Anti-EGFR Bioengineered Bacterial Outer Membrane Vesicles as Targeted Immunotherapy Agents in Triple-Negative Breast Tumor Murine Model

Abstract Cancer immunotherapy using checkpoint inhibitors is considered a promising approach in various cancers, however, around 55–87% of patients fail to respond to this treatment. Combinatorial therapy through targeted nano-size particles carrying various Toll-like receptor (TLR) agonists to the tumor site can enhance the therapeutic index by activation of intratumoral antigen-presenting cell (APC). Outer membrane vesicles (OMVs) secreted by all gram-negative bacteria present various surface-exposed immune stimulators in their native conformation and Toll-like receptor activating components. In this study, OMVs were engineered to target highly overexpressing EGFR cancer cells in vitro. Affinity-based assays with both EGFR positive and negative cells were performed, and the equilibrium dissociation constant (Kd) of the designed scFv was calculated. Then, we employed the syngeneic model of a triple-negative mouse breast cancer, 4T1, to assess the function of bioengineered OMVs in vivo. Bioengineered OMVs were able to attach to EGFR high-expression cells in vitro. Analyzing the effectiveness of these nanovesicles in a triple-negative breast tumor mice model indicated that the administration route plays an important role in stimulating anti-tumor response. Both intraperitoneal (i.p.) and intratumoral (i.t.) injections of bioengineered OMVs stimulated innate immune response by activating natural killer cells and decreasing the rate of M2 macrophage in the tumor microenvironment. Contrary to the i.p. route with significant tumor size reduction, the in i.t. route only the growth of the tumor was inhibited. Considering both in vitro and in vivo results obtained in this study indicates the potential of OMVs as an effective anti-tumor strategy in future studies.