Use of umbilical cord blood-derived human activated nk cells in the preparation of a medicament for treating systemic onset juvenile idiopathic arthritis

Abstract

This application belongs to the field of biomedical technology, specifically providing the application of human activated NK cells in the preparation of drugs for treating systemic juvenile idiopathic arthritis. Human activated NK cells derived from umbilical cord blood are intravenously infused at an optimal regimen of 3×10⁶-5×10⁶ cells / individual, once every 3 days for a total of 2 times. This significantly reduces the levels of IL-6, TNF-α, and SAA in the sJIA model, alleviates joint swelling, promotes weight recovery, reduces spleen index, and inhibits cytokine storm. The therapeutic effect can be maintained for at least 14 days without rebound. It has high safety, no obvious liver and kidney damage, and is suitable for clinical translational application.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, and in particular relates to the application of umbilical cord blood-derived human activated NK cells in the preparation of drugs for the treatment of systemic juvenile idiopathic arthritis. Background Technology

[0002] Systemic juvenile idiopathic arthritis (sJIA) is a rare autoinflammatory disease that primarily affects children and adolescents and has been included in my country's Rare Disease Catalogue. The main clinical manifestations include recurrent remittent fever, transient skin rashes, generalized lymphadenopathy, hepatosplenomegaly, and arthritis. Its core pathology involves a systemic cytokine storm triggered by immune system dysregulation. Patients exhibit significantly elevated levels of key inflammatory factors such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in their serum, which can lead to severe systemic wasting symptoms such as weight loss and growth retardation.

[0003] Currently, clinical treatment for sJIA mainly relies on biologics such as glucocorticoids and IL-6 receptor inhibitors (e.g., tocilizumab). However, long-term use of these drugs often leads to numerous problems: glucocorticoids can cause side effects such as growth inhibition and osteoporosis; biologics can lead to drug resistance, increased risk of infection, and liver and kidney damage. Especially for refractory patients, existing therapies have limited efficacy and lack safe, effective, and long-term treatment options.

[0004] Natural killer (NK) cells are core members of the innate immune system, possessing the potential to regulate immunity and suppress excessive inflammatory responses. Current research on NK cells primarily focuses on tumor immunotherapy, or has made preliminary explorations in common arthritis such as rheumatoid arthritis (RA). However, sJIA is essentially a systemic cytokine storm, with fundamentally different pathological mechanisms, inflammatory cytokine profiles, and systemic effects compared to autoimmune arthritis such as RA. To date, there are no reports of using human-derived activated NK cells to treat sJIA. Summary of the Invention

[0005] To address the aforementioned issues, this application provides the use of umbilical cord blood-derived human activated NK cells in the preparation of a drug for treating systemic juvenile idiopathic arthritis.

[0006] This application provides the use of human activated NK cells in the preparation of a drug for treating systemic juvenile idiopathic arthritis, the drug comprising: umbilical cord blood-derived human activated NK cells and an anti-IL-1β monoclonal antibody.

[0007] Furthermore, the drug is administered via intravenous infusion at a dose of 3 × 10⁻⁶. 6 -5×106 Subject weight per kg; And / or, the application frequency is specifically: once every 3 days, for a total of 2 consecutive applications.

[0008] Furthermore, the human activated NK cells are human activated NK cells derived from umbilical cord blood; And / or, the anti-IL-1β monoclonal antibody is a mouse neutralizing antibody, clone number B122, administered at a dose of 250 μg / mouse via intraperitoneal injection, concurrently with NK cell administration.

[0009] Furthermore, the drug reduces serum levels of interleukin-6, tumor necrosis factor-α, and serum amyloid A in subjects by inhibiting the activation of the NF-κB inflammatory pathway.

[0010] Furthermore, the drug was able to reduce joint swelling, restore weight, and lower spleen index in the subjects.

[0011] Furthermore, the therapeutic effect of the drug is maintained for at least 14 days without rebound after administration.

[0012] Furthermore, the therapeutic effect of the drug is superior to that of IL-6 receptor inhibitors.

[0013] Furthermore, the drug did not damage the liver and kidney function of the subjects.

[0014] Furthermore, the dosage is 3 × 10⁻⁶. 6 Subject weight per kg; And / or, the administered dose is 5 × 10 6 Subject weight per kg

[0015] Furthermore, the purity of the human-derived activated NK cells is ≥90%, and the viability is ≥95%.

[0016] Compared with the prior art, this application has the following beneficial effects: 1. This application describes the use of umbilical cord blood-derived human activated NK cells in the treatment of systemic juvenile idiopathic arthritis (sJIA), a rare autoinflammatory storm disease with a pathology completely different from rheumatoid arthritis. This application explicitly limits the application to 3×102 NK cells. 6 -5×10 6 The optimal dosing regimen of 1 person / kg individual, once every 3 days for a total of 2 doses, provides precise dosage, stable efficacy, and strong repeatability. It can significantly inhibit the core inflammatory factors IL-6, TNF-α, and SAA of sJIA, rapidly improve systemic symptoms such as weight loss, foot swelling, and splenomegaly, and has an outstanding effect on blocking cytokine storms.

[0017] 2. The application method of this application is highly safe, does not damage liver and kidney function, has no obvious adverse reactions, and is suitable for long-term use in pediatric patients. The efficacy is long-lasting, with no rebound effect for at least 14 days after administration, superior to existing first-line clinical drugs, and has good prospects for clinical translation. Detailed Implementation

[0018] The technical solutions in the embodiments of this application are clearly and completely described below. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0020] When using “including,” “having,” and “contains” as described herein, the intention is to cover non-exclusive inclusion, unless an explicit qualifying term such as “only,” “consisting of,” etc., is used, in which case another component may be added.

[0021] The terms "preferred," "more preferably," "better," and "even better" used in this application refer to embodiments of this application that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this application. That is, in this application, "preferred," "more preferably," "better," and "even better" are merely descriptions of implementations or embodiments with better effects, but do not constitute a limitation on the scope of protection of this application.

[0022] In this application, terms such as "further," "even more," and "particularly" are used for descriptive purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0023] In this application, "at least one" means one or more, such as one, two, or more. "Multiple" or "several" means at least two, such as two, three, etc., and "multi-layered" means at least two layers, such as two layers, three layers, etc., unless otherwise explicitly specified. In the description of this application, "several" means at least one, such as one, two, etc., unless otherwise explicitly specified.

[0024] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0025] Unless otherwise specified, all steps in this application may be performed sequentially or randomly. For example, the method comprising steps (a) and (b) indicates that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order; for example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc. Unless otherwise stated, singular terms may include plural forms and should not be construed as having a quantity of one.

[0026] In this application, "above" or "below" includes the number itself. For example, "below 1" includes 1.

[0027] In this application, room temperature refers to 0~40℃, including but not limited to 10~40℃, or further to 20~30℃.

[0028] Based on extensive experimental research, this application provides the application of human activated NK cells in the preparation of a drug for treating systemic juvenile idiopathic arthritis, wherein the drug comprises: umbilical cord blood-derived human activated NK cells and an anti-IL-1β monoclonal antibody.

[0029] Furthermore, the drug is administered via intravenous infusion at a dose of 3 × 10⁻⁶. 6 -5×10 6 Subject weight per kg; And / or, the application frequency is specifically: once every 3 days, for a total of 2 consecutive applications.

[0030] In some specific implementations, the dosage can be 3 × 10⁻⁶. 6 3.5×10 6 4×10 6 4.5×10 6 5×10 6 Subject weight per kg

[0031] Furthermore, the human activated NK cells are human activated NK cells derived from umbilical cord blood; And / or, the anti-IL-1β monoclonal antibody is a mouse neutralizing antibody, clone number B122, administered at a dose of 250 μg / mouse via intraperitoneal injection, concurrently with NK cell administration.

[0032] Furthermore, the drug reduces serum levels of interleukin-6, tumor necrosis factor-α, and serum amyloid A in subjects by inhibiting the activation of the NF-κB inflammatory pathway.

[0033] Furthermore, the drug was able to reduce joint swelling, restore weight, and lower spleen index in the subjects.

[0034] Furthermore, the therapeutic effect of the drug is maintained for at least 14 days without rebound after administration.

[0035] Furthermore, the therapeutic effect of the drug is superior to that of IL-6 receptor inhibitors.

[0036] Furthermore, the drug did not damage the liver and kidney function of the subjects.

[0037] Furthermore, the dosage is 3 × 10⁻⁶. 6 Subject weight per kg; And / or, the administered dose is 5 × 10 6 Subject weight per kg

[0038] Furthermore, the purity of the human-derived activated NK cells is ≥90%, and the viability is ≥95%.

[0039] The present application will be further illustrated by the following examples, but these examples do not limit the scope of the present application.

[0040] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in this application, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments whose manufacturers are not specified are conventional products that can be purchased commercially. In addition to the specific methods, equipment, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description in this application, any prior art methods, equipment, and materials similar to or equivalent to those described, used, or made by the methods, equipment, and materials in the embodiments of this application may be used to implement this application.

[0041] The embodiments of this application use the following method to prepare human activated NK cells derived from umbilical cord blood: Umbilical cord blood was collected from healthy mothers at full term. Mononuclear cells were isolated from the cord blood using density gradient centrifugation with human lymphocyte separation medium. The cells were co-cultured with inactivated trophoblast cells in KBM581 serum-free medium supplemented with IL-2 (200 IU / mL). The cells were cultured at 37°C and 5% CO2 for 12–14 days. Flow cytometry analysis showed that CD3⁻CD56⁺ ≥ 90% and viability ≥ 95%, and the cells were then used for further processing.

[0042] sJIA model construction: Six- to eight-week-old SPF-grade C57BL / 6 mice were selected and injected intraperitoneally daily with recombinant mouse IL-1β (10 μg / kg) for 7 consecutive days to induce systemic inflammation, arthritis, and splenomegaly. The model was deemed qualified based on a significant increase in inflammatory factors, obvious paw edema, and an enlarged spleen index.

[0043] RA Model Construction: Six- to eight-week-old SPF-grade DBA / 1 mice were selected. On day 0, 100 μL of bovine type II collagen-CFA emulsion was subcutaneously injected into the tail base of each mouse. On day 21, the mice were boosted with IFA emulsion to induce typical arthritis, and the model was deemed qualified.

[0044] Unified testing indicators: Weight gain, foot swelling thickness, serum IL-6, TNF-α, SAA levels, and spleen index.

[0045] Spleen index (%) = spleen wet weight (mg) ÷ body weight (g) × 100%; serum factors were detected using a mouse ELISA kit.

[0046] Example 1: Validation of the basic efficacy of human activated NK cell therapy for sJIA Mice that successfully established the model were randomly divided into a model group and an NK treatment group, with 12 mice in each group (6 males and 6 females). The model group received a tail vein injection of saline as a blank control; the NK treatment group received a tail vein injection of 3 × 10⁻⁶ mg / L saline solution. 6 One NK cell per cell (using physiological saline as the solvent) was administered to both groups once every 3 days for two consecutive times.

[0047] Relevant indicators were measured 24 hours after the last administration. Specific data are shown in Table 1 below, visually presenting the therapeutic effect of NK cells on sJIA.

[0048] Table 1. Validation data on the therapeutic effect of human activated NK cell therapy on sJIA in this embodiment. Analysis of the data in Table 1 shows that, compared with the model group, human activated NK cells have a significant therapeutic effect on sJIA, and can simultaneously improve systemic inflammation and splenomegaly symptoms, which is consistent with the data logic of the subsequent embodiments.

[0049] Example 2: Dose-effect and positive drug control experiment Mice were randomly divided into 6 groups (n=12 per group, 6 males and 6 females): normal control group, model group, positive drug group, and low-dose NK cell group (1×10⁻⁶). 6 (each animal), NK medium-dose group (3×10) 6 (5 x 10) 6 (each / each).

[0050] All groups were administered via tail vein injection, with a dosing frequency of once every 3 days for 2 consecutive times. The positive control group was given a clinically common IL-6 receptor inhibitor (administered at the mouse equivalent dose), the model group was given physiological saline, and each NK dose group was given the corresponding dose of umbilical cord blood-derived human activated NK cells.

[0051] The results showed: 3×10 6 ~5×10 6 The optimal treatment dose is defined as one unit per animal. Specific test data for each group are shown in Table 2. The optimal dose group showed significantly better results in weight recovery, inflammation suppression, and overall improvement than the positive drug group, and also demonstrated good safety.

[0052] Table 2. Dosage-effect and positive drug control experimental data of this embodiment. The results showed: 3×10 6 ~5×10 6 The optimal treatment dose is defined as one unit per animal. Specific test data for each group are shown in Table 2. The optimal dose group showed significantly better results in weight recovery, inflammation suppression, and overall improvement than the positive drug group, and also demonstrated good safety.

[0053] Example 3: Clinical Trial of Differential Therapy between sJIA and RA Models In this embodiment, conventional methods in the art were used to construct sJIA mouse models (C57BL / 6) and rheumatoid arthritis (RA) mouse models (DBA / 1), respectively. Each 12 mice (6 males and 6 females) were administered 3 × 10⁻⁶ mg / L of sJIA mouse model (C57BL / 6). 6 Treatment was administered using one NK cell per individual, with the administration method consistent with Example 1.

[0054] The results showed that the systemic inflammatory storm in the sJIA model was significantly stronger than that in RA; the improvement of sJIA by NK cells was significantly better than that in RA, indicating that this application has specific and highly effective therapeutic advantages for sJIA, and is not a generalized effect on arthritis. Specific experimental results are shown in Table 3.

[0055] Table 3. Experimental data on the differential therapeutic effects between the sJIA and RA models in this embodiment. Example 4: Long-term efficacy observation experiment In this embodiment, the sJIA mouse model was constructed using conventional methods. After successful model validation, the mice were divided into a model group, a positive drug group, and a medium-dose NK cell group (3×10⁻⁶). 6 (Number of mice per group), the dosing regimens for each group were completely consistent with Example 2; each group consisted of 24 mice, which were sacrificed in batches of 8 mice at three time points: 24h, 7d, and 14d after the last administration, to ensure that mice were available for testing at each time point. After the last administration, the body weight, paw edema thickness, serum IL-6, SAA levels, TNF-α, and spleen index of each group of mice were measured at the above three time points to observe the duration of therapeutic effect. Specific test data are shown in Table 4 below.

[0056] Table 4. Experimental data on long-term efficacy observation in this embodiment. Analysis of the data in Table 4 shows that the NK cell therapy group (3×10) 6 In the NK cell therapy group (dose per unit), serum SAA, TNF-α, IL-6, and spleen index were significantly lower than those in the model group and the positive drug group at all time points. At day 14, the spleen index remained low, showing significant improvement compared to the baseline of the sJIA model (0.89±0.11%), and body weight and foot swelling thickness remained within the normal range, with no signs of inflammatory rebound. In contrast, the model group and the positive drug group showed a significant rebound after day 7, with serum SAA, TNF-α, IL-6, and spleen index all rising sharply at day 14, showing a significant difference from the NK cell therapy group. This further demonstrates that human activated NK cells have a long-lasting anti-inflammatory effect on sJIA, reduce splenomegaly, and have a stable and reliable therapeutic effect. It also confirms that its therapeutic effect is superior to commonly used IL-6 receptor inhibitors, and its drug application is safer.

[0057] The results showed that the NK cell therapy group had a stable and sustained therapeutic effect, with no rebound of inflammation within 14 days. All indicators were significantly better than those of the model group and the positive drug group, and the therapeutic effect was stable, durable and safe.

[0058] Example 5: Validation of the synergistic effect of human activated NK cells combined with anti-IL-1β monoclonal antibody in the treatment of sJIA sJIA model mice were randomly divided into 5 groups, with 12 mice in each group (6 males and 6 females): normal control group, model control group, NK monotherapy group, anti-IL-1β monoclonal antibody group, and combination therapy group.

[0059] 1. Experimental Materials Anti-mouse IL-1β neutralizing monoclonal antibody (clone number: B122, in vivo injection grade, catalog number: BE0246, purchased from BioXCell, USA), low endotoxin, sodium azide-free, sterile, suitable for in vivo treatment in mice.

[0060] 2. Dosing regimen (completely identical to the previous example) NK cell administration: 3×10 6 Each animal (using saline as a solvent) is administered via tail vein injection, once every 3 days, for a total of 2 doses.

[0061] Anti-IL-1β monoclonal antibody administration: 250 μg / animal, intraperitoneal injection, once every 3 days, for 2 consecutive doses. Combination group: NK cells (tail vein) + anti-IL-1β monoclonal antibody (intraperitoneal) were administered on the same day, with the same frequency and dosage as above. The model group received an equal volume of physiological saline, while the normal control group received no treatment. 24 hours after the last administration, the following were measured: weight gain, paw edema thickness, serum IL-6, TNF-α, SAA levels, and spleen index. Specific test results are shown in Table 5.

[0062] The results showed that, compared with the model group, both the NK monotherapy group and the anti-IL-1β monoclonal antibody monotherapy group significantly reduced the inflammatory response, decreased the level of inflammatory factors, and improved systemic symptoms in sJIA mice; the combined treatment group showed a further significant improvement in efficacy, which was superior to either monotherapy group.

[0063] Table 5. Efficacy data of human activated NK cells combined with anti-IL-1β monoclonal antibody therapy for sJIA Analysis of the data in Table 5 shows that the combined use of human activated NK cells and anti-IL-1β monoclonal antibody produced a significant synergistic therapeutic effect in the sJIA mouse model. It could more effectively inhibit the cytokine storm, improve systemic symptoms and joint damage, and the effect was significantly better than NK cell therapy alone or anti-IL-1β monoclonal antibody therapy alone. Moreover, it had good safety and no obvious liver and kidney damage, and has good clinical translational value.

[0064] Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. The application of human-derived activated NK cells in the preparation of drugs for treating systemic juvenile idiopathic arthritis, characterized in that: The drug includes: umbilical cord blood-derived human activated NK cells and anti-IL-1β monoclonal antibody.

2. The application of the human activated NK cells according to claim 1 in the preparation of a drug for treating systemic juvenile idiopathic arthritis, characterized in that: The drug is administered via intravenous infusion at a dose of 3 × 10⁻⁶. 6 -5×10 6 Subject weight per kg; And / or, the application frequency is specifically: once every 3 days, for a total of 2 consecutive applications.

3. The application of the human activated NK cells according to claim 1 in the preparation of a drug for treating systemic juvenile idiopathic arthritis, characterized in that: The human activated NK cells are human activated NK cells derived from umbilical cord blood. And / or, the anti-IL-1β monoclonal antibody is a mouse neutralizing antibody, clone number B122, administered at a dose of 250 μg / mouse via intraperitoneal injection, concurrently with NK cell administration.

4. The application of the human activated NK cells according to claim 1 in the preparation of a drug for treating systemic juvenile idiopathic arthritis, characterized in that: The drug reduces serum levels of interleukin-6, tumor necrosis factor-α, and serum amyloid A by inhibiting the activation of the NF-κB inflammatory pathway.

5. The application of the human activated NK cells according to claim 1 in the preparation of a drug for treating systemic juvenile idiopathic arthritis, characterized in that: The drug was able to reduce joint swelling, increase weight, and decrease spleen index in the subjects.

6. The application of the human activated NK cells according to claim 1 in the preparation of a drug for treating systemic juvenile idiopathic arthritis, characterized in that: The therapeutic effect of the drug lasts for at least 14 days without rebound after administration.

7. The application of the human activated NK cells according to claim 1 in the preparation of a drug for treating systemic juvenile idiopathic arthritis, characterized in that: The therapeutic effect of this drug is superior to that of IL-6 receptor inhibitors.

8. The application of the human activated NK cells according to claim 1 in the preparation of a drug for treating systemic juvenile idiopathic arthritis, characterized in that: The drug did not damage the liver and kidney function of the subjects.

9. The application of the human activated NK cells according to claim 1 in the preparation of a drug for treating systemic juvenile idiopathic arthritis, characterized in that: The dosage is 3 × 10 6 Subject weight per kg; And / or, the administered dose is 5 × 10 6 Subject weight per kg 10. The use of human activated NK cells according to claim 1 in the preparation of a drug for treating systemic juvenile idiopathic arthritis, characterized in that: The purity of the human-derived activated NK cells is ≥90%, and the viability is ≥95%.

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