Overview
The goal of this single-arm, open-label, prospective clinical trial is to evaluate the safety, feasibility, and preliminary antitumor activity of intratumoral oncolytic virus (H101, recombinant human type 5 adenovirus) combined with immune checkpoint inhibitors (ICIs), and to explore the dynamic remodeling of the tumor immune microenvironment and potential predictive biomarkers, in patients with advanced metastatic colorectal cancer, gastric cancer, or hepatocellular carcinoma who have failed multiple prior lines of therapy.
The main questions it aims to answer are:
What are the incidence of treatment-related adverse events and serious adverse events, and the completion rate of the combination therapy (safety and feasibility)? What is the objective response rate and local control rate per Response Evaluation Criteria in Solid Tumors Version 1.1 (RECIST 1.1) (preliminary antitumor activity)? How does the combination therapy reshape the tumor immune microenvironment in terms of immune cell infiltration, subset proportions, functional status, and signaling pathways, and which differentially expressed genes or immune cell subtypes are associated with clinical response? This is a single-arm study; there is no comparator group.
Participants will:
Receive intratumoral H101 injection once per cycle (dose stratified by lesion size), followed 5-7 days later by intravenous ICI \[Anti-Programmed Cell Death Protein 1 Antibody (anti-PD-1) with or without Anti-Cytotoxic T-Lymphocyte-Associated Protein 4 Antibody (anti-CTLA-4)\], repeated every 2-3 weeks for a total of 4 cycles.
Provide paired tumor biopsy samples and peripheral blood specimens at baseline and after 4 cycles for single-cell RNA sequencing and biomarker analyses.
Undergo routine clinical assessments, imaging for tumor response evaluation, and safety monitoring with adverse event grading per Common Terminology Criteria for Adverse Events Version 5.0 (CTCAE v5.0).
Description
Background and Rationale Advanced metastatic colorectal cancer, gastric cancer, and hepatocellular carcinoma remain major causes of cancer-related mortality worldwide, particularly after failure of multiple lines of standard systemic therapies. Immune checkpoint inhibitors (ICIs) have shown limited efficacy as monotherapy in these tumors, partly due to an immunosuppressive tumor microenvironment (TME) characterized by sparse T-cell infiltration, high myeloid-derived suppressor cell (MDSC) accumulation, and dysfunctional antigen presentation. Oncolytic viruses, such as H101 (recombinant human type 5 adenovirus), can selectively replicate in tumor cells, induce immunogenic cell death, and release tumor-associated antigens, thereby converting a "cold" TME into a "hot" one. Preclinical and early clinical data suggest that intratumoral administration of oncolytic viruses may synergize with systemic ICIs by enhancing T-cell priming, increasing Programmed Death-Ligand 1 (PD-L1) expression, and remodeling the immune cell landscape. However, real-world evidence in the digestive cancer setting, especially for heavily pretreated patients, is scarce. This investigator-initiated study is therefore designed to generate preliminary data on the combination's safety, feasibility, and biological activity in a routine clinical practice context.
Study Objectives Primary objective: To assess the safety \[Treatment-Related Adverse Events / Serious Adverse Events(TRAEs/SAEs)\], feasibility (treatment completion and modification rates), and preliminary antitumor activity (ORR and LCR by RECIST 1.1) of intratumoral H101 plus ICIs.
Secondary objective: To evaluate disease control rate (DCR), progression-free survival (PFS), and longitudinal changes in the TME, including immune cell infiltration (T cells, NK cells, dendritic cells, macrophages), subset proportions, functional markers, and immune-related signaling pathways.
Exploratory objective: To identify potential predictive biomarkers of response by integrating single-cell RNA sequencing (scRNA-seq) data from paired tumor biopsies and peripheral blood, and to characterize the molecular and cellular mechanisms underlying the combination therapy.
Study Design This is a single-center, prospective, open-label, single-arm observational study conducted in a real-world clinical setting. Although the treatment is protocol-specified, all clinical decisions are made per routine multidisciplinary team (MDT) evaluation; the study does not add extra interventions beyond planned therapy but standardizes data collection, efficacy assessment, safety monitoring, and biospecimen banking. A total of 30-45 patients (10-15 per cancer type) will be enrolled.
Participant Selection Eligible patients are adults (≥18 years) with histologically or cytologically confirmed advanced metastatic colorectal cancer, gastric cancer, or hepatocellular carcinoma, who have progressed on or were intolerant to at least two prior lines of systemic therapy (including chemotherapy and/or targeted agents). Patients must have at least one measurable lesion suitable for intratumoral injection per RECIST 1.1, adequate organ function, and an Eastern Cooperative Oncology Group performance status of 0-2. Key exclusion criteria include prior treatment with oncolytic viruses or ICIs (unless washout \>6 months for ICIs), active central nervous system metastases, uncontrolled intercurrent illnesses, or known allergy to adenoviral vectors. All patients are assessed by the institutional MDT and deemed appropriate for the combination regimen.
Treatment Protocol
The treatment schedule consists of 4 cycles, each lasting 2-3 weeks, as follows:
Cycle 1: On Day 1, H101 is injected intratumorally at a dose stratified by the largest lesion diameter: ≤5 cm → 5.0×10¹¹ viral particles (vp); \>5 cm → 1.0×10¹² vp, divided into multiple injection points if necessary. On Day 5-7 (window), the first dose of ICI is administered intravenously: either anti-PD-1 monotherapy (e.g., sintilimab, toripalimab, or pembrolizumab at standard approved doses) or a combination of anti-PD-1 plus anti-CTLA-4 (e.g., ipilimumab at 1 mg/kg) per the treating physician's discretion and local label.
Subsequent cycles (2-4): H101 injection is repeated on Day 1 of each cycle, followed by ICI on Day 5-7. Dose modifications for toxicity follow standard guidelines; treatment delays of up to 2 weeks are permitted, but discontinuation is required if toxicity does not resolve to ≤ Grade 1 within that period.
Assessments and Data Collection Safety: All adverse events are recorded and graded using CTCAE v5.0 from the first dose until 30 days after the last dose. SAEs are reported within 24 hours.
Efficacy: Tumor imaging (CT/MRI) is performed at baseline and after cycle 4 (or earlier if clinically indicated). Responses are independently reviewed by two radiologists according to RECIST 1.1. PFS is calculated from the first treatment date to disease progression or death from any cause.
Biomarker sampling: Paired tumor biopsies (core needle or fine-needle aspiration from the injected lesion) and peripheral blood (10 mL) are collected at baseline (within 7 days before first H101) and at the end of cycle 4 (within 7 days after the last ICI). Samples are processed for scRNA-seq, flow cytometry, and multiplex cytokine assays.
Data management: All data are entered into a secured electronic case report form (eCRF) with unique subject codes; personal identifiers are removed to protect privacy.
Statistical Methods
Given the exploratory nature, the sample size is not based on formal power calculation but is considered adequate to provide descriptive estimates and generate hypotheses. Continuous variables will be summarized as mean (SD) or median (IQR); categorical variables as frequencies and percentages. For primary endpoints:
Safety and feasibility will be reported with descriptive statistics. ORR and LCR will be calculated with exact 95% confidence intervals (Clopper-Pearson method).
DCR will be similarly reported. PFS will be estimated using the Kaplan-Meier method, with median time and 95% CI.
For paired immune parameters (e.g., cell subset frequencies, cytokine levels), changes from baseline to post-treatment will be assessed using paired t-tests for normally distributed data or Wilcoxon signed-rank tests for non-normal distributions. All statistical tests are two-sided, with a significance level of α=0.05 (exploratory, no multiplicity adjustment).
For scRNA-seq data, after quality control and normalization, unsupervised clustering (Seurat) will be performed to identify cell populations; differential expression analysis between responder and non-responder groups, or between pre- and post-treatment, will employ the MAST or Wilcoxon rank-sum test. Gene set enrichment analysis (GSEA) and pathway analysis (GO/KEGG) will be applied to characterize functional changes. Correlations between baseline immune features and clinical outcomes will be explored using Spearman's rank correlation.
Ethical and Regulatory Considerations The study protocol has been approved by the institutional review board/ethics committee of the participating center. Written informed consent will be obtained from all patients prior to any study-specific procedures. The study will be conducted in full compliance with the Declaration of Helsinki (Fortaleza revision), Good Clinical Practice guidelines, and all applicable local regulations. Any protocol amendments will be submitted for ethical review and updated on this registry.
Expected Significance This study is among the first to prospectively evaluate the H101-ICI combination in a real-world, heavily pretreated population of three major digestive cancers. The integrated clinical and multi-omics data are expected to provide crucial evidence on the safety profile and clinical benefit of this approach, while the mechanistic explorations may uncover immune biomarkers that guide patient selection and combination strategy refinement. The findings will inform the design of larger, randomized controlled trials and ultimately contribute to the development of personalized immunotherapeutic regimens for patients with limited treatment options.
Eligibility
Inclusion Criteria:
- Patients aged 18-75 years.
- Pathologically confirmed solid tumors, including superficial metastatic lesions and intra-abdominal or intrathoracic tumors, that are amenable to intratumoral injection with a longest diameter ≥1.0 cm.
- For superficial metastatic lesions that have not received local radiotherapy, ablation, or surgical resection; if such treatments have been performed, at least 4 weeks must have elapsed since completion and the lesion must show documented progression.
- Prior systemic therapy must have been completed at least 4 weeks or 5 half-lives ago, and the patient must have recovered from previous treatment-related toxicities.
- Adequate cardiopulmonary, hepatic, and renal function to tolerate Oncorine® (H101) intratumoral injection.
- The patient and their family members are willing to participate in this study and provide written informed consent.
Exclusion Criteria:
- Peripheral blood lymphocyte count \<1.0×10⁹/L；
- Target superficial or body cavity tumors located adjacent to major blood vessels or nerves, with a high risk of puncture or injection; or lesions with active infection or ulceration；
- Known severe hypersensitivity to Oncorine® (H101) or any of its components；
- Active intra-abdominal infection or uncontrolled ascites；
- Currently receiving systemic glucocorticoids or other potent immunosuppressive agents；
- Planned receipt of other antitumor therapies during the study period (except supportive care)；
- Concurrent other malignancy or a history of prior malignancy；
- Tumors complicated by rupture with hemorrhage, massive gastrointestinal bleeding, intestinal obstruction, or other conditions requiring emergency surgery；
- American Society of Anesthesiologists (ASA) classification ≥ IV and/or Eastern Cooperative Oncology Group (ECOG) performance status score \>2；
- Active autoimmune disease, or history of autoimmune disease requiring systemic therapy; uncontrolled severe infections (e.g., HIV, active hepatitis B with HBV-DNA above the lower limit of detection, active hepatitis C); severe cardiovascular or cerebrovascular disease; history of organ transplantation; active bleeding or high bleeding risk；
- History of severe psychiatric illness；
- Pregnant or breastfeeding women；
- Uncontrolled active infection；
- Other clinical or laboratory conditions that, in the opinion of the nvestigator, render the patient unsuitable for participation in this trial.
