Indoleamine 2,3-Dioxygenase (IDO) silencing for improved antitumour vaccination

Background As tumor progresses the efficacy of vaccination is tuned down by suppressive activities. The administration of adjuvants or the silencing of specific immune regulatory molecules will optimize the function antigen presenting cells (APC) and will permit the immune response elicited to be active at the tumor site. Indoleamine 2, 3-dioxygenase (IDO) the enzyme that degrades the essential amino acid tryptophan in mammals is overexpressed in both tumor cells and APCs in tumor-draining lymph nodes, where it promotes the establishment of peripheral immune tolerance to tumor antigens. IDO seems to be an ideal target to be silenced for the optimal induction of an antitumor immune response. We plan to use plasmids coding short shRNA specific for IDO to be administered together with the plasmid coding portion of Erbb-2, or plasmids containing both the shRNA module and the oncoantigen module, in vaccination-protection tests in BALBneuT mice transgenic for the rat Erbb-2. Material and methods Retroviral vectors (pLKO.1, Open Biosystem®) including five shRNA sequences targeting IDO mRNA have been used as template to amplify the interference cassettes (pU6-shRNA-IDO) that we cloned into the Eco72I site of both pVAX1 (Invitrogen®). The gene silencing efficacy of the various interference cassettes was evaluated in a kynurenine assay using N11 microglial cells (Grant et al. 2000). The most efficacious cassettes were subcloned into a pVAX vector containing the sequence of the extracellular and transmembrane domains of rat Erbb-2 (pVAX-ratECTM) and used for vaccination of BALB-neuT mice carrying different stages of mammary carcinogenesis. Results All the five interference cassettes were able to reduce kynurenine release from N11 cells, confirming their ability to silence IDO expression. Two cassettes were chosen to be subcloned into pVAX-ratECTM, and used to vaccinate BALB-neuT mice bearing atypical hyperplasia and in situ carcinomas (weeks 10 and 12 of age) or microscopic invasive carcinomas (weeks 16 and 18). The in vivo observation of mammary cancer progression is still ongoing. Conclusions We expect that this simultaneous alteration of tumor microenvironment and induction of an immune response against Erbb-2 elicits an anti-tumor response of therapeutic significance, in that it halts the progression of lesions that cannot be inhibited by Erbb-2 vaccination alone.