Clinical use of sbcma in cerebrospinal fluid in diagnosis and monitoring of central nervous system autoimmune diseases

Abstract

Disclosed in the present invention is the use of sBCMA in cerebrospinal fluid as a biomarker in the diagnosis and monitoring of antibody-mediated central nervous system autoimmune diseases. Taking NMOSD as an example, the level of sBCMA in the cerebrospinal fluid of NMOSD patients is significantly higher than that of age- and gender-matched subjects in a control group. Furthermore, the level of sBCMA in cerebrospinal fluid is positively correlated with a nerve injury-related index NFL, a blood-cerebrospinal fluid barrier destruction-related index QAlb, and a neuroinflammation-related index sTREM2 in the cerebrospinal fluid of the NMOSD patients. This indicates that the level of sBCMA in cerebrospinal fluid can be used as a biomarker for the diagnosis of antibody-mediated central nervous system autoimmune diseases, such as NMOSD, and can also be used for assessing the severity of nerve injury, the extent of blood-cerebrospinal fluid barrier disruption, the level of neuroinflammation, etc., in patients with central nervous system autoimmune diseases.

Description

Clinical application of sBCMA in cerebrospinal fluid in the diagnosis and monitoring of autoimmune diseases of the central nervous system Technical Field

[0001] This invention belongs to the field of disease detection technology, specifically relating to the application of sBCMA in cerebrospinal fluid as a biomarker in the diagnosis and monitoring of antibody-mediated autoimmune diseases of the central nervous system. Background Technology

[0002] Autoimmune diseases are a group of illnesses characterized by immune dysfunction, in which the body exhibits an abnormal immune response to normal body components. More than 80 autoimmune diseases have been identified in humans, including systemic diseases such as systemic lupus erythematosus, as well as tissue- or organ-specific diseases such as autoimmune thyroiditis. Autoimmune diseases can affect a wide range of systems throughout the body, including the skin, joints, kidneys, and central nervous system.

[0003] Central nervous system (CNS) autoimmune diseases are characterized by the direct or indirect attack of the nervous system (including neurons, glial cells, and myelin sheath) by autoimmune cells, autoantibodies, and other immune molecules. CNS autoimmune diseases are diverse, including neuromyelitis optic spectrum disorder (NMOSD), myelin oligodendrocyte glycoprotein-IgG associated disorders (MOGAD), multiple sclerosis (MS), acute disseminated encephalomyelitis (ADEM), autoimmune encephalitis (AE), and CNS vasculitis. The pathogenesis of CNS autoimmune diseases is complex, with the immune inflammatory response caused by the direct or indirect attack of the nervous system by autoimmune cells, autoantibodies, and other immune molecules playing a crucial role in their development. Autoantibodies, in particular, play a vital role in the diagnosis and differential diagnosis of CNS inflammatory demyelinating diseases.

[0004] B lymphocytes / plasma cells are among the most important effector cells in autoimmune diseases. They attack normal cells and tissues through various means, including abnormal secretion of autoantibodies, inducing systemic immune abnormalities. The proliferation and maturation of B lymphocytes play a crucial role in the pathogenesis of CNS autoimmune diseases. B-cell maturation antigen (BCMA) is a transmembrane glycoprotein in the tumor necrosis factor superfamily, primarily expressed on the surface of mature B lymphocytes. It regulates the maturation and differentiation of B cells into plasma cells. Soluble B-cell maturation antigen (sBCMA), derived directly from membrane BCMA, has been proven to be a good biomarker for multiple myeloma and systemic lupus erythematosus (SLE). A prospective study evaluated the correlation between serum sBCMA and bone marrow plasma cell infiltration in multiple myeloma patients, confirming that sBCMA levels are a good prognostic indicator for multiple myeloma. Another retrospective cohort study revealed the potential and significance of serum sBCMA as a biomarker for SLE patients. Due to the unique structure of the central nervous system (CNS) – the blood-brain barrier – autoimmune cells cannot easily enter the CNS under normal circumstances. Therefore, unlike other autoimmune diseases, the immune response in CNS antibody-mediated autoimmune diseases may be limited, and there are no relevant clinical studies demonstrating the clinical application of sBCMA as a biomarker in the diagnosis and monitoring of CNS antibody-mediated autoimmune diseases of the central nervous system. Summary of the Invention

[0005] This invention provides a novel use of sBCMA in cerebrospinal fluid as a biomarker, namely, its application in the preparation of diagnostic kits and disease monitoring kits for antibody-mediated autoimmune diseases of the central nervous system, such as neuromyelitis optica spectrum disease (NMOSD).

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The application of reagents for detecting the biomarker sBCMA in cerebrospinal fluid in the preparation of kits includes the following aspects:

[0008] (1) Detection of sBCMA levels in cerebrospinal fluid is used to diagnose antibody-mediated autoimmune diseases of the central nervous system;

[0009] (2) Detecting sBCMA levels in cerebrospinal fluid is used to assess the severity of neurological damage in patients with antibody-mediated central nervous system autoimmune diseases.

[0010] (3) Detecting sBCMA levels in cerebrospinal fluid is used to assess the extent of blood-cerebrospinal fluid barrier disruption in patients with antibody-mediated central nervous system autoimmune diseases.

[0011] (4) Detecting sBCMA levels in cerebrospinal fluid is used to assess the level of neuroinflammation in patients with antibody-mediated central nervous system autoimmune diseases;

[0012] Specifically, the steps include: a. Measuring the sBCMA level in the cerebrospinal fluid of the subjects; b. Performing a significant difference analysis on the sBCMA level in the cerebrospinal fluid of the subjects and the sBCMA level of sex- and age-matched controls; The sBCMA level in the cerebrospinal fluid of patients with antibody-mediated central nervous system autoimmune diseases is positively correlated with the levels of NFL, QAlb, and sBCMA in their cerebrospinal fluid. Therefore, compared with the sBCMA level of the control group, the higher the sBCMA level in the cerebrospinal fluid of the subjects, the more severe their nerve damage, the more severe the degree of blood-cerebrospinal fluid barrier dysfunction, and the higher the level of neuroinflammation.

[0013] The aforementioned antibody-mediated autoimmune diseases of the central nervous system, in addition to neuromyelitis optica spectrum disorders, also include myelin oligodendrocyte glycoprotein antibody-related diseases, anti-N-methyl-D-aspartate receptor encephalitis, anti-leucine-rich glioma inactivating protein 1 encephalitis, and anti-contactin-associated protein 2 antibody-related autoimmune encephalitis.

[0014] Compared with existing technologies, this invention has the following advantages and beneficial effects: First, this invention has determined that the level of sBCMA in the cerebrospinal fluid of NMOSD patients is significantly higher than that in sex- and age-matched control subjects. Furthermore, this invention has determined a positive correlation between the concentration of sBCMA in the cerebrospinal fluid of NMOSD patients and indicators related to nerve damage and blood-cerebrospinal fluid barrier disruption. This indicates that the level of sBCMA in the cerebrospinal fluid of NMOSD patients can serve as a biomarker for the auxiliary diagnosis of NMOSD patients and can reflect the severity of nerve damage and pathological destruction in NMOSD patients. Simultaneously, this invention has determined a positive correlation between sBCMA in the cerebrospinal fluid of NMOSD patients and neuroinflammation. Therefore, sBCMA, as a biomarker, can provide a powerful auxiliary means for the diagnosis and disease monitoring of NMOSD and other antibody-mediated central nervous system autoimmune diseases (including myelin oligodendrocyte glycoprotein antibody-related diseases, anti-N-methyl-D-aspartate receptor encephalitis, anti-leucine-rich glioma inactivating protein 11 encephalitis, anti-contactin-associated protein 2 antibody-related autoimmune encephalitis, etc.), exhibiting good sensitivity and specificity. Attached Figure Description

[0015] Figure 1 shows the results of sBCMA level analysis in cerebrospinal fluid of subjects of different genders.

[0016] Figure 2 shows the results of the correlation analysis between the age of the subjects and the level of sBCMA in the cerebrospinal fluid.

[0017] Figure 3 shows the results of the sBCMA level analysis in the cerebrospinal fluid of the subjects.

[0018] Figure 4 shows the ROC curve of sBCMA levels in the cerebrospinal fluid of the subjects.

[0019] Figure 5 shows the level analysis of the neurological injury-related index NFL in the subjects (A) and the correlation analysis results between the sBCMA level in cerebrospinal fluid and NFL (B).

[0020] Figure 6 shows the analysis of QAlb levels (A) and the correlation analysis results between sBCMA levels in cerebrospinal fluid and QAlb (B).

[0021] Figure 7 shows the analysis of the neuroinflammation-related marker sTREM2 level in the subjects (A) and the correlation analysis results between the sBCMA level in cerebrospinal fluid and the neuroinflammation-related marker sTREM2 (B).

[0022] Figure 8 shows the results of sBCMA level analysis in the cerebrospinal fluid of subjects of different genders in the validation cohort.

[0023] Figure 9 shows the correlation analysis results between the age of the subjects in the validation cohort and the level of sBCMA in the cerebrospinal fluid.

[0024] Figure 10 shows the results of the sBCMA level analysis in the cerebrospinal fluid of the validation cohort subjects.

[0025] Figure 11 shows the ROC curve of sBCMA levels in the cerebrospinal fluid of the subjects in the validation cohort.

[0026] Figure 12 shows the level analysis of cerebrospinal fluid nerve injury-related index NFL in the validation cohort subjects and the correlation analysis results between sBCMA level in cerebrospinal fluid and NFL.

[0027] Figure 13 shows the level analysis of sTREM2, a neurological injury-related index in cerebrospinal fluid, and the correlation analysis results between sBCMA level and sTREM2 in cerebrospinal fluid of the validation cohort subjects. Detailed Implementation

[0028] Neuromyelitis optica spectrum disorder (NMOSD) is a rare, progressive, autoimmune-mediated inflammatory demyelinating disease of the central nervous system (CNS) primarily affecting the optic nerve and spinal cord. NMOSD is characterized by inflammatory demyelination and axonal damage in the optic nerve and spinal cord, potentially leading to blindness, paralysis, and urinary and fecal incontinence. Its pathogenesis primarily stems from plasma cells of B cells producing aquaporin 4 (AQP4) antibodies, which cross the blood-brain barrier and bind to AQP4 antigens on astrocytes. Through antibody-dependent cytotoxicity and complement-dependent cytotoxicity, this ultimately results in astrocyte damage, oligodendrocyte injury, demyelination, and neuronal loss. NMOSD commonly affects young adults, predominantly women, and is clinically characterized by severe optic neuritis and longitudinally extending, long-segment transverse myelitis, with high relapse and disability rates. The incidence rate in China is approximately 0.41 per 100,000, and the prevalence is approximately 3.31 per 100,000. Non-motorized neuropathic disease (NMOSD) is a highly relapsing and disabling disease, leaving most patients with severe sequelae, placing a significant burden on healthcare systems, families, and communities. This invention uses NMOSD as an example to reveal the important role of sBCMA in cerebrospinal fluid as a biomarker in the diagnosis and monitoring of antibody-mediated CNS autoimmune diseases.

[0029] Example 1: sBCMA in cerebrospinal fluid as a biomarker for NMOSD

[0030] Clinical study subjects: In the following examples, the clinical study subjects included 30 NMOSD patients (positive for aquaporin 4 antibody—AQP4-IgG) and 30 sex- and age-matched control subjects with negative serum AQP4 antibodies. Details are shown in the table below:

[0031] (1) Sample collection and preparation

[0032] Cerebrospinal fluid (CSF) samples were collected from clinical study subjects using sterile centrifuge tubes. The collected CSF samples were centrifuged at 500×g for 10 minutes at 4°C, and the supernatant was aliquoted into cryovials and stored at -80°C.

[0033] (2) sBCMA concentration detection method

[0034] The expression level of sBCMA in cerebrospinal fluid was detected using a human BCMA / TNFRSF17 ELISA kit (R&D Systems, DY193). Specifically, before the assay, the samples were brought back to a temperature range of 18–25°C. The samples were diluted and standards were prepared according to the manufacturer's instructions. Then, 100 μl of standards and samples were added to each well of the plate and incubated at room temperature for 2.5 hours. Next, the liquid in the plate was discarded, and the plate was washed three times with 1× washing buffer (300 μl each time, 3–5 minutes each time). Then, 100 μl of antibody was added and incubated at room temperature for 1 hour. Again, the plate was washed three times with washing buffer. Then, 100 μl of HRP was added and incubated at room temperature for 45 minutes, followed by another wash. 100 μl of substrate solution was added to each well sequentially, and the plate was incubated at room temperature in the dark for 30 minutes. After color development, 50 μl of stop solution (2 mol / L H₂SO₄) was added to each well to terminate the reaction. Finally, the OD value at 450 nm was read using an ELISA reader to determine the sBCMA level in the cerebrospinal fluid.

[0035] (3) Analysis of sBCMA levels in cerebrospinal fluid

[0036] Specifically, this invention detected sBCMA levels in the cerebrospinal fluid (CSF) of 30 NMOSD patients and a sex- and age-matched control group. The results showed no statistically significant difference in sBCMA levels between male and female subjects (P = 0.7498) (Figure 1), and no correlation was found between subject age and sBCMA levels in CSF (R = 0.031, P = 0.179) (Figure 2). Further analysis of sBCMA levels in the CSF of NMOSD patients and the control group revealed that sBCMA levels in the CSF of NMOSD patients were significantly higher than those in the control group (P < 0.0001) (Figure 3). Furthermore, as shown in Figure 4, the ROC curve results showed an area under the curve (AUC) of 0.8678, which was statistically significant (P < 0.0001). This indicates that sBCMA in CSF can serve as a biomarker to aid in the diagnosis of NMOSD, exhibiting good sensitivity and specificity.

[0037] Example 2: sBCMA in cerebrospinal fluid as a biomarker of neurological injury in NMOSD

[0038] Neurofilament light chains (NFLs) are widely recognized as biomarkers of nerve injury, capable of dynamically reflecting irreversible nerve damage and disease progression, and can serve as biological indicators for the progression and monitoring of NMOSD. In this embodiment, based on the automated SIMOAHD-X platform, ultrasensitive SIMOA technology was used to quantify the NFL levels in the cerebrospinal fluid of the clinical study subjects in Example 1. The results showed that the NFL levels in the cerebrospinal fluid of NMOSD patients were significantly higher than those in the control group (P = 0.0002). Furthermore, this embodiment analyzed the correlation between sBCMA and NFL in cerebrospinal fluid. As shown in Figure 5, there was a positive correlation between sBCMA levels and NFL in NMOSD patients (R = 0.236, P = 0.006), while there was no correlation between sBCMA levels and NFL levels in the control group (R = 0.084, P = 0.120). This suggests that elevated sBCMA levels in cerebrospinal fluid (CSF) indicate increased neurological damage, and that CSF sBCMA levels can indicate the severity of neurological injury. Therefore, CSF sBCMA can serve as a biomarker for predicting neurological damage, reflecting the degree of neurological injury in antibody-mediated CNS autoimmune diseases such as NMOSD.

[0039] Example 3: sBCMA in cerebrospinal fluid as a biomarker of blood-brain barrier damage in NMOSD

[0040] In NMOSD, the pro-inflammatory cytokine interleukin-6 can mediate disruption of the blood-brain barrier (BBB), leading to increased penetration of AQP-4 antibodies and further causing subsequent neurotoxicity. Clinical studies have shown that the degree of BBB disruption is a reliable clinical biomarker for diagnosing NMOSD and its severity. The protein concentration gradient between plasma and cerebrospinal fluid (CSF) is caused by the function of the CSF barrier. The ratio of CSF albumin (Alb) levels to serum albumin levels is called the albumin quotient (QAlb), which reflects the degree of CSF barrier disruption. The QAlb calculation formula is (CSF Alb / Blood Alb) × 1000. As shown in Figure 6, in Example 1, the QAlb levels of NMOSD patients were significantly higher than those of the control group (P = 0.0073). This study analyzed the correlation between sBCMA levels in cerebrospinal fluid (CSF) and QAlb. The results showed a positive correlation between CSF sBCMA levels and QAlb in NMOSD patients (R = 0.401, P = 0.0002), while no correlation was found in the control group (R = 0.027, P = 0.383). This suggests that elevated CSF sBCMA levels represent aggravated blood-brain barrier disruption, and CSF sBCMA levels can serve as a biomarker to indicate the severity of CSF barrier dysfunction. Therefore, CSF sBCMA levels can indicate the severity of CSF barrier disruption during the pathogenesis of antibody-mediated CNS autoimmune diseases such as NMOSD.

[0041] Example 4: sBCMA in cerebrospinal fluid as a biomarker of neuroinflammation in NMOSD

[0042] Increased levels of soluble myeloid cell triggering receptor 2 (sTREM2) expressed in cerebrospinal fluid (CSF) are associated with the risk of NMOSD and are positively correlated with neuroinflammatory responses. The expression level of sTREM2 in the CSF of clinical study subjects in Example 1 was detected using a human TREM2 ELISA kit (Abcam, ab224881). As shown in Figure 7, the sTREM2 level was significantly higher in NMOSD patients compared to the control group (P = 0.0004). Similarly, this example analyzed the correlation between sBCMA and sTREM2 levels. The results showed a positive correlation between sBCMA levels and sTREM2 in the CSF of NMOSD patients (R = 0.454, P < 0.0001). However, this correlation was not present in the control group (R = 0.003, P = 0.757). This suggests that cerebrospinal fluid sBCMA levels can reflect the degree of neuroinflammation, and that cerebrospinal fluid sBCMA levels can serve as a biomarker for predicting the level of neuroinflammation, playing a role in the early warning and auxiliary diagnosis of antibody-mediated central nervous system autoimmune diseases such as NMOSD.

[0043] Example 5: Validation of the application of sBCMA in cerebrospinal fluid as a biomarker in the diagnosis of NMOSD

[0044] To further confirm the reliability of sBCMA in cerebrospinal fluid as a biomarker for NMOSD, the following examples included a validation cohort of 15 NMOSD patients (positive for aquaporin 4 antibody—AQP4-IgG) and 15 sex- and age-matched control subjects with negative serum AQP4 antibodies. Details are shown in the table below:

[0045] Specifically, this invention further examined the sBCMA levels in the cerebrospinal fluid (CSF) of 15 NMOSD patients and 15 sex- and age-matched control subjects in the validation cohort. The results showed no statistically significant difference in sBCMA levels between male and female subjects (P = 0.1388) (Figure 8), and no correlation was found between subject age and sBCMA levels in CSF (R = 0.0899, P = 0.1074) (Figure 9). Further analysis of sBCMA levels in the CSF of NMOSD patients and control subjects revealed that sBCMA levels in the CSF of NMOSD patients were significantly higher than those in the control group (P = 0.0075) (Figure 10). Furthermore, the ROC curve results showed an area under the curve (AUC) of 0.7822, which was statistically significant (P = 0.0084) (Figure 11). This further demonstrates that sBCMA in CSF can serve as a biomarker for the diagnosis of NMOSD, exhibiting good sensitivity and specificity.

[0046] Furthermore, this invention detected the levels of neurological injury markers (NFL) and neuroinflammatory markers (sTREM2) in the cerebrospinal fluid (CSF) of subjects in the validation cohort. The results showed that the levels of NFL (P = 0.0420) and sTREM2 (P = 0.0141) in the CSF of NMOSD patients were significantly higher than those in the control group. Further, this embodiment analyzed the correlation between CSF sBCMA and NFL / sTREM2. As shown in Figure 12, the CSF sBCMA level was positively correlated with NFL in NMOSD patients (R = 0.4157, P = 0.0095), while no correlation was found between CSF sBCMA and NFL levels in the control group (R = 0.0180, P = 0.6331). This suggests that CSF sBCMA levels can serve as a biomarker for the severity of neurological injury. Figure 13 shows that the cerebrospinal fluid (CSF) sBCMA level in NMOSD patients was positively correlated with sTREM2 (R = 0.3684, P = 0.0213). However, this correlation was not observed in the control group (R = 0.0013, P = 0.9026). This suggests that the CSF sBCMA level can reflect the degree of neuroinflammation and can serve as a biomarker for neuroinflammation levels.

[0047] The above analysis indicates that sBCMA in cerebrospinal fluid (CSF) can reflect the severity of neurological damage, blood-brain barrier disruption, and central nervous system inflammatory response in NMOSD patients. Furthermore, based on the common pathogenesis of antibody-mediated central nervous system autoimmune diseases, the level of CSF sBCMA can not only provide a powerful auxiliary tool for the early diagnosis of NMOSD, but also offer excellent reference value for the clinical diagnosis and disease monitoring of other antibody-mediated central nervous system autoimmune diseases (such as myelin oligodendrocyte glycoprotein antibody-related diseases, anti-N-methyl-D-aspartate receptor encephalitis, anti-leucine-rich glioma inactivating protein 11 encephalitis, and anti-contactin-associated protein 2 antibody-related autoimmune encephalitis).

Claims

1. The application of reagents for detecting the biomarker sBCMA in the preparation of a kit, characterized in that, The kit described is used for the diagnosis of antibody-mediated autoimmune diseases of the central nervous system.

2. The application of reagents for detecting the biomarker sBCMA in the preparation of a kit, characterized in that, The kit described is used to assess the severity of neurological damage in patients with antibody-mediated central nervous system autoimmune diseases.

3. Application of reagents for detecting the biomarker sBCMA in the preparation of a kit for assessing the degree of blood-brain barrier disruption in patients with antibody-mediated central nervous system autoimmune diseases.

4. Application of reagents for detecting the biomarker sBCMA in the preparation of a kit for assessing the level of neuroinflammation in patients with antibody-mediated central nervous system autoimmune diseases.

5. The application according to any one of claims 1 to 4, characterized in that, The biomarker sBCMA is derived from cerebrospinal fluid.

6. The application according to any one of claims 1 to 4, characterized in that, Includes the following steps: 1) Measure the sBCMA level in the cerebrospinal fluid of the subjects; 2) Perform a significant difference analysis on the sBCMA level in the cerebrospinal fluid of the subjects and the sBCMA level of the sex- and age-matched control group.

7. The application according to any one of claims 1 to 4, characterized in that, The antibody-mediated autoimmune diseases of the central nervous system mentioned above are neuromyelitis optica spectrum disorders.

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