Clinical Study of Chemotherapy and Immunotherapy Combined With Lactobacillus Johnsonii in Patients With Multiple Irresectable Solid Tumors

Research Objective This study is a randomized, double-blind, controlled, prospective multicenter clinical trial, designed to evaluate the clinical efficacy and safety of chemotherapy and immune therapy combined with Lactobacillus johnsonii in patients with various advanced unresectable solid tumors.

Study Methods:

Design: Randomized, controlled, double-blind, multicenter clinical study. Duration: 6-12 months. Start Date: Upon ethical approval and completion of clinical trial registration.

Study Design:

1. Overall Design:

A prospective, multicenter, randomized controlled trial assessing the efficacy and safety of immunotherapy supplemented with Lactobacillus johnsonii in various tumor types.

Eligible patients will be randomly assigned to treatment groups after screening.

Participating Centers:

The Second Affiliated Hospital, Zhejiang University School of Medicine Zhejiang Cancer Hospital Hangzhou First People's Hospital The First Affiliated Hospital of Wenzhou Medical University 2. Sample Size Calculation:

Statistical Parameters: Two-sided test, α=0.05, power (1-β)=80%.

Sample Size per subgroup:

Subgroup A (Lung cancer): 48 cases (24 per group) Subgroup B (Breast cancer): 52 cases (26 per group) Subgroup C (Gastric/gastroesophageal junction adenocarcinoma): 56 cases (28 per group) Total Sample Size: 156 cases (calculated using PASS 15 software). 3. Randomization:

Computer-generated random sequences (1:1 allocation). Managed by independent researchers to avoid bias. Each participant receives a unique screening number (non-reusable, even if withdrawn). 4. Study Population:

Inclusion Criteria:

1. Signed informed consent. 2. Age 18-75 years. 3. Histologically confirmed unresectable/metastatic tumors:

EGFR/ALK wild-type adenocarcinoma non-small cell lung cancer (NSCLC) PD-L1+ (CPS≥1) triple-negative breast cancer (TNBC) HER2- gastric/gastroesophageal adenocarcinoma 4. ≥1 measurable lesion (RECIST v1.1). 5. Progression after PD-1/PD-L1 inhibitor therapy (defined as:

\- ≥2 doses administered. * Radiographic progression per RECIST/iRECIST/irRECIST, confirmed ≥4 weeks later. * Progression within 12 weeks of last PD-1/PD-L1 dose. 6. Adequate organ function. 7. ECOG performance status 0-1. 8. Negative pregnancy test (urine/serum β-HCG) for women of childbearing potential.

Exclusion Criteria

Subjects meeting any of the following will be excluded:

1. Prior allogeneic transplantation (cells, tissues, or solid organs). 2. History of immune-related adverse events (irAEs) from immunomodulators (e.g., PD-1/PD-L1 or CTLA-4 inhibitors) leading to permanent discontinuation or Grade 3/4 severity. 3. Recent anticancer therapy (chemotherapy/immunotherapy/biologics/experimental drugs) within: \<5 half-lives of the drug or \<21 days before starting study treatment (whichever is shorter). Exceptions: Stable hormone therapy (e.g., for prostate/breast/ovarian cancer). 4. Concomitant corticosteroid use (\>10 mg prednisone/day or equivalent) within 7 days prior to treatment, unless for physiological replacement (≤10 mg/day) or non-immunosuppressive purposes (e.g., inhaled/topical steroids). 5. Severe cardiac dysfunction, including: NYHA Class III-IV heart failure, symptomatic coronary artery disease, severe ventricular arrhythmias, myocardial infarction/unstable angina within 6 months. 6. Active autoimmune disease requiring systemic treatment (immunosuppressants/corticosteroids) in the past 2 years, except: Hormone replacement (thyroxine, insulin, physiologic corticosteroids). 7. Active severe infection requiring systemic therapy. 8. Antibiotic use completed within 2 weeks before the first dose. 9. Psychiatric or substance abuse disorders compromising protocol compliance. 10. Live vaccines administered within 28 days before treatment. 11. Active HIV or hepatitis (A/B/C) infection. 12. History of steroid-requiring (non-infectious) pneumonitis or current active pneumonitis. 13. Other malignancies requiring active treatment or progressing within 2 years (exceptions: Non-melanoma skin cancer, cervical/prostate carcinoma in situ). 14. Pregnancy or lactation. 15. Known intolerance/allergy to study drugs. 16. Other conditions deemed unsuitable by investigators.

3\. Subject Discontinuation/Withdrawal

\- Pre-randomization withdrawal: Considered a screen failure; reasons must be documented in medical records.

* Post-enrollment withdrawal: * Subjects may withdraw at any time without penalty or loss of benefits. * Reasons (if provided) should be recorded (e.g., intolerable AEs, consent withdrawal). * Protocol-defined discontinuation if:

1\. Use of prohibited immunomodulatory drugs. 2. Pregnancy or new severe illness. 3. Intolerable toxicity. 4. Subject/investigator decision. 5. Other reasons hindering continued participation. 5. Investigational Drugs and Treatment Protocol 1. Source of Investigational Drugs: Lactobacillus johnsonii and matching placebo\* are provided by Hangzhou Yuanda Biopharmaceutical Co., Ltd. 2. Treatment Regimens Subjects will receive the following designed anticancer therapies every 3 weeks (unless disease progression or unacceptable toxicity occurs). Concurrently, subjects will take oral Lactobacillus johnsonii (1\*1011 CFU CFU, twice daily, dissolved in warm water) starting from the first anticancer treatment.

Tumor-Specific Chemoimmunotherapy Protocols:

Subgroup A: EGFR/ALK Wild-Type NSCLC (Unresectable/Metastatic) * tislelizumab (200 mg IV on Day 1) * pemetrexed (500 mg/m² BSA IV on Day 1) * cisplatin (75 mg/m² BSA IV on Day 1). Subgroup B: PD-L1+ (CPS ≥1) Triple-Negative Breast Cancer (TNBC) * Toripalimab (240 mg IV, Day 1) * nab-paclitaxel (260 mg/m² BSA IV on Day 1) * carboplatin (300 mg/m² BSA IV on Day 1) Subgroup C: HER2- Gastric/Gastroesophageal Junction Adenocarcinoma * Tislelizumab (200 mg IV, Day 1) * oxaliplatin (130 mg/m² BSA IV on Day 1) * capecitabine (1000 mg/m² BSA orally twice daily, taken 30 minutes after meals on Days 1-14) 3. Investigational Drug Management Accountability: Drugs are strictly for trial use; no unauthorized redistribution. Designated staff must oversee storage, dispensing, returns, and destruction.

Documentation: Logistics records (shipment, receipt, allocation). Dispensing logs (recipient, verifier, date, dose, quantity). Reconciliation of unused drugs against prescriptions/records post-use.

Disposal: Unused drugs and packaging must be returned to the sponsor for destruction.

4\. Permitted/Prohibited Concomitant Medications Allowed: Medications for non-excluded conditions (if stable at baseline). Prohibited: Antibiotics, other probiotics, herbal medicines, or CoQ10 (may interfere with Lactobacillus johnsonii assessment).

6\. Study Procedures and Follow-up Plan Study Population: Patients with various tumors (lung cancer, breast cancer, gastric cancer) scheduled to receive immune checkpoint inhibitors. A total of 156 subjects will be enrolled across 4 medical centers (48 lung cancer, 52 breast cancer, 56 gastric cancer).

Screening Phase: Collection of medical history, demographic data, tumor stage/grade, pathological type, vital signs, physical examination, and pre-enrollment lab tests. Eligible subjects proceed to the treatment phase.

Treatment Phase: Daily administration of the study treatment per protocol. Scheduled visits for efficacy and safety assessments, including medication records, adverse events (AEs), and concomitant medications.

Follow-up Schedule:

* Timepoints: Baseline (Week 0), Weeks 6, 12, 18, and 24. * Treatment regimen: Chemotherapy combined with immunotherapy (21-day cycles). * Tumor response assessed every 2 cycles (42 days/6 weeks). * Chemotherapy discontinued after 8 cycles (6 months).

Follow-up Assessments:

Biological samples: Fecal and plasma collection at Weeks 0, 6, 12, 18, and 24 for:

* Gut microbiota composition (e.g., Lactobacillus johnsonii colonization). * Indole derivative concentrations.

Clinical evaluations:

* Tumor progression (RECIST criteria). * Treatment-related adverse events (TRAEs). * Concomitant medications. * Quality of life (QoL) scores. 7. Study Endpoints

Primary Endpoint:

• Secondary Efficacy Endpoints [Срок оценки: From enrollment to the end of treatment at 24 weeks]