Calreticulin-Targeting L-Asparaginase-Flagellin Conjugate Enhances Salmonella-Mediated Antitumor Efficacy

Targeted therapeutics have transformed cancer treatment by selectively eliminating malignant cells while limiting systemic toxicity. L-asparaginase (L-ASNase), which induces metabolic stress by depleting asparagine (Asn), is clinically used for hematological malignancies but shows limited activity against solid tumors due to poor delivery and an immunosuppressive microenvironment. We previously developed CRT3LP, a calreticulin (CRT)-targeting monobody-L-ASNase conjugate, designed to exploit immunogenic cell death (ICD); however, its therapeutic potential is constrained by insufficient immune activation. Here, we show that CRT3LFP, a multifunctional fusion protein incorporating the flagellin B subunit (FlaB) into the CRT3LP scaffold, potentially promotes M2-to-M1 macrophage polarization while maintaining tumor-selective metabolic disruption. In combination with the tumor-colonizing bacterial strain CNC018, which induces surface-exposed CRT, CRT3LFP achieves precise tumor localization and promotes M2-to-M1 macrophage polarization. This synergistic approach significantly inhibits tumor growth and reshapes the tumor microenvironment (TME), characterized by enhanced maturation of dendritic cells (DCs) and expanded CD8 ⁺ T cells. Furthermore, CD47-SIRPα blockade further potentiates this effect, leading to complete tumor eradication and the establishment of durable immune memory. Together, our findings establish CRT3LFP as a tumor-targeted immunometabolic platform that integrates metabolic deprivation with coordinated innate and adaptive immune activation to overcome resistance in solid tumors.