Work

931 A TCF7 phosphorylation checkpoint controls B7-H3 CAR-T cell persistence in solid tumors and brain metastases

Year: 2026

Type: conference-abstract

Abstract: Background CAR-T cell therapy has transformed hematologic malignancies, yet solid tumors and brain metastases remain refractory because immunosuppressive tumor microenvironments and chronic antigen exposure rapidly drive T cell exhaustion. Stem-like persistence is essential for durable CAR-T activity, and T cell factor 1 (TCF1), encoded by TCF7, is a master regulator of this state. However, wild-type TCF7 overexpression does not reliably prevent exhaustion, suggesting post-translational neutralization. Because LCK is physically coupled to CAR activation and sustained during continuous tumor engagement, we investigated whether LCK directly suppresses TCF7. We identified an LCK-phosphorylated, highly conserved tyrosine residue in TCF7 and hypothesized that blocking this phosphorylation would shield CAR-T cells from tumor-induced exhaustion.Methods We generated B7-H3 CAR-T cells expressing wild-type TCF7, a phosphomimetic Y-to-D mutant, or a phosphorylation-resistant Y-to-F mutant and evaluated function using tumor killing, expansion assays, transcriptomic profiling, and a MDA-MB-231-BrM intracardiac injection brain metastasis model. An 18-gene pTCF7-HIGH signature, derived from phosphomimetic versus wild-type cells, was evaluated in two clinical single-cell RNA-sequencing (scRNA-seq) datasets from central nervous system (CNS) CAR-T trials and in Xenium 5K spatial transcriptomic profiles from anti-PD-1-treated triple-negative breast cancer biopsies.Results In the brain metastasis model, B7-H3 CAR-T cells demonstrated robust initial anti-tumor activity but intracranial relapse occurred, consistent with insufficient durable CAR-T persistence. In contrast, the phosphorylation-resistant TCF7 mutant maintained tumor killing and preserved stemness-associated transcriptional programs compared with wild-type TCF7, whereas the phosphomimetic mutant promoted dysfunction ( figure 1). In clinical GD2-CAR-T CNS datasets, pTCF7-HIGH scores increased from pre-infusion products to post-infusion cerebrospinal fluid CAR-T cells through Day 14. Non-responder product cells showed higher pTCF7-HIGH scores than responder products (p = 1.43 × 10-10), and the signature correlated with exhausted T cell states. In a 45-patient GD2-CAR-T cohort, higher pre-infusion pTCF7-HIGH burden predicted reduced peak expansion (p = 0.049; Spearman rho = -0.378, p = 0.0109). In a separate IL13Rα2 CAR-T CNS trial, pTCF7-HIGH similarly aligned with exhausted states and reduced expansion. Xenium analysis of anti-PD-1-treated triple-negative breast cancer biopsies showed that pTCF7-HIGH T cells were enriched in non-responders by cell-level analysis (odds ratio = 3.40, 95% confidence interval 2.73–4.25, p = 3.00 × 10-31; n = 1,947 cells from 5 patients), localized closer to tumor cells, and associated with exhaustion (figure 2).Conclusions TCF7 phosphorylation is a targetable checkpoint limiting T cell persistence and anti-tumor function in solid tumors and brain metastases. Engineering phosphorylation-resistant TCF7 could improve B7-H3 CAR-T durability, while clinical CNS CAR-T and spatial triple-negative breast cancer validation further support this axis as a shared mechanism of T cell dysfunction across cellular therapy and immune checkpoint blockade.Abstract 931 Figure 1The LCK-TCF7 axis as a checkpoint for CAR-T exhaustion. (sssA) Longitudinal BLI of intracranial tumor in B7-H3 CAR-T treated mice; (B) Representative MRI of brain mets; (C) In vivo tracking of iron-labeled CAR-T cells; (D–E) BLI signal quantification; (F) Immunoblot of LCK-mediated TCF7 phosphorylationAbstract 931 Figure 2Clinical validation of the p-TCF7-HIGH signature. (A) p-TCF7-HIGH signature in CAR-T responders ornon-responders; (B) Correlation between p-TCF7-HIGH and in vivo expansion; (C) Xenium 5K spatial maps of brain mets; (D) UMAP visualization; (E) exhaustion program enrichment in p-TCF7-HIGH cells; (F) Model

Source: Journal for ImmunoTherapy of Cancer

DOI: 10.1136/jitc-2026-sitc2026.0931