Targeting cfDNA and NETs with DNAse I to augment CAR T-cell function and antitumor efficacy.

Abstract

7531 Background: Chimeric antigen receptor (CAR) T-cell therapy induces high initial response rates in B-cell malignancies; however, limited persistence and early relapse remain major clinical challenges. Accumulation of cell-free DNA (cfDNA) and neutrophil extracellular traps (NETs) within the tumor microenvironment represents a potential mechanism of CAR T-cell dysfunction through induction of exhaustion, immunosuppression and impaired expansion. We hypothesized that enzymatic degradation of extracellular DNA using DNase I could enhance CAR T-cell persistence and antitumor efficacy. Methods: The effects of cfDNA and NETs on human CD19 CAR T-cell function were evaluated using sequential killing assays, proliferation assays, and flow cytometry–based phenotyping. DNase I was assessed for its ability to reverse cfDNA/NET-mediated suppression and prevent exhaustion. In vivo efficacy, expansion, and persistence of CAR T cells were studied in xenogeneic B-cell acute lymphoblastic leukemia (Nalm-6) and Burkitt lymphoma (Raji) mouse models. Longitudinal in vivo tracking of CAR T-cell expansion was performed using bioluminescence imaging and serial peripheral blood analysis. Translational relevance was further explored through compassionate-use administration of DNase I in combination with CAR T-cell therapy in a pediatric patient with relapsed/refractory Burkitt lymphoma. Results: cfDNA and NETs suppressed CAR T-cell proliferation and cytotoxicity while promoting upregulation of exhaustion markers, including PD-1, LAG-3, and TIM-3. DNase I efficiently degraded extracellular DNA, preserved CAR T-cell effector function, improved CD8:CD4 ratios, and reduced exhaustion across multiple rounds of tumor rechallenge in vitro. In vivo, DNase I significantly enhanced CAR T-cell expansion and persistence following infusion, as demonstrated by longitudinal tracking studies. Combination therapy resulted in improved tumor control, delayed relapse upon rechallenge, and prolonged survival in both NALM-6 and Raji xenograft models. In a single pediatric patient with highly refractory Burkitt lymphoma, DNase I co-administration was associated with marked CAR T-cell expansion and progressive reduction in tumor burden after prior CAR T-cell failure. Conclusions: Extracellular DNA and NETs constitute a critical barrier to durable CAR T-cell efficacy. DNase I enhances CAR T-cell persistence and antitumor activity by eliminating cfDNA/NET-driven immunosuppression, supporting further clinical evaluation of DNase I as a combinatorial strategy to improve CAR T-cell therapy outcomes.