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 39 R/R B-NHL samples. PDLS were treated with glofitamab for 3 days and B-cell depletion was quantified to assess the ex-vivo treatment response. Comprehensive immune profiling was performed on patient samples using multiparametric flow cytometry, single-cell RNA sequencing, CODEX spatial proteomics and functional assays. High responders to glofitamab possessed CD8+ T-cells with consistently higher cytotoxic and activation signatures across effector differentiation states, while low responders showed enrichment of exhausted CD8+ T-cell with enhanced expression of exhaustion markers (TIGIT, LAG3, PD1). Furthermore, low responders exhibited 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. Analysis of pretreatment RNA-seq data from 48 R/R B-NHL patients confirmed that high Tfh abundance is associated with poor glofitamab response. In PDLS, anti-TIGIT co-treatment enhanced glofitamab efficacy in low responders, and Tfh depletion experiments confirmed that reducing Tfh activity increased B-cell depletion. Together, these findings identify CD8+ T-cell exhaustion and functionally activated Tfh cells as key factors associated with glofitamab resistance in R/R B-NHL. This work supports their potential use as predictive biomarkers for selecting patients with higher probability of response and provides a foundation for future combination therapeutic strategies.