Overcoming Self-Tolerance: Long-Term Protection Against Specific erbB2-Expressing Prostate Tumors Using Low Doses of Lentivirus-Transduced DCs.

Abstract Anti-tumor immunotherapy is difficult to achieve in vivo in part due to naturally occurring peripheral tolerance. Our laboratory has developed a potent immunotherapy strategy that uses unusually low doses of dendritic cells (DCs) to break self-tolerance in a mouse model of prostate cancer. We constructed a lentiviral vector (LV) encoding a truncated form of the true self-antigen murine erbB2 (LV/erbB2tr). This protein is the murine form of HER-2/neu, a tumor associated antigen (TAA) upregulated in 20% of primary prostate tumors and 80% of metastatic cases. With LV/erbB2tr, we achieved highly efficient gene transfer into DCs that were generated from donor murine bone marrow, as up to 47% of DCs over-expressed erbB2tr following one infection (MOI of 3). Similar transduction levels were achieved using a control enGFP LV. The purity of transduced DCs was confirmed by flow cytometry for CD11c, CD80, CD86, and I-Ab expression. We then tested the ability of two low dose inoculations of these transduced DCs to overcome self-tolerance to erbB2 and protect against specific challenge in a bilateral tumor model. Mice were vaccinated twice, two weeks apart by administration of either 2 × 105 or 2 ×103 cells (i.p.). Six weeks later, the mice were injected on one hind flank with the highly aggressive murine tumor cell line RM1 and on the opposite flank with erbB2tr-transduced RM1 (RM1-erbB2tr). We saw complete protection from RM1-erbB2tr tumors in 100% of the mice receiving 2 × 105 erbB2tr-transduced DCs (n=6). Control RM1 tumors were not rejected. No systemic auto-immune responses to the self-antigen were observed, as all mice survived and the procedure was well tolerated. Furthermore, our strategy was effective even using 100-fold fewer DCs in the inoculations. In the mice receiving 2 ×103 erbB2tr-transduced DCs, 33% (2/6 animals) were completely protected from developing RM1-erbB2tr tumors and 2 others showed near-complete protection. At regular points throughout the experiment, serum levels of anti-erbB2tr antibodies were measured. In mice receiving the 2 ×105 DC dose, a peak antibody response was seen 1 week after the second immunization (4.6-fold above controls). Long-term antibody responses were detected beyond 6-weeks post-vaccination. T cell responses are being assessed by cytokine (IFN-γ, IL-2, IL-4, IL-5, and IL-10) secretion assays. Cytokine specificity data are pending. We speculated that the antigen-specific anti-tumor effect might have been modulated, in part, by apoptosis of regulatory T (Treg) cells that help maintain peripheral tolerance. Indeed, CD4+CD25+Foxp3+ Treg cells were consistently reduced in mice vaccinated with erbB2tr-transduced DCs, as shown by flow cytometry. Together these experiments show that we can overcome tolerance to an endogenously-expressed TAA, even using very low doses of erbB2-transduced DCs. Future studies will focus on testing our DC therapy in mice with established prostate tumors for a more clinically applicable model.

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