Exploratory study to evaluate the combined effect of CBM588: Clostridium butyricum in platinum plus pemetrexed with pembrolizumab for non-small cell lung cancer—protocol of MIYABRO study

Introduction

In selecting systemic therapy for patients with non-small cell lung cancer (NSCLC), the presence or absence of oncogenic driver alterations and the programmed death-ligand 1 (PD-L1) tumor proportion score (TPS) are critically important factors. In driver oncogene mutation-negative NSCLC, treatment strategies are generally determined according to the PD-L1 TPS. Combination therapy with cytotoxic chemotherapy and immune checkpoint inhibitors (ICIs) is an important therapeutic option, particularly for NSCLC with low-to-intermediate PD-L1 expression. For non-squamous NSCLC, the global phase III KEYNOTE-189 trial demonstrated that the addition of pembrolizumab to platinum plus pemetrexed prolonged progression-free survival (PFS) (8.8 months; 95% confidence interval: 7.6–9.2) and overall survival (OS) (22.0 months; 95% confidence interval: 19.5–24.5) compared with platinum plus pemetrexed and placebo. Accordingly, this regimen is regarded as the standard first-line chemotherapy for this patient population. In the subgroup analysis of this clinical trial, consistent clinical benefit was also demonstrated in patients with a PD-L1 TPS of 0% and those with a TPS of 1–49%, thereby supporting the position of this regimen as a standard-of-care treatment option (1). However, not all patients derive clinical benefits from this regimen, and the identification of predictive biomarkers remains an important unmet need. Although tumor mutational burden (2) and microsatellite instability (3) have been reported as biomarkers associated with the response to pembrolizumab, reliable biomarkers capable of predicting the efficacy of platinum plus pemetrexed and pembrolizumab remain limited, representing a key clinical challenge.

In recent years, the interplay between the gut microbiota and host immunity has attracted increasing attention across a wide range of diseases. Although the existence of the gut microbiota has been recognized for over a century, recent advances in genomic sequencing technologies have enabled its comprehensive characterization and analysis (4). More recently, the diversity of the gut microbiota has been shown to be closely associated with antitumor immunity and is now considered an emerging hallmark of cancer (5). It influences cancer progression, antitumor immune responses, and therapeutic efficacy. The composition and abundance of microbial taxa vary substantially among individuals, and the microbiota may either promote or suppress tumor development, depending on its specific configuration. In the context of tumor immunity, previous studies have reported distinct microbiome profiles across tumor types. Moreover, microbial composition and function have been associated with the cancer type, molecular subtype, smoking history, and responsiveness to therapy (6). Furthermore, specific bacterial taxa have been shown to exert substantial effects on the efficacy of ICIs and cytotoxic chemotherapy, as well as on the severity of treatment-related adverse events. These findings expand the application of microbiota-targeted therapeutic strategies in oncology (4).

CBM588, a live biotherapeutic preparation containing Clostridium butyricum MIYAIRI, a butyrate-producing bacterium, is widely used to treat abdominal symptoms associated with intestinal dysbiosis. CBM588 is commercially available in Japan as MIYA-BM^®, which is manufactured by Miyarisan Pharmaceutical Co., Ltd., and is an approved medicinal product in Japan. The active microorganism is Clostridium butyricum MIYAIRI 588. The organism is formulated in its spore form, which enables it to tolerate exposure to gastric fluid and bile, reach the intestine, and subsequently germinate. Recently, several clinical trials have investigated the potential synergistic effects of CBM588 in combination with ICIs. In a phase I clinical trial evaluating changes in the gut microbiota induced by the addition of CBM588 to first-line ipilimumab plus nivolumab in patients with renal cell carcinoma, exploratory secondary analyses showed higher objective response rates and prolonged PFS and OS in the CBM588 combination group (7). The addition of CBM588 to cabozantinib plus nivolumab is associated with favorable objective response rates and PFS (8). Thus, combination therapy using ICIs and CBM588 has attracted increasing attention as a microbiota-targeted therapeutic strategy for cancer treatment.

In this single-arm phase II clinical study, we will explore the efficacy of adding CBM588 to platinum plus pemetrexed and pembrolizumab, as well as its association with gut microbiota. Gut microbiota analyses will assess the baseline microbial diversity and predominant bacterial taxa before treatment initiation. Longitudinal changes in the gut microbiota will also be evaluated before the initiation of the protocol treatment, cycle 3, and cycle 5. We present this article in accordance with the SPIRIT reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0742/rc).

Methods

Study design

This is a phase II, multi-institutional in Japan, single-arm, interventional, and exploratory study in treatment-naïve patients with non-squamous NSCLC and with PD-L1 TPS <50%. We plan to enroll 20 patients and evaluate the relationship between the effect of platinum (cisplatin or carboplatin) + pemetrexed + pembrolizumab with CBM588 and analysis of the gut microbiome (Figure 1). This study was registered in the Japan Registry of Clinical Trials (No. jRCT1071260004) on 6 April 2026. The study will be conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Clinical Review Board of Nagasaki University (No. CRB25-011). The investigators will explain the concept of the trial to the patients and obtain written informed consent from all participating patients.

Figure 1 Treatment schedule and timing of sample collection. Platinum (carboplatin or cisplatin) is administered during the initial treatment course in combination with pemetrexed and pembrolizumab. CBM588 was administered continuously throughout the protocol. *, stool samples are collected at predefined time points to evaluate longitudinal changes in the gut microbiota. ECOG PS, Eastern Cooperative Oncology Group performance status; NSCLC, non-small cell lung cancer; PD-L1, programmed death-ligand 1; TPS, tumor proportion score.

Inclusion criteria

• Histological or cytological confirmation of non-squamous NSCLC.
• Previously untreated advanced or recurrent non-squamous NSCLC. The presence or absence of measurable lesions is not required.
• Presence of diarrhea, abnormal bowel habits, appetite loss, abdominal pain, abdominal bloating, nausea, or vomiting at the time of informed consent.
• Age ≥18 years at the time of informed consent.
• Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.
• No active multiple primary malignancies.
• Laboratory values obtained within 14 days prior to enrollment (including the same day of the week within 2 weeks before informed consent) meeting all of the following criteria:
  • Absolute neutrophil count ≥1,500/µL.
  • Platelet count ≥100,000/µL.
  • Total bilirubin ≤1.5 mg/dL.
  • Aspartate aminotransferase ≤100 U/L (≤200 U/L in patients with liver metastases).
  • Alanine aminotransferase ≤100 U/L (≤200 U/L in patients with liver metastases).
  • Creatinine clearance ≥45 mL/min.
  • Oxygen saturation (SpO₂) ≥92% on room air.
• PD-L1 (22C3) TPS <50%.
• Negative for activating EGFR mutations (exon 19 deletion, exon 21 L858R mutation, G719X mutation, L861Q mutation, S768I mutation, and these mutations with concomitant T790M mutation) and negative or unknown status for ALK fusion, ROS1 fusion, BRAF (V600E) mutation, MET exon 14 skipping mutation, and RET fusion (unknown status includes untested cases, inadequate specimens, or unavailable test results at enrollment). Patients positive for NTRK fusion, KRAS G12C mutation, or HER2 mutation were eligible for inclusion in this study.
• No autoimmune disease and no history of chronic or recurrent autoimmune disease.
• Written informed consent for participation in this study obtained from the patient.

Exclusion criteria

• Stage IA to stage IIIA disease amenable to lobectomy.
• Subjective symptoms due to pericardial effusion.
• Symptoms of superior vena cava syndrome requiring urgent radiotherapy.
• Serious cardiac disease, such as uncontrolled angina, myocardial infarction within 3 months, or heart failure.
• Uncontrolled diabetes mellitus.
• Uncontrolled hypertension.
• Infection requiring systemic treatment.
• Women who are pregnant, possibly pregnant, within 28 days postpartum, or breastfeeding; or men who wish their partner to become pregnant.
• Psychiatric disease or symptoms that interfere with daily life and are judged to make participation in this study difficult.
• Ongoing systemic corticosteroid therapy equivalent to prednisolone ≥10 mg/day or systemic immunosuppressive therapy, administered orally or intravenously.
• Positive hepatitis B surface antigen or positive hepatitis C virus antibody.
• Positive human immunodeficiency virus (HIV) antibody.
• Interstitial pneumonia, pulmonary fibrosis, severe emphysema, or a combination thereof, diagnosed by chest computed tomography.
• Administration of CBM588 within 5 days before the date of informed consent acquisition.
• Participation in a clinical trial or another clinical study.
• Any other comorbidity judged to interfere with the administration of treatment.

Treatment plan

CBM588 is administered at a dose of 120 mg/day from the initiation of treatment until the end of the protocol. Pembrolizumab, platinum, and pemetrexed are administered intravenously on day 1 of the 3-week cycles. After four cycles of induction therapy, maintenance therapy with pembrolizumab plus pemetrexed is administered until disease progression or the development of unacceptable toxicity. Pembrolizumab is administered at a dose of 200 mg every 3 weeks as induction therapy, 200 mg every 3 weeks, or 400 mg every 6 weeks as maintenance therapy. Carboplatin is administered at a calculated dose to yield an area under the concentration-time curve of 5 mg⋅mL⁻¹⋅min. Cisplatin is administered at a dose of 75 mg/m². Pemetrexed is administered at a dose of 500 mg/m².

Endpoints and evaluation of response and safety

The primary endpoints are evaluating the overall response rate (ORR) and PFS. Secondary endpoints are evaluating OS, safety profiles, and gut microbiota biomarker analyses. Tumor response is assessed according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Because this is an exploratory study, no formal statistical significance threshold has been predefined. Safety will be evaluated according to the Common Terminology Criteria for Adverse Events (CTCAE), version 5.0.

Gut microbiota profiling

Fecal samples will be collected using the iSWAB-Microbiome^® collection kit at three time points: before initiation of protocol treatment, before cycle 3, and before cycle 5. Bacterial genomic DNA will be extracted from stool samples using the MagMAX™ Microbiome Ultra Nucleic Acid Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA).

The V3–V4 region of the bacterial 16S rRNA gene will be amplified and sequenced using an Illumina sequencing platform (MiSeq or NextSeq 1000). Raw sequencing data will be processed using the QIIME2 pipeline. Amplicon sequence variants (ASVs) will be inferred using the DADA2 algorithm for quality filtering, denoising, chimera removal, and sequence variant inference. Taxonomic assignment will be performed using the SILVA reference database.

Microbial diversity will be evaluated by calculating alpha diversity indices (e.g., Shannon index) and beta diversity metrics (e.g., Bray-Curtis dissimilarity and weighted UniFrac distance). Principal coordinate analysis (PCoA) will be used to visualize differences in microbial community composition between groups.

Biomarker candidate selection

Cross-sectional differences in taxonomic abundance between patients achieving a complete or partial response and those with stable or progressive disease, according to RECIST version 1.1, will be assessed at each sample collection time point using compositional differential abundance methods such as ANCOM-BC. Longitudinal changes in taxonomic abundance and their association with treatment response will be evaluated using mixed-effects modeling approaches such as MaAsLin2, with the patient as a random effect. These analyses will be implemented in R (R Foundation for Statistical Computing, Vienna, Austria), with adjustment for multiple comparisons where applicable.

ASVs significantly enriched in patients achieving a complete or partial response will be defined as positive biomarker candidates, and those enriched in patients with progressive disease will be defined as negative biomarker candidates. Species-level identification of these candidate taxa will be performed using published species-specific PCR assays where available, or full-length 16S rRNA gene sequencing (V1–V9) on the Oxford Nanopore MinION platform.

Quality control

The monitoring manager will be responsible for overseeing the protection of participants’ rights and welfare, ensuring that the study is conducted in compliance with the protocol, and confirming the accuracy and completeness of the collected data. Central monitoring will be performed semi-annually using CRF data submitted from each participating institution. After each monitoring activity, the monitoring manager will prepare a monitoring report summarizing the key findings, including protocol deviations, noncompliance, safety concerns, and other relevant issues, and will report these findings to the principal investigator. An independent expert will serve as the monitoring director and will be responsible for safety oversight. The monitoring director will conduct safety monitoring, including the evaluation and comparison of adverse event incidence throughout the study. No on-site audits are planned for this study.

Data sharing

Personal information of study participants is pseudonymized using a linkable anonymization procedure to protect confidentiality. Case report forms are entered and managed using REDCap, a secure electronic data capture system.

Statistical analysis plan

PFS and OS will be analyzed using the Kaplan-Meier method, ORR will be summarized using an exact binomial confidence interval, and safety outcomes will be analyzed descriptively. PFS will be defined as the time from study enrollment to documented disease progression or death from any cause, whichever occurs first. The PFS distribution will be estimated using the Kaplan-Meier method. Median PFS and its corresponding 95% confidence interval will be reported where estimable. PFS rates at clinically relevant time points may also be presented with their 95% confidence intervals. Patients without documented disease progression or death at the data cutoff will be censored at the date of their last adequate tumor assessment. OS will be defined as the time from study enrollment to death from any cause and will be estimated using the Kaplan-Meier method. Median OS and its corresponding 95% confidence interval will be reported where estimable. Patients who are alive at the data cutoff or lost to follow-up will be censored at the date on which they were last known to be alive. ORR will be defined as the proportion of patients whose best overall response is complete response or partial response according to RECIST version 1.1. ORR will be calculated using all enrolled patients as the denominator and will be reported with a two-sided 95% exact binomial confidence interval calculated using the Clopper-Pearson method. The safety analysis population will include all patients who receive at least one dose of the study treatment. Adverse events will be classified and graded according to the CTCAE version. 5.0. Safety outcomes will be summarized descriptively using the number and proportion of patients experiencing adverse events, treatment-related adverse events, serious adverse events, and grade 3 or higher adverse events. No formal statistical hypothesis testing will be performed for safety outcomes. Missing data will not generally be imputed because of the small sample size and the exploratory nature of the study. The number and reasons for missing or unevaluable observations will be summarized for each endpoint. No formal interim efficacy or futility analysis is planned. Safety will be monitored continuously throughout the study; however, such routine safety monitoring will not constitute a formal statistical interim analysis. Although no formal interim analysis is planned, the study may be prematurely terminated to protect study participants or preserve the ethical and scientific integrity of the study. Premature termination may be considered under any of the following circumstances:

• An unexpected serious illness or other serious adverse event occurs, raising concern about potential harm to the overall study population.
• A serious violation of or noncompliance with applicable laws and regulations or the study protocol is identified.
• Information is obtained that compromises, or may compromise, the ethical acceptability or scientific validity of the study.
• A significant risk to study participants is identified.
• The Certified Review Board provides an opinion recommending suspension or termination of the study.
• The Minister of Health, Labour and Welfare issues a request or recommendation to suspend or terminate the study.

Discussion

To date, no prospective clinical trial has evaluated the efficacy and safety of adding CBM588 to platinum plus pemetrexed and pembrolizumab in patients with non-squamous NSCLC whose PD-L1 TPS <50%, Recent retrospective studies have suggested that prior antibiotic exposure, proton pump inhibitor use, and administration of probiotics or microbiota-modulating agents such as CBM588 may influence the efficacy of chemotherapy plus ICIs in patients with NSCLC, and common factor underlying these clinical variables is the gut microbiota (9-13). Moreover, no study has prospectively monitored gut microbiota in this clinical setting. This study is the first prospective trial to evaluate the addition of CBM588 to chemotherapy plus pembrolizumab. Accordingly, it was designed as an exploratory, proof-of-concept study intended to assess the feasibility of this treatment strategy and to generate preliminary efficacy and safety data for a subsequent larger-scale trial. Because no reliable prospective data is currently available to determine the expected effect size of adding CBM588 in this setting, a formal power calculation based on hypothesis testing was not considered appropriate. The planned sample size of 20 patients was therefore determined primarily on the basis of study feasibility and the exploratory nature of the trial.

In this study, we will enroll patients with NSCLC and PD-L1 TPS <50%, a population in which chemotherapy plus ICIs are commonly used, to collect data on the additive efficacy and safety of CBM588. We will also explore the baseline gut microbiota status before treatment and longitudinal changes in the gut microbiota induced by CBM588. If this prospective exploratory study suggests an improvement in the objective response rate, this therapeutic approach may improve the prognosis of patients with NSCLC, particularly those with poor outcomes. These findings will provide preliminary estimates of the treatment effect and its variability, which will be used to formulate hypotheses and calculate the sample size required for a future confirmatory, adequately powered study. Additionally, gut microbiota profiling may provide further insights into the relationship between gut microbiota and cancer immunity.

Acknowledgments

We are grateful to all the oncologists, allied healthcare professionals, research concierges, and research assistants who participate in this study.

Footnote

Reporting Checklist: The authors have completed the SPIRIT reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0742/rc

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0742/prf

Funding: This study was supported by the Nagasaki University Hospital.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0742/coif). Hirokazu Taniguchi received honoraria for speaking and lectures from Daiichi Sankyo, AstraZeneca, Novartis Pharma, Chugai Pharmaceutical, MSD K.K., Amgen, and Taiho Pharmaceutical outside the submitted work. S.T. received lecture honoraria from MSD K.K., Eli Lilly Japan K.K., and Nippon Kayaku Co., Ltd. H.M. received contracts from FUJIFILM Toyama Chemical Co., Ltd., and honoraria from MSD K.K., Nichi-Iko Pharmaceutical Co., Ltd., NIPRO CORPORATION, Eli Lilly Japan K.K., Miyarisan Pharmaceutical Co., Ltd., and FUJIFILM Toyama Chemical Co., Ltd. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study will be conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was approved by the Clinical Review Board of Nagasaki University (No. CRB25-011). The investigators will explain the concept of the trial to the patients and obtain written informed consent from all participating patients.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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