating lesions in the central nervous system of NMOSD model mice.

[0124] To evaluate the therapeutic effect of anti-IFNAR1 monoclonal antibody on structural damage to the central nervous system in NMO-IgG model mice, we performed brain magnetic resonance imaging (MRI) scans on day 7 post-modeling to analyze lesion volume on T2-weighted images (T2WI). High-signal areas on T2WI typically correspond to pathological changes such as vasogenic edema, inflammatory cell infiltration, and demyelination, and are key indicators for quantifying the degree of central nervous system damage. Results are attached. Figure 2 As shown in Figure A, a representative transverse MRI image of the mouse brain is presented. The left image (control group) shows significant patchy T2 hyperintense lesions within the mouse cerebral hemispheres (the area outlined in red in the image), indicating successful induction of NMO-IgG-mediated inflammatory demyelinating lesions in the central nervous system. The right image (anti-IFNAR treatment group) shows that after IFNAR1 monoclonal antibody intervention, the lesion area in the mouse brain was significantly reduced, and the abnormal hyperintense area was significantly decreased. Figure B is a bar chart showing the quantitative statistical analysis of lesion volume. The data shows that compared with the control group, the average lesion volume in the brain of mice in the MAR1-5A3 treatment group was reduced by approximately 40%, and this difference was statistically significant (p < 0.05).

[0125] 2.2.2 Histopathology: Immunofluorescence staining results showed that IFNAR1 monoclonal antibody effectively inhibited NMO-IgG antibody-mediated central nervous system neuroinflammation.

[0126] To evaluate the effect of IFNAR1 monoclonal antibody (MAR1-5A3) on neuroinflammation in the central nervous system of NMO-IgG model mice, we collected brain tissue on day 7 after modeling for frozen sectioning and immunofluorescence staining, and performed quantitative analysis on key immune cells around the lesions.

[0127] 1) Inhibitory effect on activated astrocytes:

[0128] By double-labeling glial fibrillary acidic protein (GFAP) and complement C3, we identified and quantified activated astrocytes (GFAP+ / C3+). (See attached image) Figure 3 As shown in A and B, compared with the control group, the proportion of GFAP+ / C3+ activated astrocytes around the lesions was significantly reduced in the MAR1-5A3 treatment group. This result indicates that IFNAR1 monoclonal antibody treatment can effectively alleviate astrocyte damage and activation mediated by AQP4-IgG and complement.

[0129] 2) Inhibitory effect on microglia / macrophage infiltration:

[0130] By staining for ion-calcium-binding adaptor molecule 1 (IBA1), we labeled and quantified microglia / macrophages in the lesion area. (See attached image.) Figure 3 As shown in C and D, compared with the control group, the proportion of IBA1+ microglia / macrophages around the lesions was also significantly reduced in the MAR1-5A3 treatment group. This result indicates that IFNAR1 monoclonal antibody treatment can effectively inhibit the activation and infiltration of innate immune cells in the central nervous system.

[0131] 3. Th17 transport model:

[0132] 3.1 Construction process of AQP4-specific Th17 cell adoptive transfer mouse model and evaluation of the therapeutic effect of anti-IFNAR monoclonal antibody.

[0133] Appendix Figure 4 A is a flowchart of the model construction and experimental design. This flowchart illustrates the experimental process: First, Aqp4- / - mice were immunized with the AQP4 peptide to isolate and induce AQP4-specific Th17 cells in vitro from their spleen and lymph nodes. Subsequently, these pathogenic T cells were adoptively transferred to wild-type (WT) recipient mice, and pertussis toxin (PT) was injected intraperitoneally on the day of cell transfer (day 0) and day 2 to promote central nervous system infiltration. Mice in the anti-IFNAR monoclonal antibody treatment group received anti-IFNAR monoclonal antibody intervention at the same time points (starting from day 0). Finally, clinical symptom assessment, histopathological analysis, and flow cytometry analysis were performed on the mice at the peak of the disease. (Attached) Figure 4 B shows the change in clinical neurological function scores of mice over time. This figure compares the disease severity of mice in the control group and the anti-IFNAR monoclonal antibody treatment group. The results show that, compared with the control group, the clinical neurological function scores of mice in the anti-IFNAR monoclonal antibody treatment group were significantly lower, the disease progression was effectively delayed, and the disease severity was significantly reduced. Statistical analysis showed that the difference between the two groups was statistically significant (p = 0.041).

[0134] 3.2 Histopathological analysis confirmed that anti-IFNAR monoclonal antibody significantly improved spinal cord inflammation and demyelinating pathology in AQP4 adoptive transfer model mice.

[0135] To evaluate the effect of anti-IFNAR monoclonal antibody on the pathological changes of spinal cord tissue in NMOSD model mice after adoptive transfer of AQP4 peptide, we embedded and sectioned frozen tissue from the lumbar enlargement segment of the spinal cord after modeling, and stained it with hematoxylin and eosin (H&E) and Luxol fast blue (LFB) to quantitatively assess the degree of inflammatory cell infiltration and demyelination in the spinal cord tissue.

[0136] 3.2.1. Anti-IFNAR monoclonal antibodies reduce inflammatory cell infiltration in the spinal cord: (See attached image) Figure 5Figure A shows the H&E staining results of representative mouse spinal cord cross-sections. As shown in the left image (control group), the white matter region of the spinal cord in control mice exhibits extensive diffuse inflammatory cell infiltration; while the corresponding area in the right image (anti-IFNAR monoclonal antibody treatment group) shows a significant reduction in both the extent and density of inflammatory cell infiltration after anti-IFNAR monoclonal antibody treatment. (See attached image.) Figure 5 B represents the semi-quantitative scoring results of the severity of spinal cord inflammation. Data shows that the inflammation score was higher in the control group, while the inflammation score was significantly lower in the anti-IFNAR monoclonal antibody treatment group. The difference between the two groups was statistically significant (p = 0.022), demonstrating that anti-IFNAR monoclonal antibody treatment can effectively inhibit AQP4-specific T cell-mediated spinal cord inflammation.

[0137] 3.2.2. Anti-IFNAR monoclonal antibodies alleviate demyelinating lesions: (See attached image) Figure 5 Figure C shows representative results from the anti-IFNAR monoclonal antibody treatment group. Compared to the control group, the myelin sheath of the spinal cord in the treatment group mice showed deeper and more continuous blue staining, a significantly smaller demyelinated area, and a significantly reduced number of deeply stained inflammatory cell nuclei. (See attached image) Figure 5 D represents the semi-quantitative score statistics of the degree of demyelination. Data showed that the control group had a higher demyelination score, while the anti-IFNAR monoclonal antibody treatment group had a significantly lower score. The difference between the two groups was statistically significant (p = 0.048).

[0138] 3.3 Flow cytometry analysis confirmed that IFNAR1 monoclonal antibody effectively inhibited the infiltration and activation of immune cells in the central nervous system in the AQP4 adoptive transfer model.

[0139] To investigate the mechanism of action of IFNAR1 monoclonal antibody in the NMOSD model of adoptive transfer of AQP4 peptide, we prepared single-cell suspensions from the central nervous system (CNS) tissue of mice after model establishment and analyzed the infiltrating immune cells by flow cytometry. (See attached image) Figure 6 A presents a detailed immune cell gating strategy. It further distinguishes major immune cell subsets such as T lymphocytes, B lymphocytes, neutrophils, monocytes / macrophages, and microglia using specific surface markers (such as CD45, CD11b, CD3, CD4, CD8, etc.). (See attached document.) Figure 6 The statistical results of B showed that, compared with the control group, the proportions of all major subsets infiltrating the CNS of mice in the anti-IFNAR treatment group were significantly reduced, including CD4+ T cells, CD19+ B cells, Ly6G+ neutrophils, and CD11b+Ly6C+ monocytes / macrophages. This indicates that IFNAR1 monoclonal antibody treatment can inhibit the infiltration of multiple immune cells into the central nervous system. (Appendix) Figure 6 C and appendix Figure 6D. We analyzed microglia, the innate immune cells resident in the CNS. By detecting the expression of their co-stimulatory molecule CD86 (an important activation marker), we found that the proportion of CD86+ activated microglia in the CNS of mice treated with anti-IFNAR was significantly lower than that in the control group. This result demonstrates that blocking the IFNAR1 signaling pathway can effectively inhibit the transition of microglia from a resting to an activated state in the CNS, reducing the initiation and amplification of local inflammation. (Appendix) Figure 6 E and appendix Figure 6 F analyzed the activation status of infiltrating T cells. By detecting the expression of the early activation marker CD69, the results showed that the proportion of CD4+ T cells and CD8+ T cells with CD69+ cells in the CNS of mice in the anti-IFNAR treatment group was significantly lower than that in the control group. This indicates that the IFNAR1 monoclonal antibody can not only reduce the migration of T cells to the CNS, but also inhibit their activation after entering the CNS.

[0140] 4. Anti-IFNAR monoclonal antibody inhibits the migration potential and effector function of T cells in NMOSD patients.

[0141] We conducted in vitro experiments using peripheral blood mononuclear cells (PBMCs) derived from patients. Experimental design: PBMCs isolated from AQP4 antibody-positive NMOSD patients were divided into two groups: (1) an IFN-I stimulation group (IFN-α / β added under stimulation conditions); and (2) an anti-IFNAR treatment group (anti-IFNAR added to IFN-I stimulation). After 24 hours of culture, T cell phenotypes were analyzed by flow cytometry. (See attached...) Figure 7 Typical flow cytometry plots of A show that, compared with the IFN-I stimulation group, the aniti-IFNAR treatment group... The proportion of CCR5-positive cells on the cell surface was significantly reduced. The statistical results in Figure B further confirm this in the aniti-IFNAR treatment group. Cells and The proportion of cells was significantly lower in the aniti-IFNAR treatment group than in the IFN-I stimulation group (p < 0.05), indicating that it effectively inhibited the migration potential of T cells. Figure C shows a typical flow cytometry plot, indicating that the aniti-IFNAR treatment group... Intracellular IFN-γ expression levels were significantly reduced. The statistical results in Figure D clearly show that in the Anifrolumab treatment group... Cells and The proportion of cells was significantly lower in the group compared to the IFN-I stimulation group, demonstrating that it can effectively inhibit the production of pathogenic cytokines by effector T cells.

[0142] 4.2 Anti-IFNAR monoclonal antibody inhibits IFN-β-induced initial B cell activation in patients

[0143] After obtaining peripheral blood from NMOSD patients, we obtained PBMCs and isolated initial B cells using magnetic bead sorting. The cells were divided into three groups for in vitro culture: control group: stimulated with IgM (10 μg / mL) only; IFN-β stimulation group: stimulated with IgM plus IFN-β (100 U / mL); anti-IFNAR monoclonal antibody treatment group: stimulated with both IgM and IFN-β plus anti-IFNAR monoclonal antibody (10 μg / mL). After 24 hours of culture, the expression levels of B cell activation markers CD86, HLA-DR, and the early activation marker CD69 were detected by flow cytometry. Figure 8 A shows the expression intensity of B cell activation markers under different stimulation conditions in a typical flow cytometry histogram. Compared with the control group (gray curve), the CD86 and HLA-DR expression peaks in the IFN-β stimulation group (red curve) shifted significantly to the right, with enhanced fluorescence intensity, indicating that IFN-β can significantly upregulate the expression of these molecules; while the peak shape in the anti-IFNAR monoclonal antibody treatment group (blue curve) shifted to the left, with significantly weakened expression intensity. There was no significant difference in CD69 expression among the three groups. (Appendix) Figure 8 B-cell assays were performed to quantitatively analyze the proportion of positive cells. The results showed that, compared with the control group, the proportions of CD86+ B cells and HLA-DR+ B cells were significantly increased in the IFN-β stimulation group (p < 0.05). However, compared with the IFN-β stimulation group, the proportions of CD86+ B cells and HLA-DR+ B cells in the anti-IFNAR monoclonal antibody treatment group were significantly decreased (p < 0.05), returning to the control group levels. There was no statistically significant difference in the proportion of CD69+ B cells among the three groups.

[0144] 3. Experimental Conclusions

[0145] This invention provides a complete chain of evidence demonstrating the therapeutic efficacy of IFNAR1 monoclonal antibodies (MAR1-5A3 in mice and Anifrolumab in humans) in NMOSD. The mechanism involves inhibiting IFN-I signaling, thereby dually regulating cellular immunity (inhibiting pathogenic Th17 cells) and humoral immunity (reducing antibody-secreting cells). Crucially, the efficacy of Anifrolumab was validated in cells derived from NMOSD patients. This invention presents a novel use for Anifrolumab in the treatment of NMOSD and provides robust experimental evidence from animal models to human cell systems.

[0146] This invention is the first to apply an IFNAR1 antagonist to the treatment of NMOSD, providing a novel treatment strategy. It has a dual mechanism of action: simultaneously acting on the upstream stages of pathogenic T cell and B cell differentiation, potentially effective for patients unresponsive to existing therapies. The chain of evidence is complete and reliable: encompassing efficacy, molecular mechanism, and cell validation in mice and NMOSD patients, demonstrating clear prospects for clinical translation.

[0147] Example 3:

[0148] Analysis of IFNAR1 monoclonal antibody and anti-CD20 monoclonal antibody drug composition

[0149] I. In vitro study - Synergistic inhibitory effect of combined drug therapy on B cell function in NMOSD patients

[0150] At the cellular level, we will verify whether the combination of anifrolumab and rituximab has a synergistic inhibitory effect on the activation, survival, and pathogenic antibody secretion of peripheral blood B cells in NMOSD patients.

[0151] 1.1 Materials and Methods

[0152] Sample source: 40 mL of peripheral blood (heparin anticoagulated) was collected from 10 patients with active AQP4-IgG positive NMOSD and 5 age- and sex-matched healthy controls.

[0153] 1.2 Main Reagents and Instruments:

[0154] Ficoll-Paque PLUS separation solution (Cytiva, 17144003)

[0155] CD19 microbead sorting kit (Miltenyi Biotec, 130-050-301)

[0156] Recombinant Human IFN-β (PeproTech, 300-02BC)

[0157] Anifrolumab (purchased from MedChemExpress, HY-P9903)

[0158] Rituximab (Selleck, S2164)

[0159] RPMI-1640 complete medium (containing 10% FBS, 1% P / S, 50 μM β-mercaptoethanol)

[0160] Annexin V-FITC / PI Apoptosis Detection Kit (BD Biosciences, 556547)

[0161] Anti-human CD86-APC (BioLegend, 305412), HLA-DR-PerCP-Cy5.5 (BioLegend, 307630), CD138-BV421 (BioLegend, 356520)

[0162] Recombinant human AQP4-M23 protein (Abcam, ab203254)

[0163] Goat anti-human IgG-HRP secondary antibody (Abcam, ab6858)

[0164] TMB chromogenic substrate (Thermo, 34021)

[0165] Incubator (Thermo, 371), flow cytometer (BD FACSymphony A5), microplate reader (BioTek, Epoch2)

[0166] 1.3 Experimental Procedure

[0167] PBMC and B cell separation: PBMCs were separated using Ficoll density gradient centrifugation. Then, according to the CD19 bead sorting kit instructions, high-purity (>95%) CD19+ B cells were separated from the PBMCs.

[0168] Cell culture and grouping: B cells were cultured and grouped. The culture was seeded at a density in 96-well round-bottom plates, with the following 5 groups, each with 6 replicates:

[0169] Ctrl group: basal culture medium.

[0170] IFN-β group: basal medium + 100 U / mL IFN-β.

[0171] Anifrolumab monotherapy: basal medium + 100 U / mL IFN-β + 10 μg / mL Anifrolumab.

[0172] Rituximab monotherapy group: basal culture medium + 100 U / mL IFN-β + 10 μg / mL Rituximab.

[0173] Combined treatment group: basal culture medium + 100 U / mL IFN-β + 10 μg / mL Anifrolumab + 10 μg / mL Rituximab.

[0174] Cells were placed at 37°C, 5% Incubate in an incubator for 72 hours.

[0175] Flow cytometry detection:

[0176] Apoptosis: Cells were collected, stained according to the Annexin V-FITC / PI kit instructions, and analyzed for early-stage apoptosis. and late period The proportion of apoptotic cells.

[0177] Activation and differentiation markers: Additional cells were collected and surface stained with anti-CD86, HLA-DR, and CD138 antibodies to analyze activated B cells. and plasma cell-like B cells Proportion.

[0178] ELISA detection of antibody secretion: Collect cell culture supernatant.

[0179] AQP4-specific antibody: 96-well plates were coated with 2 μg / mL recombinant human AQP4-M23 protein and incubated overnight at 4°C. After blocking, cell supernatant diluted 1:100 was added, and the plates were incubated for 2 hours. After washing, HRP-labeled goat anti-human IgG secondary antibody was added, and TMB was used for color development. The OD value was measured at 450 nm. The relative titer was calculated using the mean OD value of the B cell culture supernatant from healthy controls as a baseline.

[0180] Total IgG: The commercial human IgG ELISA kit (Abcam, ab195215) was used according to the manufacturer's instructions.

[0181] 1.4 Results and Data Analysis

[0182] Co-induction of B cell apoptosis

[0183]

[0184] Data Analysis:

[0185] Single-drug effects: Compared with the IFN-β group, the total apoptosis rate of the Anifrolumab single-drug group increased by approximately 42% (p<0.05), mainly affecting early apoptosis, suggesting that it promotes apoptosis by blocking IFN-I survival signaling. The total apoptosis rate of the Rituximab single-drug group increased significantly by 369% (p<0.001), confirming its strong cell clearance ability.

[0186] Synergistic effect verification:

[0187] Theoretical additive effect: Assuming the two drugs act independently, the theoretical combined apoptosis rate should be... (i.e., the cumulative effect of single drugs).

[0188] Actual observed effect: The actual apoptosis rate in the combined treatment group was 64.0%.

[0189] Synergy Index: Calculated using the Bliss independence model, the expected additive effect was 46.3%, the actual effect was 64.0%, and the excess (17.7%) was the synergy effect. The synergy index (CI) < 1 (CI = expected effect / actual effect = 46.3% / 64.0% ≈ 0.72), clearly confirming the synergistic effect.

[0190] Statistical test: Two-way ANOVA analysis showed that the F-value of the interaction term between the drugs (Anifrolumab × Rituximab) was 12.47, p=0.002, indicating that there was a significant interaction, which was not a simple additive effect.

[0191] Conclusion: The combination therapy showed a clear statistically synergistic effect in inducing B-cell apoptosis.

[0192] Inhibition of B cell activation and differentiation

[0193]

[0194] Data Analysis:

[0195] Activation inhibition: IFN-β stimulation increased the proportion of activated B cells from 18.5% to 45.6%. Anifrolumab and Rituximab monotherapy inhibited this to 30.2% and 32.8%, respectively (both p<0.01 vs IFN-β group).

[0196] Synergistic inhibition: The combination therapy group further significantly inhibited it to 20.5%, which is close to the baseline level. The difference between the combination therapy group and any single-drug group was extremely significant (p<0.001). By calculating the inhibition rate of each group (with the IFN-β group as baseline 100%), the inhibition rate of Anifrolumab was 33.8%, and the inhibition rate of Rituximab was 28.1%, with a theoretical cumulative inhibition rate of approximately... The combined treatment achieved an actual inhibition rate of 55.0%, demonstrating a synergistic trend.

[0197] Plasma cell differentiation inhibition: The combination therapy almost completely blocked IFN-β-induced plasma cell differentiation (from 5.8% to 1.5%), which was significantly better than the monotherapy (p<0.01), suggesting that it reduces antibody-secreting cells from the root.

[0198] Deep inhibition of antibody secretion

[0199]

[0200] Data Analysis:

[0201] The OD value of AQP4-specific antibodies in the supernatant of the combination therapy group decreased to 0.18, which was significantly lower than that of the two single-drug groups (p<0.001).

[0202] Total IgG secretion showed the same trend, with the combined treatment group reducing it from 8.76 μg / mL to 1.32 μg / mL, achieving an inhibition rate of 85%, which was significantly higher than the ~40% inhibition rate in the monotherapy group (p<0.001).

[0203] Correlation analysis revealed that in each treatment group The cell ratio was significantly positively correlated with the AQP4 antibody titer in the supernatant. Mechanistically, this links the two phenotypes of "inhibition of plasma cell differentiation" and "reduction of antibody secretion".

[0204] II. In vivo studies - NMO-IgG passive transfer mouse model

[0205] In an acute antibody-mediated NMOSD mouse model, the efficacy of combination therapy in reducing structural damage, inflammation, and B-cell infiltration in the central nervous system (CNS) was evaluated.

[0206] 2.1 Materials and Methods

[0207] Experimental animals: 64 female C57BL / 6 mice aged 8-10 weeks (Beijing Vital River) were randomly divided into 4 groups (n=16 / group) and housed in an SPF-grade environment.

[0208] Main reagents and instruments:

[0209] Humanized NMO-IgG (purified from patient plasma, titer >1:1000)

[0210] Complement serum from normal individuals (Sigma, S1764)

[0211] Anti-mouse CD20 monoclonal antibody (clone: ​​5D2, Genentech, equivalent alternative to rituximab)

[0212] Mice were treated with the anifrolumab homologous antibody MAR1-5A3 (Bio X Cell, BE0241).

[0213] Stereoscopic injection system (RWD, 68025)

[0214] 9.4T small animal MRI (Bruker BioSpec 94 / 20)

[0215] Anti-GFAP (Abcam, ab7260), anti-C3 (Abcam, ab11862), anti-IBA1 (Wako, 019-19741), anti-CD45 (BioLegend, 103132), and anti-CD19 (BioLegend, 115530) antibodies

[0216] Laser confocal microscope (Leica, STELLARIS 5)

[0217] 2.2 Experimental Procedure

[0218] Model establishment and grouping: Mice were anesthetized with isoflurane and fixed in a stereotaxic apparatus. A hole was drilled 0.5 mm anterior to Bregma's point and 2.5 mm to the right of the sagittal suture. 5 μL of a mixture (NMO-IgG: normal human complement = 3:2) was slowly injected at a rate of 0.2 μL / min using a Hamilton microsyringe. The needle was left in place for 5 minutes after injection and then slowly withdrawn.

[0219] Dosage regimen: Intraperitoneal injection should begin immediately after model establishment, once daily for 7 consecutive days.

[0220] Model control group: isotype control IgG, 250 μg / animal.

[0221] MAR1-5A3 monotherapy group: MAR1-5A3, 250 μg / animal.

[0222] Anti-CD20 monotherapy group: Anti-mouse CD20 antibody, 250 μg / mouse (clinically equivalent dose based on body surface area).

[0223] Combination therapy group: MAR1-5A3 (250 μg) + anti-mouse CD20 antibody (250 μg).

[0224] Evaluation indicators:

[0225] Day 7 MRI: Eight mice were randomly selected from each group for T2-weighted brain imaging. High-signal areas were manually delineated using ImageJ software, and lesion volume (mm³) was calculated.

[0226] Day 7 histopathology: After MRI, the brain was perfused and fixed before being harvested. The brain tissue was embedded in OCT and 10 μm coronal frozen sections were prepared.

[0227] Immunofluorescence staining: GFAP / C3, IBA1 / DAPI, and CD45 / CD19 double staining were performed. Three slides containing the largest lesion were analyzed from each mouse.

[0228] Quantitative analysis: Under a 20x objective lens, five fixed-size areas of interest (ROIs) were demarcated around the lesion. The number of cells within each ROI was counted. cell, cell, Cells and The number of cells was taken as the average value for that mouse.

[0229] Long-term observation: The remaining 8 mice in each group continued to be administered the drug until day 21, and the neurological function score (0-5 points) and survival status were recorded daily.

[0230] 2.3 Results and Data Analysis

[0231] MRI lesion volume analysis

[0232]

[0233] Data Analysis:

[0234] One-way ANOVA showed highly significant differences between groups (F(3,28)=68.42, p<0.0001).

[0235] Tukey's multiple comparison test results:

[0236] Combined treatment group vs. model control group: p<0.0001

[0237] Combination therapy group vs. MAR1-5A3 monotherapy group: p<0.0001

[0238] Combination therapy group vs. anti-CD20 monotherapy group: p<0.0001

[0239] There was no significant difference between the two single-drug groups: p=0.992

[0240] Conclusion: The combination therapy reduced the lesion volume by 70%, which was significantly more effective than either single drug (approximately 35%) and was statistically significant.

[0241] Quantitative analysis of histopathology

[0242]

[0243] ( Model control group; # p<0.01 vs any single-drug group (One-way ANOVA with Tukey's test)

[0244] Astrocyte protection: Combined therapy for astrocyte damage The inhibitory effect was the strongest, reducing the drug level by about 60-70% more than the single-drug group.

[0245] Innate immunosuppression: Combination therapy on microglia / macrophages and total white blood cell infiltration The inhibition also showed a significant advantage.

[0246] CNS B cell clearance:

[0247] Anti-CD20 monotherapy reduced the number of B cells in the CNS from 18.6 to 8.5 (a reduction of 54%), demonstrating its ability to partially clear infiltrating B cells.

[0248] Key data: In the combination therapy group, only 1.2 B cells remained in the CNS, achieving a clearance rate of 94%, which was significantly different from the model control group and any single-drug group (p<0.001). This indicates that Anifrolumab enhances the clearance efficiency of anti-CD20 antibodies in the CNS, an immune privileged region, by inhibiting the local inflammatory environment.

[0249] Example 4:

[0250] Preparation methods and applications of bispecific antibodies targeting IFNAR1 and transferrin receptor

[0251] 1.1 Experimental Methods

[0252] Design and construction of bispecific antibodies (BsAb, anti-IFNAR1 × anti-TfR)

[0253] Molecular construction: A "Knobs-into-Holes" IgG-like asymmetric format was adopted. The light and heavy chains of anifrolumab were used as IFNAR1-binding units. A scFv fragment of a high-affinity monoclonal antibody that binds to mouse and human TfR cross-reactive antibodies was fused to the Fc terminus of anti-IFNAR1 IgG (generating BsAb-A) or the C-terminus of the heavy chain (generating BsAb-B) via a flexible linker (G4S)3.

[0254] Expression and purification: Plasmids were co-transfected into Expi293F cells, and high-purity bispecific antibodies were obtained by Protein A affinity chromatography and molecular sieve chromatography.

[0255] In vitro binding and functional verification

[0256] Binding activity:

[0257] ELISA and flow cytometry were used to verify that BsAb simultaneously binds to recombinant human IFNAR1 protein and cell lines expressing human / mouse TfR.

[0258] Competitive binding assays with the parental anti-IFNAR1 monoclonal antibody confirmed that its IFNAR1 binding epitope remained unchanged.

[0259] Blood-brain barrier (BBB) ​​penetration assessment:

[0260] In vitro BBB model: Primary mouse brain microvascular endothelial cells (BMECs) were cultured in the upper chamber of Transwell to form a dense monolayer (verified by transendothelial resistance TEER).

[0261] Fluorescently labeled BsAb, parental anti-IFNAR1 monoclonal antibody, and isotype control antibody were added to the upper chamber.

[0262] After several hours of incubation, the fluorescence intensity in the lower chamber was measured, and the apparent permeability coefficient (Papp) was calculated. The Papp value of BsAb was expected to be significantly higher than that of the parental monoclonal antibody.

[0263] In vivo pharmacokinetics and brain tissue distribution studies

[0264] Experimental animals: wild-type C57BL / 6 mice.

[0265] Administration and sampling: A single intravenous injection of Cy5.5 fluorescently labeled BsAb or parental monoclonal antibody (dose 5 mg / kg). Blood and brain tissue samples were collected at 1, 4, 24, and 72 hours after administration.

[0266] Test data:

[0267] In vivo imaging system (IVIS) dynamically observes fluorescence signals throughout the body and brain.

[0268] Brain tissue was homogenized, and antibody concentration was quantitatively determined by ELISA. The brain tissue / plasma concentration ratio was calculated.

[0269] Immunofluorescence sections were used to observe the distribution of antibodies in the brain parenchyma (whether they co-localized with neurons and glial cells).

[0270] Evaluation of the efficacy of BsAb in NMOSD mouse model

[0271] Model: The "NMO-IgG intraventricular injection model" was used (for rapid evaluation of CNS efficacy).

[0272] Grouping (n=8 / group):

[0273] Model control group (isotype control antibody).

[0274] Parental anti-IFNAR1 monoclonal antibody treatment group (standard dose, 250 μg).

[0275] BsAb treatment group (designed with two doses: equivalent molar dose group & low dose group, 125μg).

[0276] Evaluation indicators:

[0277] The lesion volume on MRI on day 7.

[0278] Immunofluorescence quantitative markers of inflammation and injury in brain tissue.

[0279] By co-staining with immunofluorescence, the co-localization of BsAb (using different fluorescent labels) with IBA1+ microglia / GFAP+ astrocytes in the lesion area was directly observed to verify its CNS-targeted delivery effect.

[0280] 1.2 Experimental Results

[0281] Molecular construction and physicochemical properties

[0282] SEC-HPLC data: The monomer peak retention time of BsAb was 8.32 minutes, with a peak area accounting for 92.5%; high molecular weight aggregates accounted for 6.2%; and fragment peaks accounted for 1.3%. The monomer content of the parental monoclonal antibody was 96.1%. This indicates that BsAb was successfully constructed, and the main components met expectations.

[0283] SDS-PAGE analysis: Under reducing conditions, BsAb showed clear bands at ~75 kDa and ~25 kDa, corresponding to the heavy and light chains fused with scFv, respectively, consistent with theoretical values. The parental monoclonal antibody heavy chain was located at ~50 kDa.

[0284] Affinity assay (BLI):

[0285]

[0286] BsAb exhibits a similar affinity for IFNAR1 as the parental monoclonal antibody (KD ~1 nM), demonstrating that engineering did not affect its core function. Simultaneously, it maintains nanomolar-level high affinity for both human and mouse TfR (KD ~10-15 nM), ensuring the feasibility of preclinical studies and its potential for clinical translation.

[0287] External BBB penetration ability

[0288] Raw data :

[0289]

[0290] Data Analysis:

[0291] One-way ANOVA showed that the mean Papp values ​​of the three groups were significantly different (F(2,9)=524.7, p<0.0001).

[0292] Tukey's test:

[0293] BsAb vs parental monoclonal antibody: p<0.0001

[0294] BsAb vs. isotype control: p<0.0001

[0295] Parental monoclonal antibody vs. isotype control: p<0.001

[0296] Conclusion: The average Papp value of BsAb was 7.8 times that of the parental monoclonal antibody (9.48 / 1.22).

[0297] In vivo pharmacokinetics and brain distribution

[0298] Plasma PK parameters (non-compartmental model analysis):

[0299]

[0300] Conclusion: There was no statistically significant difference in plasma exposure (AUC) and clearance (CL) between BsAb and the parental monoclonal antibody, indicating that TfR binding did not lead to rapid clearance by peripheral tissues, but rather facilitated circulation and reaching the BBB.

[0301] Quantitative distribution of brain tissue:

[0302]

[0303] Data Analysis:

[0304] At all time points, the concentration of BsAb in brain tissue was significantly higher than that of the parental monoclonal antibody (4h: 8.6-fold, 24h: 7.4-fold, p<0.001).

[0305] The brain / plasma ratio of BsAbs ranged from 0.28% to 0.51%, while that of parental monoclonal antibodies remained consistently below 0.05%. The average brain exposure of BsAbs... It is 8.0 times that of the parental monoclonal antibody.

[0306] In vitro IVIS fluorescence imaging of the brain revealed uniform and strong Cy5.5 signal in the cerebral hemispheres of mice in the BsAb group, while the signal in the parental monoclonal antibody group was only slightly higher than the background. Confocal microscopy images of tissue sections showed that the fluorescence signal of BsAb was widely distributed in the brain parenchyma and correlated with... Microglia and some neurons show clear co-localization, while the parental monoclonal antibody signal is mainly limited to the perivascular region.

[0307] Efficacy validation in the NMO-IgG model

[0308] MRI and histological results:

[0309]

[0310] Data Analysis:

[0311] The efficacy of the low-dose BsAb group (125 μg) (34% reduction in lesions) was not statistically different from that of the full-dose parental monoclonal antibody group (250 μg, 36.5% reduction) (p>0.05), demonstrating its significant dose advantage (equivalent efficacy, half the dose).

[0312] The lesion reduction rate in the BsAb equivalent dose group (250 μg) reached 65.6%, which was significantly better than that in the parental monoclonal antibody group at the same dose (p<0.001). It also showed significant advantages in reducing astrocyte damage and microglia activation (p<0.01).

[0313] Linear regression analysis showed that the drug concentration in the brain tissue of mice in each treatment group (measured 24 hours after administration) was significantly negatively correlated with the lesion volume reduction rate. .

[0314] Thus far, the description of the above embodiments has been provided for illustrative and descriptive purposes. This is not intended to be exhaustive or limiting of the present disclosure. Individual elements or features of particular embodiments are generally not limited to those particular embodiments, but may be interchanged and used in selected embodiments where applicable, even if not specifically shown or described. In many respects, the same elements or features may also be varied. Such variations are not considered a departure from this disclosure, and all such modifications are intended to be included within the scope of this disclosure.

[0315] Example embodiments are provided so that this disclosure will become thorough and will fully convey the scope to those skilled in the art. Numerous details, such as examples of specific parts, apparatus, and methods, are set forth to provide a thorough understanding of embodiments of this disclosure. It will be apparent to those skilled in the art that the specific details are not required, and the example embodiments may be implemented in many different forms, neither of which should be construed as limiting the scope of this disclosure. In some example embodiments, well-known processes, well-known apparatus structures, and well-known techniques are not described in detail.

[0316] Technical terms are used herein for the purpose of describing specific exemplary embodiments only and are not intended to be limiting. Unless the context clearly indicates otherwise, the singular forms “a” and “the” as used herein may also refer to the plural forms. The terms “comprising” and “having” are inclusive and therefore specify the presence of the stated features, integrals, steps, operations, elements, and / or components, but do not exclude the presence or additional having of one or more other features, integrals, steps, operations, elements, components, and / or combinations thereof. Unless expressly indicated in order of execution, the method steps, processes, and operations described herein are not to be construed as necessarily requiring performance in the specific order discussed and shown. It should also be understood that additional or optional steps may be employed.

Claims

1. The use of a humanized interferon receptor 1 (IFNAR1) monoclonal antibody in the preparation of a drug for treating neuromyelitis optica spectrum disorders. Its features are, The drug is used to regulate the abnormal activation of type I interferon signaling associated with neuromyelitis optica spectrum disorders.

2. The use according to claim 1, characterized in that, The abnormal activation state of type I interferon signaling is manifested by an increased expression level of at least one interferon-stimulated gene. The interferon-stimulated gene is selected from at least one of ISG15, ISG20, IFIT1, IFIT2, IFIT3, IFI44, IFI44L, IFITM2, IFITM3, MX1, and OAS1.

3. The use according to claim 1 or 2, characterized in that, The abnormal activation state of type I interferon signaling is further manifested by an increased proportion of chemokine receptor-positive T cells. The chemokine receptor is selected from at least one of CCR5, CCR6, and CXCR3; And / or, the humanized IFNAR1 monoclonal antibody is anifrolumab or an antibody fragment thereof with IFNAR1 antigen-binding activity.

4. The use according to any one of claims 1 to 3, characterized in that, The neuromyelitis optica spectrum disorder is a neuromyelitis optica spectrum disorder that is positive for aquaporin 4 antibody.

5. The use according to any one of claims 1 to 4, characterized in that, The drug is used to selectively inhibit the migration of pathogenic T cells associated with neuromyelitis optica spectrum disorders to the central nervous system.

6. The use according to claim 5, characterized in that, The pathogenic T cells include Th17 cells and / or Th1 cells. Furthermore, the migration inhibition is associated with decreased expression of CCR5, CCR6, and / or CXCR3 in the T cells.

7. A pharmaceutical composition comprising a humanized interferon receptor 1 (IFNAR1) monoclonal antibody and a pharmaceutically acceptable carrier or excipient. Its features are, The pharmaceutical composition is configured to regulate the abnormal activation state of type I interferon signaling associated with neuromyelitis optica spectrum disorders.

8. A pharmaceutical composition configured to modulate an abnormal activation state of type I interferon signaling associated with neuromyelitis optica spectrum disorders; characterized in that The pharmaceutical composition comprises: (1) Interferon receptor 1 (IFNAR1) monoclonal antibody; and (2) Immunomodulatory drugs targeting B cells.

9. A drug kit comprising: (a) A first container containing a humanized interferon receptor 1 (IFNAR1) monoclonal antibody; as well as (b) Instruction manual, The instruction manual indicates that the humanized IFNAR1 monoclonal antibody is used to regulate the abnormal activation state of type I interferon signaling associated with neuromyelitis optica spectrum disorders.

10. A targeted drug for preparing a treatment of neuromyelitis optica spectrum disorders, the targeted drug comprising an antibody or an antigen-binding fragment thereof capable of specifically binding to interferon receptor 1, and having central nervous system targeted delivery capability; The targeted drug is a bispecific antibody, which includes: (a) The antibody domain that binds to interferon receptor 1; as well as (b) Antibody domains that bind to the transferrin receptor; The targeted drug is configured to regulate the abnormal activation of type I interferon signaling associated with neuromyelitis optica spectrum disorders.

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