A Study of BEN301 Injection in theTreatment of Autoimmune Diseases

A Phase I Exploratory Study on the Safety and Efficacy of BEN301 Injection in the Treatment of Autoimmune Diseases

The study aims to investigate the safety, tolerability, and preliminary clinical efficacy of BEN301 Injection in patients with autoimmune diseases. In patients with autoimmune diseases, Treg cells are typically deficient or dysfunctional. CAR-Treg cell therapy represents a promising strategy for the treatment of autoimmune diseases and may be applicable to a broad spectrum of autoimmune conditions. Preclinical studies have shown that BEN301 Injection not only effectively suppresses the aberrant activation of T and B cells in SLE models, but also significantly reduces total lgG secretion and the production of SLE-specific autoantibodies, particularly anti-double-stranded DNA (dsDNA) antibodies. Moreover, no significant treatment-related toxicities were observed. Treg cell therapy has already demonstrated favorable efficacy in multiple indications, including organ transplantation and autoimmune diseases, with a well-established safety and tolerability profile. Meanwhile, CAR-Treg cell therapy has been actively explored in various autoimmune conditions; several products have shown therapeutic potential, and some patients have already benefited from treatment. These findings highlight the promising prospects of CAR-Treg cell therapy in the management of autoimmune diseases.

Inclusion Criteria:

1. Voluntary participation in the study, providing written informed consent, and willingness and ability to comply with the protocol.
2. Age 18-70 years (inclusive) at the time of signing informed consent.
3. Expected survival ≥ 12 months.
4. Disease-Specific Criteria

1. Systemic Sclerosis (SSc)

   • Diagnosis of Systemic Sclerosis according to the 2013 ACR/EULAR Classification Criteria for SSc, with a total score ≥ 9.
   • Disease duration (defined as time from onset of first non-Raynaud's phenomenon manifestation) ≤ 6 years. If disease duration > 6 years at screening, the participant may be enrolled if the investigator determines potential benefit and following discussion with the medical monitor of the BEN301 manufacturing site.
   • Evidence of active disease at screening meeting at least one of the following criteria: o Modified Rodnan Skin Score (mRSS) ≥ 10 and ≤ 35;
   • Increase in mRSS ≥ 3 points compared to the most recent assessment within 6 months;o Increase in mRSS ≥ 2 points with involvement of a new body region compared to the most recent assessment within 6 months;
   • Increase in mRSS ≥ 1 point with involvement of two new body regions within 6 months;
   • Progressive Interstitial Lung Disease (ILD) meeting the following standards: Inadequate response or intolerance to at least one prior treatment (including cyclophosphamide, methotrexate, mycophenolate/mycophenolic acid, nintedanib, rituximab, or tocilizumab);
   • AND at least one of the following:Evidence of progression on High-Resolution Computed Tomography (HRCT); ORForced Vital Capacity (FVC) < 80% predicted, Diffusing Capacity of the Lung for Carbon Monoxide (DLCO) < 80% predicted, evidence of FVC decrease (absolute) ≥ 10%, or FVC decrease 5%-9% with DLCO decrease ≥ 15%.• Inadequate response or intolerance to conventional therapies (glucocorticoids and immunosuppressants).

2. Rheumatoid Arthritis (RA)

   • Diagnosis of RA according to the 1987 ACR or 2010 ACR/EULAR classification criteria.
   • Inadequate response, intolerance, or contraindication to at least one conventional synthetic DMARD (csDMARD) and at least one targeted synthetic DMARD (tsDMARD) or biologic DMARD (bDMARD), failing to achieve remission or low disease activity.
   • Moderate-to-severe active RA at screening, defined as ≥ 4 tender joints (of 68 assessed) and ≥ 4 swollen joints (of 66 assessed).
   • Erythrocyte Sedimentation Rate (ESR) > 28 mm/hr and/or C-reactive protein (CRP) > 10 mg/L.• Positive Rheumatoid Factor (RF) and/or Anti-Citrullinated Protein Antibody (ACPA).• Duration of RA diagnosis > 6 months.

3. Primary Sjögren's Syndrome (pSS)

   • Meets the 2016 ACR/EULAR Classification Criteria for Sjögren's Syndrome.• High disease activity index: ESSDAI ≥ 6 points.
   • Inadequate response or intolerance to conventional therapies (glucocorticoids and immunosuppressants).
   • Positive for Anti-Sjögren's Syndrome A (SSA) and/or Anti-Sjögren's Syndrome B (SSB) antibodies.

4. Idiopathic Inflammatory Myopathies (IIM)

   • Suspected or confirmed diagnosis of Polymyositis (PM) or Dermatomyositis (DM) according to the 2017 EULAR/ACR Classification Criteria.

   • Moderate-to-severe PM or DM, defined as meeting both Part A and Part B below: Part A: Manual Muscle Testing (MMT-8) total score < 142. Part B: At least 2 of the following 5 items: Physician Global Assessment (PhGA, 10-cm VAS) ≥ 2 cm; Patient Global Assessment (PtGA, 10-cm VAS) ≥ 2 cm; Health Assessment Questionnaire-Disability Index (HAQ-DI) > 0.25; Elevation of any muscle enzyme (CK, LDH, AST, ALT) ≥ 1.3 × Upper Limit of Normal (ULN); Myositis Disease Activity Assessment Tool (MDAAT) global extramuscular activity (10-cm VAS) ≥ 2 cm.

   • Alternatively, presence of active disease: evidence of active muscle inflammation on MRI or muscle biopsy within 12 weeks.

     5. Adequate Organ Function Within 7 days prior to screening (without transfusion, hematopoietic growth factors within 2 weeks, or medication correction):

   • Bone Marrow: o Hemoglobin (Hb) ≥ 80 g/L;o Absolute Neutrophil Count (ANC) ≥ 1.5 × 10⁹/L;o Platelet count (PLT) ≥ 50 × 10⁹/L.• Renal Function: Creatinine Clearance (CrCl, calculated by Cockcroft-Gault formula, see Appendix 1) ≥ 50 mL/min (without hydration assistance).

   • Hepatic Function: Serum ALT and AST ≤ 2.5 × ULN (exceptions for CK-induced elevations may be determined by the investigator); Total bilirubin ≤ 1.5 × ULN.• Pulmonary Function: Oxygen saturation ≥ 92% on room air without supplemental oxygen; no clinically significant pleural effusion.

   • Cardiac Function: Left ventricular ejection fraction (LVEF) ≥ 40%; no pericardial effusion; ECG at screening showing no clinically significant abnormalities.

   • Coagulation: Fibrinogen ≥ 1.5 g/L; Activated Partial Thromboplastin Time (APTT) ≤ 1.5 × ULN; Prothrombin Time (PT) ≤ 1.5 × ULN.4. Biological Requirements

   • Presence of CD19+ B cells in peripheral blood.• Adequate venous access for leukapheresis, without contraindications to leukapheresis.5. Reproductive Criteria

   • Women of childbearing potential (WOCBP) must have a negative serum pregnancy test at screening. (Women who are surgically sterile or postmenopausal for at least 2 years are considered non-childbearing).

   • Sexually active WOCBP and male participants must agree to use highly effective contraception from the time of signing informed consent until 1 year after the last study treatment, and must not donate eggs/sperm for assisted reproduction during this period.

Exclusion Criteria:

1. Severe Renal Disease

   • History of severe nephritis or nephrotic syndrome.
   • Severe is defined as:

   Prior receipt of any of the following treatments within specified timeframes: Prior kidney transplantation; Dialysis or plasmapheresis within 3 months prior to screening; Glucocorticoid pulse therapy (defined as ≥500 mg/day prednisone or equivalent) within 1 month prior to screening.Requirement for any of the following treatments during the study period: Use of prohibited medications per protocol; Need for dialysis or plasmapheresis; Need or planned kidney transplantation.

2. Severe Cardiovascular Disease

   • History of severe cardiovascular disease, including but not limited to: symptomatic chronic heart failure requiring intervention, acute myocardial infarction or coronary artery bypass grafting within 6 months, unstable angina, history of severe arrhythmia, or history of stroke.
   • New York Heart Association (NYHA) Class III-IV heart failure.• QTcF interval > 450 ms (male) or > 470 ms (female) on screening ECG (Fridericia formula: QTcF = QT / (RR^0.33)).

3. SSc-Specific Exclusions• Receipt of oral, intramuscular, or intravenous glucocorticoids (>10 mg/day prednisone or equivalent) within 1 week prior to leukapheresis. (Note: Investigators may consider dose reduction prior to leukapheresis/infusion provided the participant's disease is controlled).

   • Receipt of immunosuppressants within 1 week prior to leukapheresis, including but not limited to methotrexate, hydroxychloroquine, cyclosporine A, azathioprine, mycophenolate, sirolimus, colchicine, or D-penicillamine.

4. RA-Specific Exclusions • Use of anti-TNF agents within 8 weeks prior to dosing (or 4 weeks for etanercept).

   • Use of any JAK inhibitor within 2 weeks prior to dosing.
   • Use of other biologics or non-B-cell depleting investigational drugs within fewer than 5 half-lives prior to dosing.

5. pSS-Specific Exclusions

   • Diagnosis of secondary Sjögren's syndrome (defined as overlap with other autoimmune or systemic inflammatory diseases, e.g., RA, SLE, scleroderma, or idiopathic inflammatory myopathy).
   • Severe systemic involvement assessed by the investigator, including: o Serious vasculitis involving kidneys, digestive system, heart, lungs, or CNS (excluding cutaneous vasculitis);o Active CNS or PNS involvement requiring high-dose glucocorticoids;o Severe renal involvement (e.g., GFR < 60 mL/min);o Severe pulmonary involvement (e.g., dyspnea at rest, or FVC < 60% or DLCO < 40%);o Myopathy requiring high-dose glucocorticoids;o Lymphoma.
   • Use of sodium hyaluronate eye drops, artificial tears, artificial saliva, or sialogogues (e.g., pilocarpine) within 7 days prior to dosing; use of immunosuppressants (e.g., cyclophosphamide, leflunomide, methotrexate, azathioprine) within 4 weeks prior to dosing; use of JAK inhibitors or other kinase inhibitors within 2 weeks prior to dosing; use of targeted biologics (e.g., Telitacicept, Belimumab, Abatacept, Adalimumab) within 6 weeks prior to dosing.
   • Requirement for medications causing dry mouth/dry eyes during the study.

6. IIM-Specific Exclusions

   • Participants with a record of Inclusion Body Myositis (IBM), juvenile myositis, drug-induced PM/DM, cancer-associated PM/DM (defined as cancer diagnosed within 3 years of PM/DM diagnosis), or non-inflammatory myopathies (e.g., muscular dystrophies).

   • Patients with PM/DM who have a high risk of malignancy.• Permanent muscle weakness due to causes other than PM/DM (e.g., stroke) as judged by the investigator.• Any of the following prior treatments/procedures: o Topical corticosteroids or topical immunomodulators (e.g., tacrolimus) for IIM-associated rash within ≤ 1 week prior to dosing;o JAK inhibitors within 2 weeks prior to dosing;o Cyclophosphamide, rituximab, or other anti-CD20 antibodies within 12 weeks prior to dosing;o Other investigational products within 4 weeks or 5 half-lives (whichever is longer) prior to dosing;o Combination therapy with >1 immunosuppressant, >1 antimalarial, or immunosuppressants plus antimalarials within 2 weeks prior to dosing.

7. Other Autoimmune Diseases

   • History of other autoimmune diseases excluding the target indications, including Eosinophilic Granulomatosis with Polyangiitis (EGPA), Henoch-Schönlein purpura, cryoglobulinemic vasculitis, anti-glomerular basement membrane disease, Behçet's disease, or Takayasu arteritis.

8. Malignancy

   • History of malignancy within 5 years prior to enrollment, including patients with tumor-associated PM/DM. Exceptions include: surgically removed and cured non-melanoma skin cancer, cervical carcinoma in situ, localized prostate cancer, low-stage bladder cancer, ductal carcinoma in situ of the breast, or patients cured with no evidence of recurrence in the past 2 years requiring no treatment.

9. Hypersensitivity

   • Known allergy, hypersensitivity, intolerance, or contraindication to BEN301 or any component of the study medications; or history of severe allergic reactions.

10. Prior Cell Therapy• Prior receipt of autologous/allogeneic hematopoietic stem cell transplantation or CAR-T cell therapy.

11. Recent Procedures/Vaccinations• Major surgery, live vaccine administration, or participation in other clinical trials within 1 month prior to screening.

12. Active Infection

    • Uncontrolled active infection requiring systemic antibiotics, antivirals, or antifungals within 2 weeks prior to screening (prophylactic treatment excluded); active tuberculosis (excluded if T-SPOT positive).

13. Viral Serology

    • Positive serology for Hepatitis B (HBsAg positive regardless of HBV-DNA level), Hepatitis C (HCVAb positive), HIV (HIV antibody positive), or Syphilis (confirmed TPPA positive) at screening.

14. Prohibited Medications• Use of medications/treatments prohibited by the protocol during screening.

15. Investigator's Discretion• Any other condition deemed unsuitable for study participation by the investigator, such as certain psychiatric disorders, dementia, suicidal ideation, or poor compliance.