Abstract 788: Uptake and cross-presentation of the leukemia associated antigens neutrophil elastase (NE) and proteinase-3 (P3) increases susceptibility of solid tumors to PR1-targeted immunotherapy

Abstract

Abstract We have shown that the HLA-A2-restricted nonapeptide PR1 (VLQELNVTV) is a leukemia-associated peptide derived from P3 and NE. Furthermore, immunologic and clinical responses to PR1 peptide vaccination occur in patients with acute (AML), chronic (CML) myeloid leukemia and myelodysplastic syndrome. We have also shown that PR1 expression results from NE and P3 cross-presentation by B-cells and dendritic cells. Because NE and P3 are present in lung and breast cancer tumor tissue, we investigated whether uptake of NE and P3 might result in PR1 expression on solid tumors, which could induce susceptibility of non-hematopoietic tumors to PR1-targeted immunotherapy. We studied melanoma, breast, ovarian and lung cancer cell lines to determine whether NE and P3 are cross-presented. Using flow cytometry, we show that numerous malignant cell lines take up 5 to 10 °g/ml of soluble NE and P3 and was dose- and time-dependent, consistent with a receptor-mediated mechanism. In addition to soluble NE and P3, uptake of granulocyte-associated NE and P3 was also demonstrated; however, uptake of soluble NE and P3 were 3- and 2-fold higher, when compared to granulocyte-associated proteins. Within 4 hours of uptake, confocal microscopy showed that soluble P3 localized to endosomes and lysosomes, suggesting the possibility of lysosomal degradation of P3, a common pathway for MHC-I processing of cross-presented proteins. With the use of 8F4, a monoclonal antibody that is specific for a PR1/HLA-A2 conformational epitope, we show that PR1/HLA-A2 is expressed by 12-hours on P3- and NE-pulsed HLA-A2+ melanoma and breast cancer cell lines. Because 8F4 mediates complement-dependent cytotoxicity (CDC) of AML, we studied whether PR1 expression induced susceptibility of P3- and NE-pulsed tumor cells to killing by 8F4 and PR1-CTL. Twelve hours after pulsing the HLA-A2+ breast cancer cell lines MDA-MB-453 and MDA-MB-231 with P3 or NE, up to 40% and 60% of pulsed breast cancer cells were lysed by PR1-CTL and 8F4, respectively. We conclude that PR1 is cross-presented by solid tumors following receptor-mediated uptake of soluble NE and P3 at concentrations observed in tumors and inflammatory sites. Importantly, this cross-presentation increased susceptibility of embryological distinct tumors to PR1-specific immunotherapy. Together, these data suggest that cross-presentation of extracellular inflammatory proteins that are normally not expressed in the tumor may be a mechanism shared by many tumors, which could be exploited by immunotherapy strategies targeting these proteins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 788. doi:10.1158/1538-7445.AM2011-788