PAUL MARCOUX, Fabien Gava, Marie Tosolini, Pauline Gravelle, Christina Schniederjohann, S. Quertinmont, Neus Serrat, Fanny Bouquet, Sylvia Herter, Karin Tarte, Mikaël Roussel, Pierre Sesques, Caroline Bret, C. N. Rossi, Pierre Aubert, Franck Morschhauser, Guillaume Cartron, Wolfgang Huber, Sascha Dietrich, Loïc Ysebaert, Pierre Brousset, Peter‐Martin Bruch, Patricia Pérez-Galán, Christine Bezombes, Camille Laurent
ABSTRACT: Bispecific antibodies (bsAbs) such as glofitamab represent a promising therapeutic approach for relapsed/refractory B-cell non-Hodgkin lymphoma (R/R B-NHL), but resistance mechanisms remain poorly understood. This study aimed to identify predictive markers of bsAb resistance based on the response of 3-dimensional 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, codetection by indexing spatial proteomics, and functional assays. High responders to glofitamab possessed CD8+ T cells with consistently higher cytotoxic and activation signatures across effector differentiation states, whereas low responders showed enrichment of exhausted CD8+ T cells with enhanced expression of exhaustion markers (T-cell immunoglobulin and ITIM domain [TIGIT], LAG3, and PD1). Furthermore, low responders exhibited elevated functional CD4+ T follicular helper (Tfh) cells in close proximity to malignant B cells, thus promoting their survival through interleukin-21 and C-X-C motif chemokine ligand 13 signaling pathways. Analysis of pretreatment RNA-sequencing data from 48 patients with R/R B-NHL confirmed that high Tfh cell abundance is associated with poor glofitamab response. In PDLS, anti-TIGIT cotreatment enhanced glofitamab efficacy in low responders, and Tfh cell depletion experiments confirmed that reducing Tfh cell 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.