A Clinical Study of AFN50 in the Treatment of Autoimmune Diseases

The study employs a "3+3" dose-escalation design with a total of 9-18 subjects enrolled. The objective is to evaluate the safety, tolerability, and primary efficacy of AFN50 injection in relapsing and refractory autoimmune disease. The primary endpoint is the incidence and severity of treatment-related adverse events, with a total follow-up period of approximately 24 months.

Inclusion criteria

• General Inclusion Criteria 1.1 Must be capable of understanding and voluntarily providing written informed consent.

1.2 Age 18 to 69 years (inclusive), any gender. 1.3 Adequate bone marrow, coagulation, cardiac, pulmonary, hepatic, and renal function at screening:

Bone Marrow Function:

• Absolute neutrophil count (ANC) ≥1.0 × 10⁹/L (no use of granulocyte colony-stimulating factor (G-CSF) within 7 days before screening; for long-acting G-CSF, a 14-day interval is required); ② Haemoglobin (Hb) ≥90 g/L (no red blood cell transfusion within 14 days before screening; use of recombinant human erythropoietin is permitted); ③ Platelet count (PLT) ≥75 × 10⁹/L; absolute lymphocyte count (ALC) ≥0.5 × 10⁹/L.

Coagulation Function: International normalised ratio (INR) or activated partial thromboplastin time (APTT) ≤1.5 × the upper limit of normal (ULN).

Cardiac Function: Left ventricular ejection fraction (LVEF) ≥50% as shown by echocardiography (ECHO).

Pulmonary Function: Dyspnea ≤ CTCAE Grade 1, and pulse oxygen saturation (SpO₂) >92% on room air.

Hepatic Function: Alanine aminotransferase (ALT), aspartate aminotransferase (AST) ≤2.5 × ULN; total bilirubin ≤1.5 × ULN.

Renal Function: Creatinine clearance (Cockcroft-Gault formula) ≥50 mL/min, without requiring fluid support.

1.4 Baseline oxygen saturation >92% without supplemental oxygen. 1.5 Non-pregnant/non-lactating subjects. Women of childbearing potential must have a negative serum or urine pregnancy test report (women who have undergone surgical sterilisation or are postmenopausal for at least 2 years are not considered to be of childbearing potential) and must be willing to use contraception for 12 months following drug infusion.

Disease-Specific Inclusion Criteria：

• Relapsed/Refractory Systemic Lupus Erythematosus (SLE) 1.1 Meet the 2019 European Alliance of Associations for Rheumatology/American College of Rheumatology (EULAR/ACR) classification criteria for SLE.

1.2 SLEDAI-2K score ≥6; if the score includes low complement and/or anti-dsDNA antibodies, the SLEDAI-2K clinical symptom score after excluding these two items must be ≥4.

1.3 History of SLE for at least 6 months, with disease remaining active or relapsing despite receiving stable standard therapy for at least 8 weeks (drug doses stable for the past 2 weeks).

1.4 Oral glucocorticoids (prednisone or equivalent) at a daily dose ≥7.5 mg and ≤30 mg; if combined with immunosuppressants, there is no minimum daily dose requirement.

1.5 At least two immunosuppressants (including hydroxychloroquine) have been used in a standardised manner.

1.6 Screening tests meet: positive serum antinuclear antibody (ANA), and/or positive anti-double-stranded DNA (anti-dsDNA) antibody, and/or hypocomplementemia (low C3 and/or low C4).

1.7 Definition of relapsed/refractory: Ineffective to conventional therapy or recurrence of disease activity after remission. Definition of conventional therapy: Stable use, alone or in combination, of the following drugs: non-steroidal anti-inflammatory drugs (NSAIDs), antimalarials, glucocorticoids, immunosuppressants (including but not limited to cyclophosphamide, methotrexate, azathioprine, mycophenolate mofetil, leflunomide, tacrolimus, cyclosporine), targeted drugs (including but not limited to belimumab, telitacicept, eculizumab, rituximab).

• Relapsed/Refractory Sjögren's Syndrome (SS) 2.1Meet the 2002 AECG criteria or the 2016 ACR/EULAR classification criteria for primary Sjögren's Syndrome.

2.2 Definition of relapsed/refractory: Ineffective to conventional therapy or recurrence of disease activity after remission. Definition of conventional therapy: Use of glucocorticoids (≥1 mg/kg/day) and cyclophosphamide, plus any one or more of the following immunomodulatory drugs for >6 months: antimalarials, azathioprine, mycophenolate mofetil, methotrexate, leflunomide, tacrolimus, cyclosporine, and biologics including rituximab, belimumab, telitacicept, etc.

• Refractory Myasthenia Gravis (MG) 3.1 Meet the diagnostic criteria for generalized myasthenia gravis as per the Chinese guidelines for the diagnosis and treatment of MG (2025 edition).

3.2 Myasthenia Gravis Foundation of America (MGFA) clinical classification: Type II, III, or IV.

3.3 Positive serology for acetylcholine receptor antibody (AChR-Ab), or muscle-specific tyrosine kinase antibody (MuSK-Ab), or low-density lipoprotein receptor-related protein 4 antibody (LRP4-Ab) at screening, or documented history of positive AChR-Ab, MuSK-Ab, or LRP4-Ab.

3.4 Myasthenia Gravis Activities of Daily Living (MG-ADL) score ≥6, with ocular-related scores comprising less than 50% of the total score.

3.5 Myasthenia Gravis (QMG) score ≥8, with at least 4 individual item scores being ≥2 points.

3.6 Definition of refractory: Ineffective to conventional therapy or disease reactivation following remission.

• Relapsed/Refractory or Progressive Diffuse Cutaneous Systemic Sclerosis (dcSSc) 4.1 Meet the 2013 ACR classification criteria for systemic sclerosis, with diffuse cutaneous involvement.

4.2 Presence of interstitial lung disease: evidence of interstitial changes with ground-glass opacity on chest HRCT and forced vital capacity (FVC) or diffusing capacity of the lung for carbon monoxide (DLCO) <70% of predicted value on pulmonary function tests.

4.3 Definition of relapsed/refractory: Ineffective to conventional therapy or disease reactivation following remission.

4.4 Definition of conventional therapy: Use of glucocorticoids (≥1 mg/kg/day) and cyclophosphamide, plus any one or more of the following immunomodulatory drugs for >6 months: antimalarials, azathioprine, mycophenolate mofetil, methotrexate, leflunomide, tacrolimus, cyclosporine, and biologics including rituximab, belimumab, telitacicept, etc.

4.5 Definition of progressive disease:

• Skin progression: Increase in modified Rodnan skin score (mRSS) >10%.
• Lung disease progression: Decrease in FVC by 10%, or decrease in FVC by 5% accompanied by a decrease in DLCO by 15% (OMERACT progression).
• Relapsed/Refractory or Progressive Idiopathic Inflammatory Myopathy (IIM) 5.1 Meet the 2017 EULAR/ACR classification criteria for idiopathic inflammatory myopathies (including DM, PM, ASS, and NM).

5.2 For patients with muscle involvement: Manual Muscle Testing-8 (MMT-8) score below 142 and at least two abnormal findings among the following five core measures (Physician Global Activity (PhGA), Patient Global Activity (PtGA) or extra-muscular disease activity score ≥2; Health Assessment Questionnaire (HAQ) total score ≥0.25; muscle enzyme levels 1.5 times the upper limit of normal).

5.3 Positive myositis antibodies. 5.4 Definition of relapsed/refractory: Ineffective to conventional therapy or recurrence of disease activity after remission. Definition of conventional therapy: Use of glucocorticoids (≥1 mg/kg/day) and cyclophosphamide, plus any one or more of the following immunomodulatory drugs for >6 months: antimalarials, azathioprine, mycophenolate mofetil, methotrexate, leflunomide, tacrolimus, cyclosporine, and biologics including rituximab, belimumab, telitacicept, etc.

5.5 Definition of progressive disease: Rapidly progressive interstitial pneumonia occurring within a short period.

• Relapsed/Refractory Anti-Neutrophil Cytoplasmic Antibody (ANCA)-Associated Vasculitis (AAV) 6.1 Meet the 2022 ACR/EULAR diagnostic criteria for ANCA-associated vasculitis, including microscopic polyangiitis, granulomatosis with polyangiitis, and eosinophilic granulomatosis with polyangiitis.

6.2 Positive ANCA-related antibodies (MPO-ANCA or PR3-ANCA). 6.3 Birmingham Vasculitis Activity Score (BVAS) ≥15 points (total 63 points), indicating active vasculitis.

6.4 The BVAS assessment must include at least one major item, at least three minor items, or at least two renal items (hematuria and proteinuria).

6.5 Definition of relapsed/refractory: Ineffective to conventional therapy or recurrence of disease activity after remission. Definition of conventional therapy: Use of glucocorticoids (≥1 mg/kg/day) and cyclophosphamide, plus any one or more of the following immunomodulatory drugs for >6 months: antimalarials, azathioprine, mycophenolate mofetil, methotrexate, leflunomide, tacrolimus, cyclosporine, and biologics including rituximab, belimumab, telitacicept, etc.

Exclusion criteria

• General Exclusion Criteria 1.1 Individuals with positive hepatitis B surface antigen (HBsAg) and/or hepatitis B core antibody (HBcAb) with detectable hepatitis B virus (HBV) DNA or titer above the detection threshold; positive hepatitis C virus (HCV) antibody with detectable HCV RNA or titer above the detection threshold; positive human immunodeficiency virus (HIV) antibody; detectable cytomegalovirus (CMV) DNA or level above the detection limit; positive syphilis antigen or antibody.

1.2 Presence of other uncontrolled active infections. 1.3 History of major organ transplantation (e.g., heart, lung, liver, kidney) or bone marrow/hematopoietic stem cell transplantation.

1.4 Received any mRNA-LNP product or other LNP-based therapy within the past 2 years and has a history of allergy to LNPs or their components.

1.5 Received live vaccination within the past 30 days. 1.6 History of any severe cardiovascular disease within 6 months prior to screening, including: New York Heart Association (NYHA) Class III or IV heart failure, myocardial infarction, unstable angina, uncontrolled or symptomatic atrial arrhythmia, any ventricular arrhythmia, or other clinically significant cardiac disease.

1.7 Pregnant or lactating women. 1.8 Individuals with asthma or a history of severe allergies. 1.9 According to the investigator's judgment, the individual is unlikely to complete all protocol-required study visits or procedures, including follow-up or compliance with study participation requirements.

1.10 Other unspecified reasons that, in the opinion of the investigator, render the patient unsuitable for enrollment.

Disease-Specific Exclusion Criteria

• Relapsed/Refractory Systemic Lupus Erythematosus: Concurrent neuropsychiatric lupus; thrombotic thrombocytopenic purpura (TTP)/microangiopathy (TMA).
• Relapsed/Refractory Sjögren's Syndrome: Concurrent liver cirrhosis; concurrent aplastic anaemia (AA), myelodysplastic syndrome (MDS), or other myeloproliferative disorders (MPD); drug-induced thrombocytopenia; TTP/TMA.
• Relapsed/Refractory Myasthenia Gravis: Presence of an uncontrolled myasthenic crisis within 2 weeks prior to screening.
• Relapsed/Refractory or Progressive Diffuse Cutaneous Systemic Sclerosis: NYHA Class IV cardiac function; presence of moderate to severe pulmonary hypertension (mean pulmonary artery pressure >40 mmHg by echocardiography); FVC <45% of predicted value; DLCO <40% of predicted value; significant abnormalities on HRCT not attributable to SSc; persistent unexplained hematuria (>5 red blood cells per high-power field) or creatinine clearance <40 mL/min; prior history of autologous stem cell transplantation; signs of renal crisis; active gastric antral vascular ectasia.
• Relapsed/Refractory or Progressive Idiopathic Inflammatory Myopathy: Drug-induced myositis; inclusion body myositis; cancer-associated myositis (myositis occurring within 2 years of cancer diagnosis).
• Relapsed/Refractory ANCA-Associated Vasculitis:

Estimated glomerular filtration rate (eGFR) <15 mL/min/1.73 m²; if the participant has alveolar haemorrhage requiring invasive mechanical ventilation expected to last beyond the screening period; requiring dialysis or plasma exchange during the screening period; or prior kidney transplantation.