Baseline phosphoproteomic signatures in peripheral blood and prediction of durable response to CAR T-cell therapy in B-cell malignancies.

Abstract

7038 Background: Despite the success of Chimeric Antigen Receptor (CAR) T-cell therapy, most patients fail to achieve durable remission. T-cell "fitness" is a critical, yet undercharacterized, determinant of efficacy. Standard immune and transcriptomic profiling do not measure the dynamic signaling networks driving cellular function. We hypothesized that baseline phosphoproteomic signatures in peripheral blood mononuclear cells (PBMCs) prior to CAR T-cell could serve as functional biomarkers for T-cell fitness, predicting durable response. Methods: We performed unbiased LC-MS/MS-based phosphoproteomics (KScan) on baseline PBMCs from 59 patients (39 Aggressive Lymphoma [DLBCL], 20 Multiple Myeloma [MM]) treated with CD19- or BCMA-directed CAR-T. Kinase Substrate Enrichment Analysis (KSEA) inferred upstream kinase activity. The primary endpoint was Complete Response (CR) at 6 months. We also analyzed a subset of DLBCL patients (n=6) who achieved CR at Day 30 but progressed (POD) by Month 6 ("early relapse") to identify markers of transient versus durable response. Results: We quantified 7,602 phosphopeptides. In the DLBCL cohort (20 CR, 17 POD), distinct phosphorylation in kinase signaling networks separated durable responders from non-responders, specifically implicating dysregulated stress response and MAPK signaling in resistance. KSEA revealed dysregulation in the MAPK/ERK, Casein Kinase (e.g., CSNK1E), and Cyclin-Dependent Kinase activity, linking baseline proliferative potential to long-term efficacy. Notably, the "early relapse" group (Day 30 CR / Month 6 POD) exhibited a unique profile, characterized by differential phosphorylation of proteins involved in apoptotic regulation and chromatin remodeling (e.g., FADD, ARID1A, ACIN1, KMT2A), suggesting these intrinsic defects may limit CAR T-cell persistence. Correlation analysis further integrated these signaling features with CD3+, CD4+, and CD8+ T-cell counts, validating their biological relevance. In the MM cohort, the signaling landscape was heavily dominated by immune cell composition. KSEA revealed that kinase activity in CR patients strongly correlated with the CD4:CD8 ratio and absolute CD3+ counts, validating that the captured phosphoproteomic variance reflects the underlying T-cell phenotype required for successful expansion and cytotoxicity. Conclusions: Baseline PBMC phosphoproteomics is a powerful tool for predicting CAR T-cell efficacy. We identified specific signatures—distinct from broad immune cell counts—that differentiate durable responders from patients at risk of primary refractory disease or early relapse. These targets highlight avenues for pharmacological conditioning to enhance T-cell fitness. These data support integrating functional proteomic biomarkers into patient stratification strategies to optimize outcomes.