Exploratory Clinical Study on the Safety and Efficacy of Anti- CD19/BCMA CAR-NK Cell Injection for the Treatment of Refractory Pediatric Rheumatic Diseases

Exploratory Clinical Study on the Safety and Efficacy of Anti- CD19/BCMA CAR-NK Cell Injection for the Treatment of Relapsed/Refractory Pediatric Rheumatic Diseases

A single arm, open-label pilot study is designed to determine the safety and effectiveness of anti-CD19/BCMA CAR-NK cell injection in patients with refractory pediatric rheumatic diseases.

Common Inclusion Criteria:

1. . Gender unrestricted, age ≥5 years;
2. . The patient or their legal guardian agrees to participate in this clinical trial and signs the informed consent form, indicating their understanding of the trial's purpose and procedures and willingness to participate;
3. . Peripheral blood B cells confirmed by flow cytometry to express CD19, with a B cell count >5 cells/uL;
4. . If previously treated with B cell-targeted therapy, peripheral blood B cell count at screening has returned to normal or above the pre-treatment level;
5. . Echocardiography indicates basically normal cardiac structure and left ventricular ejection fraction (LVEF) ≥55%; electrocardiogram shows no significant abnormalities;
6. . Liver function: aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤3.0×ULN, total bilirubin (TBIL) ≤2.0×ULN;
7. . Renal function: estimated glomerular filtration rate (eGFR) ≥30 mL/min/1.73m²; (If eGFR <30 mL/min/1.73m² and/or undergoing renal replacement therapy, the subject may be considered for enrollment after the investigator's assessment that the benefit outweighs the risk and with full informed consent from the patient/guardian);
8. . Pulmonary function: No severe pulmonary lesions, blood oxygen saturation (SpO₂) ≥92%;
9. . Female subjects of childbearing potential must have a negative urine pregnancy test and agree to use effective contraception during the trial until 1 year after infusion.

Polyarticular Juvenile Idiopathic Arthritis (pJIA):

1. . Onset before age 16, disease duration ≥6 weeks, diagnosed as polyarticular JIA according to the 2001 International League of Associations for Rheumatology (ILAR) classification criteria, and positive for rheumatoid factor (RF) and/or anti-citrullinated peptide/protein antibody (ACPA): positive on ≥2 occasions at least 3 months apart during the first 6 months of illness;
2. . Judged by the investigator to have active disease despite adequate standard-dose treatment prior to screening, meeting the following adequacy of treatment conditions: a. Treatment with conventional disease-modifying antirheumatic drugs (DMARDs) for at least 6 months, with stable doses of 2 DMARDs for ≥12 weeks; b. Treatment with at least 2 biologic agents, with stable doses for ≥12 weeks;
3. . Juvenile Arthritis Disease Activity Score-27 (JADAS-27) >8.5;
4. . Must have at least 5 active joints (defined as the presence of joint swelling; or in the absence of swelling, the presence of limited range of motion accompanied by pain on motion and/or tenderness) as per the American College of Rheumatology (ACR) definition at both screening and baseline;
5. . No occurrence of macrophage activation syndrome within 1 month prior to screening.

Sjögren's Syndrome (SS):

1. . Diagnosed with childhood-onset primary SS at least 24 weeks prior to signing the ICF, according to the 2002 American-European Consensus Group (AECG) classification criteria / 2016 EULAR/ACR classification criteria and the 2021 Japanese classification criteria for childhood primary SS;

2. . Meet the classification criteria for SS, and Intolerance or inadequate response to glucocorticoids (prednisone 1-2 mg/kg/day or equivalent doses of other corticosteroids) and at least 2 immunosuppressants, with a duration of glucocorticoid treatment of at least 6 months;

3. . EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI) score ≥5 in at least 1 of the following 8 domains at screening: constitutional, lymphadenopathy, glandular, articular, cutaneous, renal, hematological, and serological;

4. . EULAR Sjögren's Syndrome Patient Reported Index (ESSPRI) score ≥5 at screening;

5. . Positive for anti-SSA/Ro antibody.

   Juvenile Dermatomyositis (JDM):

6. . Diagnosed with JDM at least 24 weeks prior to signing the ICF according to the 2017 EULAR/ACR classification criteria;

7. . Meet the classification criteria for Refractory JDM (RJDM), and had Intolerance or inadequate response to glucocorticoids (prednisone 1-2 mg/kg/day or equivalent doses of other corticosteroids) and at least 2 immunosuppressants, with a duration of glucocorticoid treatment of at least 6 months;

8. . Patients with anti-synthetase syndrome who are anti-synthetase antibody positive and meet the criteria for RJDM can be directly enrolled;

9. . Patients with immune-mediated necrotizing myopathy who are signal recognition particle (SRP) or 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) antibody positive and meet the criteria for RJDM can be directly enrolled.

Systemic Sclerosis (SSc):

1. . Meet the 2013 ACR/EULAR classification criteria for SSc, with first non-Raynaud's phenomenon occurring at age <18 years and disease duration ≤60 months;
2. . Positive for antinuclear antibody (ANA) or any SSc-specific antibody;
3. . Modified Rodnan Skin Score (mRSS) ≥15 (total score 51);
4. . Meet the definition of treatment-refractory disease: inadequate response to glucocorticoids (≥0.5 mg/kg/day) and cyclophosphamide and at least 1 other immunomodulatory drug for over 3 months;
5. . Diagnosed with refractory UCTD-ILD: UCTD typically refers to patients with symptoms and signs suggestive of CTD and serologic evidence of autoimmunity, but not fulfilling classification criteria for any defined CTD. Presence of ILD-related clinical manifestations: e.g., dry cough, exertional dyspnea, bibasral crackles, clubbing, OR chest high-resolution CT consistent with ILD features (often symmetric, subpleural), OR pulmonary function tests showing impaired diffusion capacity and restrictive ventilatory defect. All patients must have no improvement in symptoms like dyspnea or cough after at least 1 month of glucocorticoid therapy (prednisone ≥1 mg/kg/day or equivalent).

Systemic Lupus Erythematosus (SLE):

(1. Diagnosed with childhood-onset SLE according to the 2012 Systemic Lupus International Collaborating Clinics (SLICC) or 2019 EULAR/ACR classification criteria for SLE; (2). Must meet one of the following adequacy of treatment conditions:

• After treatment with glucocorticoids (≥1 mg/kg/day prednisone or equivalent) and one or more immunomodulators (including cyclophosphamide, MMF, azathioprine, methotrexate, cyclosporine, tacrolimus, sirolimus, leflunomide, telitacicept, belimumab, and rituximab) for 3 months (3M);
• Patients intolerant to conventional therapy may be considered for enrollment after the investigator judges benefit outweighs risk and with full informed consent from the patient/guardian;
• OR inability to taper glucocorticoids to ≤5 mg/day after 6 months (6M) of conventional therapy; (3). Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) score >6; (4). No occurrence of macrophage activation syndrome within 1 month prior to screening.

Mixed Connective Tissue Disease (MCTD):

(5). Diagnosed with childhood-onset primary MCTD at least 24 weeks prior to signing the ICF according to the Sharp 1986 MCTD classification criteria adapted for children; (6). Meet the above childhood MCTD classification criteria, and also had Intolerance or inadequate response to glucocorticoids (prednisone 1-2 mg/kg/day or equivalent doses of other corticosteroids) and at least 2 conventional immunosuppressants, with a duration of standardized glucocorticoid treatment of at least 6 months; (7). Childhood version of the Mixed Connective Tissue Disease Activity Index (MDAI) score ≥8 at screening; (8). Patient-reported outcome (PRO) score for MCTD characteristic involvement dimensions ≥5 at screening; (9). High-titer positive for anti-U1-RNP antibody (titer ≥1:1000), and negative for anti-Sm antibody.

Exclusion Criteria:

1. . History of malignancy (except for basal cell or squamous cell skin cancer or carcinoma in situof the cervix that has been excised and cured for at least 5 years), or current malignancy.

2. . Known allergy, hypersensitivity, intolerance, or contraindication to CD19/BCMA CAR-NK cells or any component of the drugs that may be used in the study (including fludarabine, cyclophosphamide, and tocilizumab), or subjects who have experienced a severe allergic reaction in the past.

3. . Evidence of severe active viral or bacterial infection, or uncontrolled systemic fungal infection at screening or baseline visits, or subjects with active or uncontrolled infection requiring parenteral antimicrobial therapy.

4. . Subjects with cardiac insufficiency classified as Class III or IV according to the New York Heart Association (NYHA) functional classification (see Appendix).

5. . Subjects with congenital heart disease, or history of acute myocardial infarction within 6 months prior to screening, or severe arrhythmia (including multifrequent ventricular premature beats, supraventricular tachycardia, ventricular tachycardia, etc.); or combined with moderate to large pericardial effusion, severe myocarditis, etc.; or unstable vital signs requiring vasopressors to maintain blood pressure.

6. . Positive hepatitis B surface antigen (HBsAg) or hepatitis B core antibody (HBcAb) with detectable hepatitis B virus (HBV) DNA levels above the normal reference range in peripheral blood at screening; OR positive hepatitis C virus (HCV) antibody with detectable HCV RNA levels above the normal reference range; OR positive human immunodeficiency virus (HIV) antibody; OR positive syphilis test; OR positive cytomegalovirus (CMV) DNA test.

7. . History of severe herpes infection, such as herpes encephalitis, ocular herpes, or disseminated herpes; signs of herpes or varicella-zoster virus infection (particularly varicella, herpes zoster) within 12 weeks prior to screening.

8. . Current active tuberculosis or history of active tuberculosis, or subjects whose interferon-gamma release assay for tuberculosis infection cannot yield a negative result during the screening period.

9. . Subjects with interstitial lung disease (ILD), meeting any of the following conditions are excluded: Forced vital capacity (FVC) <50% of predicted value at screening, OR diffusing capacity of the lungs for carbon monoxide (DLCO) <40% of predicted value; Requiring long-term oxygen therapy or non-invasive ventilation; Acute exacerbation of interstitial pneumonia/acute respiratory failure within the past 6 months, or hospitalization due to ILD requiring intravenous pulse corticosteroid therapy; Rapidly progressive ILD as judged by the investigator.

10. . Subjects with pulmonary arterial hypertension (PAH), meeting any of the following conditions are excluded: Results from right heart catheterization or echocardiography (non-invasive estimation) meeting any of the following: a. Estimated systolic pulmonary artery pressure (sPAP) >50 mmHg (assessed in conjunction with tricuspid regurgitation velocity); b. Diagnosis of WHO functional class III or IV PAH.

    Hospitalization within the past 6 months due to acute exacerbation of PAH or right heart failure; Requiring intravenous prostacyclin analog therapy during the screening period; Presence of signs of right ventricular dysfunction, including but not limited to ascites, hepatic congestion, peripheral edema, accompanied by significantly elevated BNP/NT-proBNP levels and clinically unstable symptoms.

11. . History of epilepsy or other active central nervous system diseases.

12. . Subjects with acquired or congenital immunodeficiency diseases.

13. . History of any clinically significant cardiac, endocrine, hematological, hepatic, immunological, metabolic, urological, pulmonary, neurological, dermatological, psychiatric, renal disease, or other major condition that, in the investigator's judgment, precludes the administration of KN5601.

14. . Solid organ or hematopoietic stem cell transplantation within 3 months prior to screening; OR acute graft-versus-host disease (GVHD) of grade 2 or higher within 2 weeks prior to screening.

15. . Vaccination with a live vaccine within 4 weeks prior to screening.

16. . Having received the following treatments within the specified time frames prior to the baseline visit: B cell depletion therapy within 26 weeks; Within 24 weeks prior to randomization: Anti-CD40 monoclonal antibody, belimumab, abatacept, anti-tumor necrosis factor alpha (anti-TNFα) biologics, immunoglobulin, plasmapheresis; Within 12 weeks prior to randomization: JAK inhibitors or other kinase inhibitors, unless explicitly permitted by the protocol; Use of traditional Chinese medicines, proprietary Chinese medicines, or health products containing Tripterygium wilfordii(Lei Gong Teng), Tripterygium hypoglaucum(Kunming Shan Hai Tang), Colquhounia coccineavar. mollis(Huo Ba Hua Gen), or white peony root (Paeonia lactiflora, Bai Shao) within 4 weeks; Within 3 half-lives of prior therapy, OR within 4 weeks, OR until the expected pharmacodynamic effects have returned to baseline levels (whichever is longer); B cell count below the lower limit of normal or baseline value (whichever is lower) following prior B cell depletion therapy.

17. . Participation in any clinical trial within three months.

18. . Any other condition that, in the opinion of the investigator, may increase the risk to the subject or interfere with the trial results.