Abstract 3: Multiomics analyses reveal functional DNA, methylation pattern changes in clinical transgenic T-cell receptor cell therapy products
Authors: Protti Giulia , Peters Cole , Kawakami Moe , Kidd Conner , Subramanyam Varsha , Lechien Analynn , Dinh Andrew , Ribas Antoni , Pellegrini Matteo , Nowicki Theodore S. 
Published: March 2024

Background: Transgenic T-cell receptor T-cells (TCR-T) directed against tumor-specific antigens are associated with robust initial clinical responses. However, these responses are often not durable, and patients relapse frequently. Therefore, there is a significant need to understand how these cells change at the epigenomic level over time following infusion into the patient.

Methods: We analyzed MART-1 TCR-T products from 8 patients treated under a clinical trial at UCLA (NCT00910650). Samples from the baseline infusion product and samples from day +30 recovered from peripheral circulation were sorted for TCR+ cells via flow cytometry, and were then subjected to whole genome bisulfite sequencing (WGBS), ATACseq, and RNAseq in parallel. Differential chromatin accessibility and gene expression by the ATACseq and RNAseq datasets, respectively, were analyzed via DESeq2, with P adjusted less than 0.1. WGBS datasets were analyzed via Metilene, and differentially methylated regions (DMRs) were defined as those regions with CpG loci containing a methylation difference &gt;= 10% and a P adjusted &lt; 0.05. Functional DMRs were defined as those repressed over time (increased DNA methylation with decreased chromatin accessibility/gene expression relative to infusion product), or activated over time (decreased DNA methylation with increased chromatin accessibility/gene expression relative to infusion product). Functional DMRs were then analyzed via EnrichR to evaluate gene ontology and pathways significantly enriched in gene lists.

Results: We collectively identified three genes with repressed DMR activity and 49 genes with activated DMR activity compared to baseline infusion products. Our findings revealed that genes associated with repressed DMRs included IL2RA, FBLN7, and ZNRF1. These genes were collectively associated with a decrease in T-cell activation, cytokine signaling, and migration activity, potentially impacting the functional capabilities of T-cells in the post-infusion environment. Conversely, genes associated with activated DMRs included PDCD1 (PD1), FGR, KIR3DL1, KIR2DL4, and KLF3, with pathway enrichment analysis indicating a significant shift in the T-cell landscape towards reduced anti-tumor activity, decreased T-cell proliferation, T-cell aging, and the promotion of immune escape tumor phenotypes.

Conclusions: This comprehensive multi-omics approach showcases an atlas of genes in clinical TCR-T cell therapeutics which are subject to functional changes in DNA methylation over time in vivo. These findings shed light on the complex interplay between DNA methylation, chromatin accessibility, and gene expression in the context of TCR-T cell therapy, with implications for the development of more effective immunotherapeutic strategies.

Citation Format: Giulia Protti, Cole Peters, Moe Kawakami, Conner Kidd, Varsha Subramanyam, Analynn Lechien, Andrew Dinh, Antoni Ribas, Matteo Pellegrini, Theodore S. Nowicki. Multiomics analyses reveal functional DNA, methylation pattern changes in clinical transgenic T-cell receptor cell therapy products [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3.