In vivo expression of CD3/CD19 bispecific T-cell engager and α-PD-L1-Fc enables effective and durable immunotherapy for CD19+/PD-L1+ leukemia.

Approximately 50% of B-ALL patients exhibit Blinatumomab (CD3/CD19 bispecific T-cell engager, BiTE) resistance, often with high tumor PD-L1 expression, limiting therapeutic efficacy. Combining Blinatumomab with PD-L1 blockade is promising but hindered by continuous infusion, short half-life, and high costs. We developed an economical, non-viral, single-dose intramuscular plasmid gene delivery for sustained (≥4 weeks) in vivo co-expression of CD3/CD19 BiTE and PD-L1 antibodies. This platform integrates T-cell-mediated cytotoxicity, immune checkpoint blockade, and antibody-dependent cytotoxicity. This strategy induced substantial leukemia cell regression, achieving an 80% complete remission (CR) rate in CD19⁺/PD-L1⁺ Nalm-6 xenografts and a 40% CR rate in the CD19 antigen-loss immune escape model. Anti-leukemic efficacy correlated with enhanced CD3⁺, CD8⁺ T-cell, CD3⁺CD56⁺ NKT-like cell expansion, and increased granzyme B/IFN-γ levels. Furthermore, in vivo-produced antibodies promoted B-ALL patient-derived CD3⁺ T-cell proliferation and CD19⁺ tumor cell clearance. This study presents a cost-effective, durable, and potent in vivo immunotherapy integrating T-cell engagement and PD-L1 blockade, offering a promising translational approach for B-ALL.