An ideal vaccine carrier should deliver the target antigen to antigen- presenting cells (APCs), while, at the same time, promoting cellular activation. In this respect, we have hypothesized that utilization of the chemoattractant peptides as carriers for TAAs would fulfill these requirements, as they can both target and attract APCs, and in some cases (e.g., MBD2/mDF2b and HBD3) can also activate cells via TLRs. As we reported in several our papers (Biragyn et al. 1999;2001;2002, 2004;2007;Schiavo et al., 2006), chemoattractants are indeed potent inducers of antitumor immunity against variety of tumors in mice by targeted delivery of TAAs to chemokine receptors on APCs. The vaccine did not require use of any adjuvants, but it was essential that tumor antigen was fused physically with a functionally active chemokine, since immunizations with unlinked free chemokine plus antigen did not induce immune responses. The breadth of the chemokine-based vaccines was in their ability to efficiently utilize the MHC processing pathways to elicit CD4+ T helper and CD8+ T cell responses. These results were published in Blood in two subsequent papers (Biragyn et al., Schiavo et al., 2006). 1) Based on these results, we have developed chemokine-based vaccine for human use. The vaccine targets an embryonic antigen OFA-iLRP, a highly conserved the 37-kDa oncofetal immature laminin receptor, that is specifically and highly expressed in a number of human malignancies. Our data indicate that their delivery to iDCs expressing CCR6 elicit potent therapeutic and protective antitumor immune responses against A20 lymphoma in mice. A20 tumor is the most difficult to cure model that closely mimics human B cell malignancy. Although OFA-ILRP shares the same protein backbone with 67-kDa laminin receptor protein, the vaccine did not induce any harmful autoimmunity in mice. Thus, we propose that the vaccine would also be effective for treatment of human malignancies which express OFA-iLRP. The study has been recently successfully completed and published (Biragyn et al., 2007). OFA-iLRP vaccine has been thoroughly tested in mice and is ready to be used in human clinical trials. 2) SPANX-B (a sperm protein associated with the nucleus on the chromosome X -B) is novel antigen with unknown function. We have demonstrated that SPANX-B is cancer-testis antigen. The survey of a panel of human primary 145 tumors on a multi-tumor tissue microarray has revealed that SPANX-B is abundantly expressed in ovarian, colon, breast and lung cancers and melanoma. Moreover, at least in melanoma, its expression pattern was associated with the metastatic stage. We have also demonstrated that SPANX-B is an immunogenic antigen, as healthy humans contained circulating SPANX-B -specific T cell precursors that could be readily expanded to generate both helper CD4+ T cells and cytolytic CD8+ T cells. We have defined at least two immunodominant HLA-A2 restricted epitopes that can be used to elicit SPANX-B specific CD8+ CTLs. The CD8+ T cells, which were raised using these peptides, recognized and efficiently killed the HLA-matched human primary melanomas. On the other hand, we have also determined at least one HLA-DR-restricted and immunodominant epitope, which was used to generate the SPANX-B specific CD4+ T cells. Thus, SPANX-B is an immunogenic and clinically relevant antigen that has significant therapeutic potency as vaccine target. The results of this study has been recently published (Almanzar et al., 2009). However, we were not so lucky with vaccines expressing Plac1, as they did not protect mice against 4T1 cancer challenge, and we also did not elicit CTLs in vitro using mouse and human cells. Thus, clinical relevance of Plac1 remains debatable. 3) Chemokine receptors are also used by a small family of antimicrobial peptides (AMPs), namely defensins. For example, human and murine b-defensins attract immature DCs utilizing chemokine receptor CCR6 (Yang et al., 1999;Biragyn et al., 2001). In fact, we have been first to discover that antimicrobial peptide mDF2b also functions as an endogenous immunomodulatory protein that activates APCs via TLR4 (Biragyn et al., 2002). A human counterpart for mouse mDF2b, which activates human DCs, is not found yet. However, others have recently confirmed our finding by demonstrating that human b-defensin 3 can also activate TLR1 and TLR2 (Funderburg et al., 2007). We report here that, besides DC maturation, mDF2b induces atypical death of APCs by exhibiting characteristic features of apoptosis and necrosis. Although mDF2b-treated APCs produced a number of potentially cytotoxic cytokines, the process was mediated by membrane-bound TNF. However, it did not utilize its apoptosis-inducing TNFR1 receptor, as mDF2b also efficiently killed APCs with deficient TNFRI. Instead, it was using TNFRII (a second TNF receptor that does not have death domains). To do this, mDF2b up regulated surface expression of both TNF and TNFRII presumably to facilitate their engagement and signaling. In support, mDF2b failed to induce cell death of APCs from mice with non-functional TNFRII or TNFa. Taken together, we have demonstrated that mDF2b induces two distinct processes separated by time: first, as we previously reported (Biragyn et al., 2002), it activates APCs inducing TLR-4/MyD88 signaling cascade that manifests in maturation of iDCs within one day. However, starting from the third day, the activated cells get self-destruction signaling from up regulated expression of membrane-bound TNF and TNFRII. This is p38, ERK1/2 and the NF-kB dependent process, as mDF2b failed to promote death in APCs from mice with non-functional p50 NF-kB, or in w.t. DCs treated in presence of inhibitors of NF-kB activity such as TPCK. The biological meaning of our observation remains unknown, though we speculate that it might be an important and general step for eliminating harmful long-term effects of activated or over-activated DCs and macrophages. These data have been recently published (Biragyn et al., 2008).
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