FOLLICULAR LYMPHOMA PATIENT‐DERIVED ORGANOIDS FOR BISPECIFIC T‐CELL ENGAGER IMMUNOTHERAPY (2023)

Introduction: The follicular lymphoma tumor microenvironment (FL TME) is critical for FL maintenance and associated with clinical outcomes (Tobin et al. JCO, 2019). Novel immunotherapies including bispecific T-cell engagers require an intact TME for immune effector function and cannot be modeled in traditional B-cell monocultures. Organoid systems in non-lymphoid cancers have been proposed as accurate ex vivo models of the TME. We therefore developed a robust patient-derived lymphoma organoid (PDLO) system utilizing primary biopsies from FL patients and assessed response to bispecific T-cell mediated immunotherapy. Methods: Surgical lymph node biopsies were obtained at diagnosis or relapse, mechanically dissociated into single cell suspensions, cryopreserved, and later prepared as PDLOs on a high-throughput 96-well platform previously developed with human tonsils (Wagar et al. Nature Med, 2021, Figure A). PDLOs were assessed at serial timepoints by a multi-modal approach including flow cytometry, CAPP-Seq DNA sequencing, bulk RNA sequencing, immune cell deconvolution with CIBERSORTx (Newman et al. Nat Biotech, 2019), and repertoire profiling of B- and T-cell receptors. For treatment experiments, bispecific antibody (CD19:CD3; HD37 (Anti-CD19); L2K-07 (Anti-CD3); InvivoGen) or corresponding unconjugated monospecific antibodies were added on D4 at a final concentration of 100 ng/mL. Results: We studied 11 primary specimens including 3 with evidence of transformation (tFL). FL PDLOs formed aggregate clusters similar to healthy tonsil organoids with most viable for 21+ days. Although variable across samples, the proportion of malignant B-cells by flow cytometry remained largely stable (Figure B) as did CD4/CD8/Tfh T-cell subsets (Figure C,D). We assessed mutations pre-culture and at D7/14/21, with mutational stability over 21 days suggesting against strong selective pressure in untreated cultures (Figure E). To assess immunotherapy response, we treated at D4 with CD19:CD3 bispecific antibody or unconjugated anti-CD19 and anti-CD3 as “Control”. At D11 treatment with CD19:CD3 induced significant B-cell killing as assessed by flow cytometry and CIBERSORTx, along with expansion in CD8+ effector and regulatory T-cells (Figure F). Although total CD4+ T-cells remained stable, an increase in activated subsets was seen during treatment (Figure G,H). Confirming lymphoma killing, monoclonal BCR was reduced in bispecific-treated cultures (Figure I). Treated PDLOs showed significant RNA upregulation of TIGIT, LAG3, CTLA4, and PD1 (Figure J) consistent with T-cell exhaustion associated with continuous bispecific exposure (Philipp et al. Blood, 2022). Data for CD20:CD3 and cell-specific correlates of response will be presented. Keywords: immunotherapy, microenvironment, patient-derived xenograft (PDX) models Conflicts of interests pertinent to the abstract M. Diehn Consultant or advisory role: Foresight Diagnostics Stock ownership: Foresight Diagnostics L. Wagar Consultant or advisory role: VCreate Research funding: Roche A. A. Alizadeh Employment or leadership position: Foresight Diagnostics Consultant or advisory role: Adaptive Biotechnologies, Genentech, Karyopharm, Roche, Chugai, Gilead, Celgene, BMS Stock ownership: Foresight Diagnostics, Syncopation, Gilead, Cibermed Research funding: BMS