Vaccine 28 (2010) 3522–3530 Contents lists available at ScienceDirect Vaccine journal homepage: www.elsevier.com/locate/vaccine A TLR9 agonist enhances therapeutic effects of telomerase genetic vaccine Antonella Conforti a , Barbara Cipriani a , Daniela Peruzzi a , Sridhar Dharmapuri a , Ekambar R. Kandimalla b , Sudhir Agrawal b , Federica Mori a , Gennaro Ciliberto a,1 , Nicola La Monica a,2 , Luigi Aurisicchio a,∗,3 a I.R.B.M. P. Angeletti, Via Pontina km 30,600, 00040 Pomezia, Italy b Idera Pharmaceuticals, Cambridge, MA, USA article info Article history: Received 6 August 2009 Received in revised form 18 February 2010 Accepted 10 March 2010 Available online 21 March 2010 Keywords: Cancer vaccine TERT IMO Innate immunity abstract The telomerase reverse transcriptase (TERT) is an attractive target for cancer vaccination because its expression is reactivated in most tumors. In this study, we have evaluated the ability of a genetic vaccine targeting murine TERT (mTERT) based on DNA electroporation (DNA-EP) and adenovirus serotype 6 (Ad6) to exert therapeutic effects in combination with a novel TLR9 agonist, referred to as immune modulatory oligonucleotide (IMO), as an adjuvant. IMO was administered to mice at the same time as vaccine. IMO induced dose-dependent cytokine secretion and activation of NK cells. Most importantly, vaccination of mice with IMO in combination with mTERT vaccine conferred therapeutic benefit in tumor bearing animals and this effect was associated with increased NK, DC and T cell tumor infiltration. These data show that appropriate combination of a DNA-EP/Ad6-based cancer vaccine against TERT with IMO induces multiple effects on innate and adaptive immune responses resulting in a significant antitumor efficacy. © 2010 Elsevier Ltd. All rights reserved. 1. Introduction Telomerase is a ribonucleoprotein comprising an RNA com- ponent and a catalytic protein component (telomerase reverse transcriptase, TERT) [1,2]. Telomerase activity has been detected in cancerous cell lines and a diverse range of tumor types [3]. Con- versely, telomerase is inactive or only transiently expressed at low levels in normal human tissues and normal somatic cell cultures. The observation that telomerase is over-expressed in most tumor types and barely detectable in most normal cells makes TERT a tumor associated antigen (TAA) and a suitable target for cancer immunotherapy. In vivo electroporation of plasmid DNA (DNA-EP) and replication-defective recombinant adenoviruses (Ad) have proven a very efficacious platform to induce strong antibody and cellu- lar immune responses against a wide variety of antigens, including tumor TAAs [4]. Combinations of these modalities of immuniza- tion have been shown to induce superior immune reactions as compared to single vaccines [5–7]. However, the vaccination per se ∗ Corresponding author. Tel.: +39 06 91093233; fax: +39 06 91093654. E-mail address: luigi aurisicchio@libero.it (L. Aurisicchio). 1 Current address: University of Catanzaro, Dept of Experimental and Clinical Medicine “G. Salvatore”, Campus Germaneto 88100 Catanzaro, Italy. 2 Current address: Idera Pharmaceuticals, Cambridge, MA, USA. 3 Current address: Biogem IRGS, Dept of Molecular Oncology via Camporeale 83031 Ariano Irpino (AV), Italy. may have limited impact on late-stage patients because tumors can rapidly induce immuno-suppression in the host, compromising the response to the vaccine via a variety of complex mechanisms [8]. Thus, combination therapies are needed to synergize with cancer vaccines [9]. One of the most promising targets for therapeutic immune acti- vation is TLR9, which detects unmethylated CpG dinucleotides present in viral and prokaryotic genomes, that are generally methy- lated in host DNA [10,11]. TLR9 stimulation first activates innate immunity with a predominantly Th1-type cytokine response. In the second, adaptive phase of the immune response, TLR9 acti- vation enhances antigen-specific humoral and cellular responses following concomitant exposure to a wide variety of antigens in both prophylactic and therapeutic vaccines in animal models [10,11]. Several mechanisms contribute to the strong adjuvant activity of TLR9 agonists, including stimulation of antigen-specific B cells, inhibition of B-cell apoptosis, enhanced immunoglobulin G (IgG) class switch, and the generation of cytotoxic T lympho- cyte (CTL) [12]. The activation of immune responses through TLR9 can promote tumor regression either directly, through the antitu- mor activity of factors such as IFN and TRAIL [13], or indirectly, through the activation of natural killer (NK) cell-mediated tumor killing [14]. TLR9 agonists induce the functional maturation of den- dritic cells through the up-regulation of co-stimulatory molecules for T lymphocytes, the secretion of cytokines important for the ini- tiation of adaptive responses such as type I interferons and IL-12, and the modulation of the chemokine receptor repertoire allowing migration into lymphoid organs [15]. 0264-410X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2010.03.020