Background: Trophoblast cell surface antigen 2 (TROP2) is widely overexpressed in epithelial cancers and associated with poor prognosis, representing an attractive target for cancer treatment. Recent clinical studies reported favorable outcomes of TROP2-targeting antibody–drug conjugates. However, heterogeneity of human cancers and development of resistance encourage the exploration of alternative cytotoxic payloads. In this regard, bacterial immunotoxin is a viable treatment option owing to the unique and potent mechanism of action. Methods: We first constructed three immunotoxins by fusing deimmunized Pseudomonas Exotoxin A (PE24) to novel TROP2-targeting single-chain variable fragments identified through phage display, which were screened for high binding and cytotoxic activity against tumor cell lines with distinct TROP2 expression. The lead immunotoxin T3PE was engineered by introducing extra disulfide bonds and an albumin-binding domain (ABD) to generate disulfide-stabilized T3PE (dsT3PE and dsT3PE_ABD_CC). Then, both variants were characterized in stability, cell binding, internalization, pharmacokinetic profiles, and in vitro and in vivo antitumor activities. Results: T3PE bound strongly to TROP2 (EC₅₀ = 0.3824 μg/mL) and exerted potent cytotoxicity against NCI-N87 cells (EC₅₀ = 0.09074 μg/mL), but it displayed poor serum stability and was prone to aggregation. Introduction of disulfide bonds stabilized the immunotoxins while preserving the potent cytotoxic activity, and fusion of ABD extended the plasma half-life 6-fold in mice. Additionally, five doses of either dsT3PE or dsT3PE_ABD_CC at 3 mg/kg could significantly reduce tumor progression in the NCI-N87 mouse xenograft model. Conclusions: Together, these results demonstrated the therapeutic potential of these novel anti-TROP2 immunotoxins against TROP2-overexpressing tumors.

Background: The efficacy of chemotherapy in triple-negative breast cancer (TNBC) is limited by intrinsic resistance and the tumor microenvironment (TME). Accumulating evidence reveals a mechanistic connection between programmed death-ligand 1 (PD-L1) and c-x-c motif chemokine receptor 4 (CXCR4), which dominate stroma barriers, immune escape, and cancer metastasis. Earlier studies have shown that dual suppression of c-x-c motif ligand 12 (CXCL12)/CXCR4 and programmed cell death-1 (PD-1)/PD-L1 pathways regulates extracellular matrix (ECM) deposition, activation of cancer-associated fibroblasts (CAFs), and epithelial–mesenchymal transition (EMT) of pancreatic cancer cells. Methods: We combined BsNb PX4, a bispecific nanobody targeting PD-L1 and CXCR4, with paclitaxel or gemcitabine in multiple tumor cell lines and human peripheral blood mononuclear cell (hPBMC)-reconstituted xenograft mouse models. Antitumor activity was assessed by CCK-8, flow cytometry, and ELISA, and immune cell infiltration and TME remodeling were examined by immunofluorescence, immunohistochemistry, cytokine assays, and RNA-seq. Results: In MDA-MB-231 cells, BsNb PX4 synergistically enhanced paclitaxel-induced growth inhibition and apoptosis via G₂/M cycle arrest. This combinatorial strategy profoundly remodeled tumor immunity by expanding CD8⁺ T cells and depleting Foxp3⁺ CD4⁺ regulatory T cells (Tregs), while concurrently restoring T-cell cytotoxicity and skewing the cytokine balance toward an antitumor state, with elevated IFN-γ and reduced TGF-β1. Notably, compared with paclitaxel monotherapy, the combination significantly elevated intratumoral CD8⁺ T-cell infiltration, decreased Treg abundance, and exerted robust inhibitory effects on tumor growth and metastasis in humanized TNBC xenografts. Conclusions: These findings reveal that dual blockade of PD-L1 and CXCR4 acts synergistically with chemotherapy by triggering tumor cell apoptotic effects and reversing the immunosuppressive microenvironment, thereby emerging as a promising therapeutic strategy for TNBC.