Mycobacterial fusion protein REA eliciting anti-tumor activity through activation of innate immunity

Mycobacterial components are widely used as immunoadjuvants. Mycobacterium bovis bacillus Calmette-Guérin (BCG) remains the gold standard for treating high-risk, non-muscle-invasive bladder cancer, and its cell wall skeleton has been used to treat several cancers. Therefore, we investigated the anti-tumor activity of mycobacterial fusion protein Rv2299cD2D3-ESAT-6-Ag85B (REA) and its potential role as a mycobacterial-derived immunomodulator. REA exhibited potent antitumor effects, driving robust innate immune activation that secondarily engages adaptive immune responses against tumor-derived antigens. In vitro, REA-activated DCs enhanced T cell cytokine production and cytotoxicity, inducing apoptosis of LLC cells. Concurrently, NK cells stimulated by REA-activated DCs or REA alone showed increased NKG2D, IFN-γ, and granzyme B expression, along with decreased NKG2A, further promoting tumor cell apoptosis. In vivo, REA formulated with the adjuvant markedly inhibited LLC tumor growth after two subcutaneous injections, accompanied by strong infiltration of activated NK⁺ and CD8⁺ T cells in tumors and peripheral organs. Importantly, the depletion of either NK⁺ or CD8⁺ T cells abolished REA-mediated tumor suppression. Moreover, REA enhanced the efficacy of cisplatin to improve tumor control and immune activation, with increased cytokine secretion in the tumor and spleen. Its anti-tumor efficacy extended beyond that of LLC, showing benefits in MC38 colon and MBT-2 bladder carcinoma models. REA also suppressed LLC lung metastasis and provided prophylactic protection against tumor establishment. Collectively, these findings indicated that REA is a promising immunostimulatory agent with therapeutic potential against cancer.