Dendritic Cells (DC) Generated from AML Blasts Express Leukemia Associated Antigens Eliciting Specific Cytotoxic T Cell Responses in the Autologous Host after DC Vaccination.

Abstract

Several groups including ours demonstrated the generation of DC from AML blasts (AML-DC). FISH analysis has been employed to assess the origin of AML-DC from AML blasts. For clinical application, this approach is not feasible because of the restriction of AML-DC in number. Therefore we established an alternative system to prove the origin of the AML-DC, using quantitative real-time PCR to test the mRNA expression of the leukemia associated antigens (LAAs) preferentially expressed antigen in melanoma (PRAME), proteinase 3, the receptor for hyaluronic acid mediated motility (RHAMM/CD168) and the Wilms tumor gene 1 (WT-1). An elevated PCR signal for PRAME was detected in 7/12 AML-DC preparations when compared with AML blasts, for RHAMM/CD168 in 6/12 AML-DC preparations, but only in 2/12 respectively 1/12 AML-DC for WT-1 and proteinase 3. All preparations showed a strong expression of at least one of the LAAs examined. The stronger PCR signals after DC generation for PRAME and RHAMM/CD168 characterize these two LAAs as favourable target structures for immunotherapies. AML-DC positive for RHAMM/CD168 mRNA tested also positive for the protein as demonstrated by immunocytochemistry. In PRAME mRNA positive AML-DC, the described PRAME derived decamer epitope peptide ALYVDSLFFL was recognized by specific T cells as proven by chromium-51 release assay, thus proving that the mRNA assessment for RHAMM/CD168 and PRAME has an immunological significance.For five patients, AML-DC were generated under good manufacturing practice (GMP) conditions. 5x10E6 AML-DC were injected s.c. in the vicinity of inguinal lymph nodes four times at a biweekly interval. No severe adverse effects were observed after DC vaccination. One patient with AML FAB M4 required blood transfusions and remained in stable condition for several months, but eventually died from pneumonia 13 months after the DC vaccinations. A 70 year-old women with a secondary AML received a complete course of AML-DC vaccinations. During the period of 4 vaccinations, the blast level dropped from 8% in the PB to 0% and no side effects were noted. Two patients died from cranial hemorrhage after the first vaccination due to thrombocytopenia caused by the AML. One patient is still under DC vaccination. ELISPOT analysis of the first two patients revealed a significant increase in interferon gamma and granzyme B releasing CD8+ T cells recognizing a leukemic blast lysate as well as specifically RHAMM derived peptides, when compared to the initial T cell frequency.Potentiation of such an AML-DC vaccine might become feasible by the addition of adjuvants such as CPG-rich oligodeoxyribonucleotides (CPG-ODN) or lipopolysaccharides (LPS). We therefore investigated the presence of receptors for such adjuvants, i.e Toll-like receptors (TLRs) on AML-DC. Quantitative mRNA expression of TLR-2, 4 and 9 in AML-DC and DC generated of monocytes from healthy volunteers did not display any significant difference. In summary, DC could be generated from AML blasts and preserved or even upregulated LAA expression. DC vaccination was well tolerated and resulted in an enhanced and specific response of cytotoxic T cells. The adequate expression of TLRs renders potentiation of the AML-DC vaccine described here by e.g. CPG-ODN possible.