TRP2/HepB human IgG1 DNA stimulated similar frequency but higher avidity responses to peptide-pulsed DC. Other studies have failed to show protection from established tumors in TRP2 peptide immunized mice but peptide-pulsed DC induced tumor rejection 30. If the technology Protein Tyrosine Kinase inhibitor described here can be transferred into a clinical setting, it would allow a vaccine to be manufactured that is superior to DC vaccination. It would

also overcome the variability, expense and patient specificity problems associated with conventional DC-based therapies. Previous studies have shown xenogeneic DNA immunization breaks tolerance to self epitopes but using syngeneic DNA is only successful if Ag is linked to a foreign immunogenic protein

31, if it is encoded within a viral vector 32 or if various adjuvants are used 33, 34. The generation of therapeutic find more anti-tumor immunity has also been demonstrated in the absence of regulatory T cells 35. Enhanced responses of TRP2/HepB human IgG1 DNA immunization compared to syngeneic Ag DNA suggests that epitope removal out of the whole Ag context overcomes the inhibition by any regulatory elements within that whole Ag sequence. How does immunization with TRP2/HepB human IgG1 DNA enhance avidity? In vitro stimulation of splenocytes, from B16 GM-CSF-immunized mice with low doses of TRP-2 180–188 peptide generates high-avidity responses. These results indicate that a repertoire of T cells specific for the TRP2 180–188 epitope exists and that they can be modulated to high functional avidity 27. It is therefore possible that TRP2/HepB human IgG1 DNA to may be working by providing a low dose of Ag to stimulate high-avidity responses. The difference in responses generated from TRP2 human IgG1 DNA compared to the protein equivalent suggests that the direct transfection of skin APC plays a role in the generation of these immune responses. The gene gun was initially believed to stimulate CTL by direct transfection

of skin APC but has more recently been shown to also induce CTL via cross presentation 36, 37. We have also shown that the FcγR is important in generating high-avidity but not high-frequency responses from the DNA vaccination. It is of interest that there is often low and high-frequency groups within the immunized mice (see Fig. 3A). This probably reflects the degree of direct versus cross presentation. If immunization fails to transfect a significant number of APC they will have a lower response than mice with efficient APC transfection. This is a parameter which is hard to control with either gene gun or electroporation and is not enhanced with the use of cytokines such as GM-CSF or adjuvants such as imiquimod (result not shown). Reports in the literature have previously demonstrated that vaccine induced T-cell responses can be enhanced by Ab 38–40. A recent elegant study by Saenger et al.