Repeated Administration of Cytosine-Phosphorothiolated Guanine-Containing Oligonucleotides Together with Peptide/Protein Immunization Results in Enhanced CTL Responses with Anti-Tumor Activity

Abstract

The development of therapeutic anti-cancer vaccines designed to elicit CTL responses with anti-tumor activity has become a reality thanks to the identification of several tumor-associated Ags and their corresponding peptide T cell epitopes. However, peptide-based vaccines, in general, fail to elicit sufficiently strong CTL responses capable of producing therapeutic anti-tumor effects (i.e., prolongation of survival, tumor reduction). Here we report that repeated administration of synthetic oligonucleotides containing foreign cytosine-phosphorothiolated guanine (CpG) motifs increased 10- to 100-fold the CTL response to immunization with various synthetic peptides corresponding to well-known T cell epitopes. Moreover, repeated CpG administration allowed the induction of CTL to soluble protein even in the absence of additional adjuvant. Our results indicate that the potentiating effect of CpG in CTL responses required the participation of Th lymphocytes. Repeated CpG administration resulted in overt splenomegaly and lymphadenopathy with a significant increase in the numbers of CTL precursors and dendritic cells. Protein vaccination in combination with repeated CpG therapy was effective in delaying tumor cell growth and extending survival in mice bearing melanoma tumors. These findings support the contention that repeated administration of CpG-oligonucleotides enhances the effect of peptide and protein vaccines leading to potent anti-tumor responses, presumably through the induction of Th1 and dendritic cells, which are essential for optimal CTL responses. The immunostimulatory properties of CpG motifs may be key in inducing a consistent long term immunity to tumor-associated Ags when using peptides or proteins as T cell-inducing vaccines.

Characterization of the MUC1.Tg/MIN transgenic mouse as a model for studying antigen-specific immunotherapy of adenomas

“…Although some studies have showed induction of MUC1-specific antibodies in animal models and cancer patients [Reviewed in 10], our studies demonstrating the induction of CTL responses corroborate numerous reports in which CpG-ODN alone [33,37,48,49] or in combination with GM-CSF [24] were used as biologic adjuvants in peptide vaccines. A likely explanation of our results is that MHC class I-restricted MUC1 peptides which stimulate CD8 + T cells are acting in concert with CpG-ODN and GM-CSF in the vaccine cocktail to promote strong T H1 type responses resulting in the generation of efficient CTL anti-tumor responses [24,33,37,48,50]. In addition, inclusion of CpG-ODN in the vaccination cocktail in our study did not cause destruction of the lymphoid follicles or prevent isotype switching in treated mice (data not shown) as was reported by Heikenwalder, et al [51].…”

“…The MHC class I peptides confer MUC1 specificity leading to the induction of CTL-mediated anti-tumor responses; the Hepatitis B core antigen-derived MHC class II pan T helper peptide was included to boost CD8 + anti-tumor responses [30,31]; CpG-ODN was included to promote DC activation via its interaction with Toll-like receptor (TLR)-9 [32,33] to enhance CTL responses, expansion and survival [33][34][35][36][37]. GM-CSF promotes antigen presentation and DC recruitment to vaccine sites [38][39][40][41] and is commonly used as a biological adjuvant in preclinical and clinical immunotherapy studies [42,43].…”

Therapeutic anti‐tumor immunity triggered by injections of immunostimulating single‐stranded RNA

“…Indeed, as seen in the treated mice (Fig. 6) and reported by others [12,29], the repeated injection of very stable phosphorothioate CpG DNA oligonucleotides leads to uncontrolled immune stimulation (evidenced by splenomegaly and potentially leading to autoimmunity). On the contrary, stabilized RNA that do not induce the proliferation of B cells [18] and that have a half-life of approximately 110 h in serum [19] are naturally cleared with reliable and reproducible kinetics from the site of injection, thereby offering a controllable therapeutic window.…”

Maximizing CD8+ T cell responses elicited by peptide vaccines containing CpG oligodeoxynucleotides

“…TRP-2 is a non-mutated protein that is expressed by normal melanocytes in C57BL/6 mice [24]; therefore, it is subject to self-tolerance. Amino acids 257−264 of the ovalbumin protein (OVA 257−264 ) form an immunogenic epitope that is presented by H-2 K b [11]. Amino acids 366−374 of the influenza A nucleoprotein (NP 366−374 ) [2] and amino acids 49−57 of the human papillomavirus E7 protein (E7 49−57 ) [25] form immunogenic epitopes presented by H-2 D b .…”