A Magnetic Bead-Amplified SPR/SERS Dual-Modality Assay for EV-Associated TGF-β1 as a Companion Diagnostic for CAR-T Therapy
Sailing He,
Faten Bashar Kamal Eddin,
Abdur Rahim,
Wen Lei,
Qunyi Guo,
Kaixin Zheng,
Raheel Ahmed Janjua and
Wenda Luo
Chimeric antigen receptor (CAR)-T cell therapy has achieved remarkable success in hematologic malignancies, yet its efficacy in solid tumors remains limited by the immunosuppressive tumor microenvironment. The recent clinical breakthrough of GPC3-specific dnTGFβRII-armored CAR-T (C-CAR031) has demonstrated encouraging responses, but a substantial fraction of patients still fails to achieve durable remission. A key resistance mechanism is the overwhelming burden of TGF-β1 in the tumor microenvironment, which may overwhelm the dnTGFβRII ``armor'' and lead to CAR-T exhaustion. Here, we report a magnetic bead-amplified phase-interrogated surface plasmon resonance (SPR) biosensor for the quantitative detection of TGF-β1 on tumor-derived extracellular vesicles (TEVs) in patient plasma. The platform combines anti-TGF-β1 antibody-conjugated magnetic beads for EV enrichment with a ZnSe/GO SPR chip functionalized with anti-TGF-β1 for real-time, label-free detection. The assay achieved a limit of detection (LOD) of 0.051 pg/mL with a linear range spanning 10-1-105 pg/mL (R2 = 0.9894). In vitro, CAR-T + Raji co-culture significantly increased TGF-β1+ EVs compared with all control groups, peaking at 24 h. Orthogonal surface-enhanced Raman scattering (SERS) validation at 72 h independently confirmed this trend, exhibiting characteristic R6G reporter peaks at ~1367 cm-1 and ~1520 cm-1, and strictly following the hierarchy of CAR-T + Raji > Raji > CAR-T > CT + Raji > Healthy controls. Clinically, four CAR-T patients showed marked elevations in plasma EV-associated TGF-β1 (7.2-21.3 fold over baseline), with peak concentrations of 77.97, 69.22, 49.02, and 57.30 pg/mL for P1-P4, respectively. This platform provides a companion diagnostic tool to identify patients with high TGF-β1+ EV burden, specifically those whose ``armor'' is likely to be overwhelmed, thereby guiding combination therapy or EV clearance strategies. In this retrospective cohort of four patients who all developed Grade 1 CRS, the EV-associated TGF-β1 signal rose before clinical onset in the retrospectively aligned trajectories; prospective validation in larger, higher-grade CRS cohorts is required in future studies.