Cytokine‐modified Mycobacterium smegmatis as a novel anticancer immunotherapy

Intravesical administration of live M. bovis BCG organisms for carcinoma in situ of the urinary bladder is the most successful immunotherapy for solid malignancy. Nevertheless BCG‐therapy is associated with significant toxicity and is ineffective in 30–40% of cases. Recently it has been proposed that cytokine‐modified mycobacteria may give greater efficacy. As any immunotherapy involving administration of live BCG organisms (wild‐type or recombinant) is likely to have associated toxicity (notably in the immunocompromised), we examined the anti‐tumour potential of the closely related nonpathogenic organism, Mycobacterium smegmatis, and a TNFα gene‐modified recombinant M. smegmatis. When wild‐type M. smegmatis were delivered to immunocompetent C57Bl/6 mice bearing the transplantable MB49 bladder tumour, efficacy comparable to live BCG was observed with 10–20% long‐term survival. However, this effect was lost in both Nude and Beige mice, lacking functional T and NK cells, respectively. Recombinant M. smegmatis secreting TNFα, however, gave a 70% durable tumour‐free survival. Lymphocytes from draining lymph‐nodes and spleens of these mice exhibited pronounced IFNγ production to mycobacterial‐antigen and tumour‐lysate, indicating a bias towards cell‐mediated immunity. This was further supported by histopathological examination of the tumour site, which revealed significantly increased numbers of CD3+ lymphocytes in animals receiving the recombinant vaccine, but not in those receiving wild‐type bacteria. Importantly, tumour rejection following M. smegmatis/TNFα was independent of T lymphocytes, as athymic Nude mice efficiently eradicated MB49 tumours. In contrast, the therapeutic efficacy of M. smegmatis/TNFα was reduced in animals deficient in NK cytolytic function, suggesting a role for NK‐cells in initial tumour destruction. Furthermore the absence of NK‐function in Beige mice did not prevent the establishment of tumour‐protective memory. No toxicity was observed with wild‐type or recombinant M. smegmatis in immunocompetent, T‐deficient or NK‐deficient models. We demonstrate for the first time that recombinant mycobacteria expressing mammalian cytokines have markedly increased anti‐tumour properties. The lack of toxicity suggests that M. smegmatis is a “safe” vehicle for use in immunocompromised patients. © 2004 Wiley‐Liss, Inc.