Focused acoustic vortex-mediated targeted aggregation of engineered bacteria for in situ antibody production to enhance immunotherapy

Immune checkpoint blockade (ICB)-based immunotherapy is limited by its poor tumor targeting and rapid clearance of antibodies. Here, we developed the acoustic vortex (AV) tweezers that actively aggregated microbots for spatiotemporally controlled in situ explosive anti-PD-L1 production to achieve localized ICB-based immunotherapy. Engineered bacteria were conjugated with multifunctional nanoparticles combining chemotherapy, sonodynamic therapy, and plasmid-encoded anti-PD-L1, forming microbots capable of ultrasound-mediated aggregation and deep tumor penetration. AV tweezers with a larger annular focal region compared with conventional ultrasound, actively induced microbot aggregation in tumor-associated vasculature, while mild hyperthermia triggered expression and secretion of interferon-gama (IFN-γ), promoting M2-to-M1 macrophage polarization. The release of doxorubicin (DOX) and chlorin e6 (Ce6) induced immunogenic cell death, synergistically enhancing anti-tumor responses. The localized antibody production factory achieved high intratumoral anti-PD-L1 level while limiting systemic exposure, thereby overcoming major limitations of conventional ICB therapy. Hence, this strategy established a programmable and dual-targeting platform for precise tumor immunotherapy. Statement of Significance 1. Focused Acoustic vortex (AV) selectively aggregated engineered bacteria in tumor-associated vascular to achieve in situ antibody production for precise immunotherapy. 2. Engineered bacteria overcame the delivery challenges of nanomedicines in tumors for enhanced sonochemotherapy. 3. Single AV irradiation leads to improved uniform drug bioaccumulation. 4. Mild hyperthermia regulated by AV led to the secretion of IFN-γ, promoting M2-to-M1 macrophage polarization and reshaping the tumor microenvironment.