SMP-105, cell-wall skeleton purified from Mycobacterium bovis BCG Tokyo 172, activates innate immunity through TLR2/MyD88 pathway and prevents tumor metastasis into draining lymph nodes

3554 [Background] Mycobacterium cell walls have diverse adjuvant activity, and in particular, the cell wall skeleton (CWS) of Mycobacterium bovis BCG has been expected as a medicinal product for tumor immunotherapy. The anti-tumor activity of CWS has been investigated using animal models for many years, but in these studies CWS was administered directly into the tumor or admixed with the tumor. Therefore, we cannot estimate the potent activity of CWS correctly because of the influence of local inflammation during administration. Also, the activation mechanism of innate immunity by CWS was controversial because immune responses were affected by the species or strains of CWS and its purity. [Purpose] We optimized a method for preparing CWS from BCG Tokyo 172 strain (SMP-105), analyzed the activity of SMP-105 for macrophages and determined which TLRs participated in its recognition. We established a proper tumor model not affected by local inflammation at injected sites, and investigated the anti-tumor activity of SMP-105. Also, we compared the activity of SMP-105 with live BCG, which is widely used in superficial bladder carcinoma, in order to estimate the potential of SMP-105. [Methods] SMP-105 was prepared from M. bovis BCG Tokyo 172 strain. Activity for the production of cytokines from macrophages was measured using a mouse macrophage cell line RAW264.7. Toll-like receptors for SMP-105 were investigated with a reporter-gene assay, TLR-knocked-down RAW264.7 cells, and macrophages prepared from TLR-KO mice. The anti-tumor activity of SMP-105 and live BCG was evaluated with a lymph node metastasis model using strain 2 guinea pigs bearing line 10 hepatoma. [Results] SMP-105 induced TNF-α in a dose-dependent manner, while live BCG induced TNF-α in lower concentrations than SMP-105. The activation of NF-κB was dependent on TLR2 but not TLR4. The dependency on TLR2 was confirmed using TLR-knocked-down macrophages and TLR-KO macrophages. Weekly administration of 60 μg of SMP-105 and live BCG at a separate site from the implanted tumor suppressed tumor metastasis in draining lymph nodes. [Conclusion] SMP-105 activated macrophages through the TLR2/MyD88 pathway, and SMP-105 showed comparative anti-tumor activity to live BCG. SMP-105 is a promising immunotherapeutic drug.