Patient-Derived Lymphoma Spheroids Reveal Predictive Markers of Glofitamab Resistance in Relapsed/Refractory B-NHL

Bispecific antibodies (bsAbs) such as glofitamab represent a promising therapeutic approach for relapsed/refractory B-cell non-Hodgkin's lymphoma (R/R B-NHL), but resistance mechanisms remain poorly understood. This study aimed to identify predictive markers of bsAbs resistance based on the response of 3D patient-derived lymphoma spheroids (PDLS) established from R/R B-NHL samples. PDLS were treated with 0.1 nM glofitamab for 3 days and B-cell depletion was quantified as measure of ex vivo treatment response. Comprehensive immune profiling were performed on patient samples using multiparametric flow cytometry, single-cell RNA sequencing, CODEX spatial proteomics and functional assays. This study revealed that high responders to glofitamab possessed more activated CD8+ T-cells with higher cytotoxic signatures, while low responders showed enriched exhausted CD8+ T-cell populations with enhanced expression of exhaustion markers (TIGIT, LAG3, PD1). Moreover, low responders demonstrated elevated functional CD4+ T-follicular helper (Tfh) cells in close proximity to malignant B-cell thus promoting their survival through IL21 and CXCL13 signaling pathways. Functional experiments have shown that adding anti-TIGIT co-treatment enhanced glofitamab efficacy in low responder samples, and Tfh depletion improved cytotoxic T-cell function. Our findings identify CD8+ T-cell exhaustion and functionally activated Tfh cells as key resistance mechanisms to glofitamab in R/R B-NHL. Targeting these pathways represents a promising strategy to overcome bsAb resistance and improving therapeutic outcomes.