ABSTRACT: Blinatumomab (Blina), a CD19×CD3 bispecific T cell engager, is approved for the treatment of relapsed/refractory and measurable residual disease (MRD)-positive B-cell precursor acute lymphoblastic leukemia (BCP-ALL), yet many patients do not respond. The immune determinants of treatment failure remain poorly understood. We profiled peripheral blood T cells and the immune milieu of 34 patients with BCP-ALL receiving Blina using flow cytometry (n=19), single-cell CITE-seq (n=13), and ex vivo Blina-induced cytotoxicity (n=26) and serum proteomics (n=17). Patients with overt disease who achieved complete remission, or those with MRD who achieved MRD-negativity after the first cycle, were considered responders (R), the remainder were classified as non-responders (NR). At baseline, R had higher frequencies of CD8⁺ effector memory T cells (TEMs; median 18.6 vs 10.9%, p=0.047) and Granzyme K⁺CD8⁺TEMs (8.8 vs 5.1%, p=0.048). Compared to NR, CD8⁺TEMs from R expressed higher levels of cytotoxic genes and ZNF683, but less CXCR4. Longitudinally, NR exhibited expansion of TIM3⁺CD8⁺ T cells (time×response β=1.91, p=0.025) and impaired T cell function. The Blina-induced peak of TIM3⁺CD8⁺ T cells correlated positively with baseline CXCR4 (r=0.88, p=0.03) and inversely with ZNF683 (r=–0.89, p=0.03) expression in CD8+TEMs. Non-responders exhibited higher circulating levels of immunosuppressive proteins, including CSF-1, ICOSLG, and Galectin-9, throughout the first treatment cycle. To our knowledge, this is the largest study to determine factors of treatment resistance to blinatumomab. A pre-existing immunosuppressive milieu and subsequent treatment-induced T-cell exhaustion are associated with treatment failure and might serve as biomarkers for risk stratification as well as potential targets to enhance Blina efficacy in BCP-ALL.
