Piia Halmi

 Two main challenges in vaccine and immunotherapy technology are to increase the potential to generate potent defences against chronic diseases evading the immune system, and to develop effective immunity after single injections of vaccine. An antitumor vaccine has to have an ability to induce robust and sustained tumor-specific T cells responses. The initiation of these responses is dependent on the presentation of the antigen by dendritic cells (DCs). Recent strategies for developing preventative and therapeutic vaccines are focused on the ability to deliver antigen to DCs in a targeted and prolonged manner. A main interest of cancer therapy today is to develop mechanisms for induction of T-cell responses through presentation of antigens by DCs. DCs can be easily generated ex vivo from peripheral blood monocytes or bone marrow/circulating haematopoietic stem cells cultured in the presence of cytokines. DCs are the most effective antigen-presenting cells (APCs) and are able to prime CD4+ and CD8+ T cells, and ex vivo experiments of these cells have allowed the development of DC-based vaccines. These kinds of vaccines do not give long-term cures, and general limitations of using these vaccines are pour traffic of DCs to spleen and draining lymph nodes, and the clearance of survived DCs by host cytotoxic T-cells (CTLs). The critical parameter for outcome of DC-based vaccination is the number of DCs reaching the lymph node. No quality criteria are suggested for their capacity to migrate. DCs could serve as an early state treatment of cancer such as the adjuvant setting(Banchereau & Steinman, 1998; Banchereau et al., 2001; Banchereau & Palucka, 2005; Cayeux et al., 1999; Chang et al., 2002; Eggert et al., 1999; Fong et al., 2001; Kugler et al., 2000; Martin-Fontecha et al., 2004; Murphy et al., 1999; Nair et al., 2002; Nencioni & Brossart, 2004; Nestle et al., 1998; Thurner et al., 1999; Timmerman et al., 2002).

There are several types of investigations today trying to increase the expression of antigen by DCs in vivo. In vivo DC-targeting strategies use free antigen, protein fusions or viral gene therapy. Approaches using protein antigens conjugated to DC-specific antibodies, heat-shock proteins or viral replicon particles have produced effective results in vivo. Success of these strategies depends on overcoming biological delivery challenges and the weak immunogenicity of many antigens. Immunogenic properties and physiological transport barriers are investigated to achieve more-efficient antigen delivery to DCs (Bonifaz LC et al., 2004; Davis NL et al., 2002; Hauser H et al., 2004; Mahnke et al., 2005). One approach consists of pulsing DCs in vitro with antigenic peptides before transferring them back to the patient (Nestle et al., 1998; Thurner et al., 1999; Zitvogel et al., 1996). Another approach analogous for the latter one consists of transducing DCs with an antigen-recombinant viral vector. Transduction results to be more efficient method than pulsing (Brossart et al., 1997; Germain & Margulies, 1993; Song et al., 1997).

One strategy is the transduction of haematopoietic stem cells (HSCs) with genes encoding antigen, followed by transplantation of these gene-modified cells into irradiated mice. Antigen-encoding genes are introduced into the HSCs, which results in an effective delivery of antigens to the DC progenitors. Lentiviral vector technology is used as an effective transduction method, and adding autologous donor lymphocyte infusions (DLI), Flt-3L, and an activating antibody to CD40, a large numbers of DCs in vivo are produced, and the maturation of DCs enhances protective effect of antitumor vaccines. This tripartite strategy provides a potent antigen-specific immunotherapy for an aggressive established tumor (Cui et al., 2003).

Another investigated strategy is to evaluate the potential of lentiviral vectors as in vivo-administered T cell vaccines. Because it’s expensive and laborious to generate and manipulate ex vivo DCs, a cell-free, easily administered vaccine would be even more efficient. Esslinger et al. (2003) observed that lentiviral vector administration transduced DCs that appeared in the draining lymph node and in the spleen. This vaccine induces potent T cell responses up to 40% antigen-specific cells among the CD8+ subset and has high levels of specific cytotoxicity. A decisive factor for efficient T cell priming by lentiviral vector could be the targeting of DCs in situ and their migration to secondary lymphoid organs (Esslinger et al., 2003).

Receptor for hyaluronan-mediated motility (RHAMM) is overexpressed in various tumors. RHAMM has been identified as an immunogenic antigen by serologic screening of cDNA expression libraries. A plasmid for transduction of in vitro-transcribed mRNAs into DCs to efficiently transport the intracellular protein RHAMM into MHC class II compartments by adding a late endosomal/lysosomal sorting signal to the RHAMM gene was constructed. Mice immunization with modified RHAMM mRNA-transfected DCs induced killing activity against RHAMM-positive tumor cells in splenocytes. An anti-CD4 or anti-CD8 antibody was administered to mice after immunization. CD8+ T cell depletion had no effect. Depletion of CD4+ T cells diminished the induction of tumor cell-killing activity in splenocytes. Later DC/RHAMM was administered to mice on days 3, 7 and 10 after EL4 tumor inoculation, which inhibited tumor growth compared to control DCs. Antibody-mediated depletion of CD4+ T cells completely abrogated the therapeutic effect of DC/RHAMM, whereas depletion of CD8+ T cells had no effect. This indicates that DCs transfected with a modified RHAMM mRNA targeted to MHC class II compartments can induce CD4+ T cell-mediated antitumor activity in vivo (Fukui et al., 2006).

DCs pulsed with tumor antigen ex vivo have applications in tumor immunotherapy. van Broekhoven et al (2004) have investigated a crude preparation of plasma membrane vesicles (PMV) from the highly metastatic murine melanoma (B16-OVA) and a surrogate tumor antigen (OVA), and their direct targeting to DCs in vivo to elicit functional effects. B16-OVA-derived PMV was incorporated with a metal-chelating lipid (3(nitrilotriacetic acid)-ditetradecylamine), which forms a recombinant hexahistidine-tagged forms of single chain antibody fragments to the DC surface molecules CD11c and DEC-205. The flow cytometry and fluorescence confocal microscopy experiments demonstrate that the modified PMV containing OVA or OVA peptide antigen target DCs in vitro and in vivo. In syngeneic mice these stimulate a strong B16-OVA-specific CTL responses in splenic T cells and a marked protection due to a simultaneous delivery of both antigen and the DC maturation against tumor growth. This administration of the DC-targeting vaccine to mice induced a dramatic immunotherapeutic effect and prolonged disease-free survival (van Broekhoven et al., 2004).Drug resistance is a major cause of chemotherapy failure in patients with cancer. New target to reverse this resistance is the characterization of the molecular pathways involved in drug resistance. These target proteins are often overexpressed in human glioma and they are tumor antigens, which implicate the development of immunotherapy as a therapeutic strategy. Dendritic cells (DCs) stimulate antibody and cell-mediated immune responses against tumor-associated antigens. Ex vivo-generated and tumor antigen-loaded DCs have been used in clinical vaccination protocols, which have been effective in some glioma patients. Immunotherapy followed by chemotherapy could significantly increase 2-year survival in malignant glioma patients. The sensitivity of tumor cells to chemotherapy could increase using DC vaccination (Liu et al., 2006).