The SERPENTINE trial: A phase II clinical study decoding immune responses by scRNA-seq and TCR-seq analysis in patients with MSS metastatic colorectal cancer (mCRC) with and without liver metastases treated with durvalumab and tremelimumab.

3607 Background: Immune checkpoint inhibitor (ICI) therapy aims to instruct immune cells to efficiently recognize and eliminate cancer cells. It has shown efficacy in MSI colorectal (CRC), and, although there is no approval yet, a subset of patients with MSS CRC included in clinical trials respond to ICI, indicating a significant knowledge gap in our current understanding of immunological mechanisms in MSS CRCs. The relationship between the cellular and molecular responses to ICI in the tumor microenvironment (TME) and the clinical outcome remains poorly understood. We sought to address this gap in understanding through a series of high-throughput functional genomics analyses. Methods: Patients (pts) with refractory MSS CRC were treated with durvalumab (Dur) 1500mg plus tremelimumab (Trem) 300mg at Week 0, followed by Dur 1500 mg Q4W. We characterized the cellular makeup of the metastatic TME using single-cell RNA-seq, and cataloged T-cells clonotypes within metastases and in the blood using single-cell and deep TCR-seq. We collected data for each patient before and upon treatment initiation to explore the dynamic changes of the TME and blood in response to ICI. To facilitate direct comparisons between metastatic and primary TMEs, we integrated our dataset with an external single-cell RNA-seq dataset from primary CRC tumors. Results: 24 pts were included in the analysis (18 with no liver metastases (NLM), 6 with liver metastases (LM)). Around 200,000 single-cell profiles were collected from biopsies taken from 6 pts with LM and 10 pts with lung metastases. Disease Control Rate (DCR) was 39% for MSS CRC without LM (2 PR + 5 SD) and 0% for those with LM (p=0.13). Robust gene expression changes within the immune compartment of the TME were observed upon treatment, irrespective of overall treatment outcome, yet specific biological programs were differentially altered across patient response groups. Differences in the composition of TMEs in the lung and liver metastases were found with an enrichment of T regulatory and IFN-responder CD4 populations in the lung vs more CD8+ T cell subtypes in the liver. Tumor TCR analysis revealed that, following ICI treatment, liver metastases displayed a high diverse TCR repertoire, whereas lung metastases were less diverse and showed marked clonal expansion, suggesting greater T cell activity within the lung lesions (p=0.035). Conclusions: Prospective deep immune repertoire profiling detected profound differences in TME according to dissemination patterns, which might explain differences in treatment outcomes. Despite low overall efficacy with Dur and Trem in MSS patients, a trend to better response rate and DCR was observed in pts without LM. We propose immune cell tracking as powerful tool to quantify the efficacy of ICI in real time. Clinical trial information: NCT06680739 .