235 Defining the role of TCF1 in CAR T cell memory and persistence

Abstract

<h3>Background</h3> Poor CAR T cell persistence is a major factor limiting durable responses to CAR T cell therapy. TCF1 is a transcription factor that regulates the expression of memory-related genes and its expression correlates with clinical CAR T persistence, making it a promising target for enhancing CAR T efficacy. Despite TCF1’s established role in memory and stemness, overexpression of TCF1 alone does not enforce a memory-like phenotype or improve CAR T potency,¹ indicating that the function and relevance of TCF1 in human CAR T cells is unclear. These results highlight the need to more carefully interrogate the role of TCF1 in engineered human T cells. Prior work has shown that TCF1 is regulated in a context-specific manner by the presence or absence of its co-activator, β-catenin. Our central hypothesis is that TCF1 activity is governed by direct binding of TCF1 to β-catenin in CAR T cells, which can be leveraged to enforce memory programs and enhance CAR T cell potency. <h3>Methods</h3> To test whether TCF1/β-catenin binding is sufficient to enforce CAR T memory, we engineered and tested several variants of a chimeric TCF1 factor (TCF1fusion) in which β-catenin is covalently linked to TCF1. TCF1fusion expression and activity was assessed by Western blot and a TCF1 reporter assay, respectively. Human T cells were co-transduced to express a CAR and TCF1-related factors, phenotyped using flow cytometry and qRT-PCR. CAR T cell antitumor function was assessed <i>in vitro</i> by tumor cell co-culture experiments and <i>in vivo</i> using a leukemia xenograft tumor model in immunocompromised NSG mice. <h3>Results</h3> TCF1fusion expression results in as much as &gt;40-fold increased TCF1 reporter activity and increased expression of endogenous TCF1 target genes, including memory-associated markers CCR7 and IL-7Rα, compared to full-length TCF1 and negative controls. Interestingly, TCF1fusion-CAR T cells exhibit impaired expansion and antitumor activity both <i>in vitro</i> and in leukemia xenograft models, suggesting that constitutive TCF1 activity is deleterious to CAR T cell. <h3>Conclusions</h3> Our results suggest that TCF1/β-catenin binding is sufficient to promote high expression of putative TCF1 target genes and a memory-like phenotype, but might lock CAR T cells into a memory state, thereby blunting CAR T antitumor function. Our findings generate new insight into human TCF1 biology and inform ongoing approaches to leverage TCF1 to enhance the efficacy of CAR T cell therapeutics and other cancer immunotherapies. <h3>Reference</h3> Doan AE, <i>et al</i>. FOXO1 is a master regulator of memory programming in CAR T cells. <i>Nature</i> 2024;<b>629</b>:211–218. <h3>Ethics Approval</h3> Children’s Hospital of Philadelphia Institutional Animal Care and Use Committee approval has been obtained for all mouse experiments (IAC 24-000232).