Identification and Characterization of T-Cell Epitopes Deduced from RGS 5 , a Novel Broadly ExpressedTumorAntigen

Purpose: Identification of tumor-associated antigens and advances in tumor immunology resulted in the development of vaccination strategies to treat patients with malignant diseases. In a novel experimental approach that combined comparative mRNA expression analysis of defined cell types with the characterization ofMHC ligands by mass spectrometry, we found that regulator of G protein signaling 5 (RGS5) is extensively up-regulated in a broad variety of malignant cells, and we identified two HLA-A2^ and HLA-A3^ binding peptides derived from the RGS5 protein. Interestingly, RGS5 was recently shown to be involved in tumor angiogenesis. Experimental Design:We usedmonocyte-derived dendritic cells pulsed with these novel antigenic peptides or transfected with RGS5-mRNA for the in vitro induction of CTLs, generated from healthy donors, to analyze the presentation of RGS5-deduced epitopes by malignant cells. Results:The generated CTL lines elicited an antigen-specific and HLA-restricted cytolytic activity against tumor cells endogenously expressing theRGS5 protein. Furthermore, wewere able to induce RGS5-specific CTLs using peripheral blood mononuclear cells from a patient with acute myeloid leukemia capable of recognizing the autologous leukemic blasts while sparing nonmalignant cells. Conclusions:These results indicate that the RGS5 peptides represent interesting candidates for the development of cancer vaccines designed to target malignant cells and tumor vessels. The identification of relevant tumor-associated antigens (TAA) capable of mediating tumor cell elimination is a central issue in the development of immunotherapeutic strategies to treat malignant diseases. Novel experimental approaches that are currently applied combine the information of all transcribed genes (‘‘transcriptome’’) of defined cell types with the analysis of MHC ligands by mass spectrometry for the identification of tumor antigens (1–5). In a first step, comparative expression profiling of tumor tissues and the corresponding normal tissue by DNA oligonucleotide microarray technology is done for the identification of genes selectively expressed or overexpressed in the tumor cells. This is followed by isolation and characterization of MHC class I ligands identified in themalignant tissue bymass spectrometry– based peptide sequencing. Newly identified MHC ligands encoded by genes identified by the microarray technique are considered as potential targets in immunotherapeutic strategies. By applying this approach to renal cell carcinoma (RCC) tissues, RGS5 was found to be extensively up-regulated (1). G protein–linked receptors form the largest family of cell surface receptors and are found in all eukaryotes. G proteins are attached to the cytoplasmatic face of the cellular plasma membrane, where they serve as relay molecules, functionally coupling the receptors to enzymes or ion channels in this membrane. In the inactivated state, G proteins exist as heterotrimers consisting of a, h, and g subunits. The a subunit binds guanosine 5¶-diphosphate, which is replaced by GTP upon stimulation by an activated receptor. This exchange causes the G protein trimer to dissociate into an a subunit and a hg dimer. Both subunits transduce signals to a variety of G protein effectors, including adenylyl cyclase, voltage-sensitive Ca and K channels, phosphatidylinositol 3-kinase, phospholipases C-h and A2, cyclic guanosine 3¶,5¶-monophosphate phosphodiesterase, and, indirectly, mitogen-activated protein kinase (6–8). The key function of regulators of G protein signaling (RGS) is to bind to G protein a subunits and to stimulate their intrinsic GTPase activity. The hydrolysis of GTP to guanosine 5¶-diphosphate thereby is accelerated, and the inactive heterotrimer is more rapidly restored. Thus, RGS proteins inhibit the biological activity of G proteins (9–11). Seki et al. isolated RGS5 from a neuroblastoma cDNA library (12). The amino acid sequence deduced from the cDNA possessed all consensus motifs of the RGS domain and showed closest homology to mouse RGS5. It was ascertained that RGS5 attenuates angiotensin II– , endothelin-1–, and Cancer Therapy: Preclinical Authors’ Affiliations: Department of Oncology, Hematology, Immunology, Rheumatology, and Pulmology and Department of Immunology, Institute for Cell Biology, University of Tu« bingen,Tu« bingen, Germany Received 8/30/06; revised 2/16/07; accepted 3/21/07. Grant support: Deutsche Forschungsgemeinschaft, Deutsche Krebshilfe, and Promotionskolleg of University of Tu« bingen. The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Note: C.N. Boß and F. Gru« nebach contributed equally to this work. Requests for reprints: Peter Brossart, Department of Internal Medicine II, Oncology, Hematology, Immunology, Rheumatology, and Pulmology, University of Tu« bingen, Otfried-Mu« ller-Str. 10, 72076 Tu« bingen, Germany. Phone: 49-7071/29 82726; Fax: 49-7071/29-5709; E-mail: peter.brossart@med.uni-tuebingen.de. F2007 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-06-2156 www.aacrjournals.org Clin Cancer Res 2007;13(11) June1, 2007 3347 Cancer Research. on January 28, 2018. © 2007 American Association for clincancerres.aacrjournals.org Downloaded from platelet-derived growth factor–induced ERK-2 phosphorylation (13–15). Interestingly, it was recently shown that RGS5 is overexpressed in pericytes of newly developing tumor vessels, indicating that RGS5 plays an important role during tumor angiogenesis (16–18). We found RGS5 expressed in a broad variety of malignant cells. Thus, targeting RGS5 may affect both tumor cells and tumor vessels. In the present study, we examined the possible function of this molecule as a novel TAA using antigen-specific CTLs that were generated by in vitro priming with monocyte-derived dendritic cells as antigen-presenting cells. These dendritic cells were either loaded with RGS5 peptides or electroporated with pure full-length RGS5 in vitro transcript (IVT) that coded for the entire RGS5 protein. We show here that the CTLs generated from several healthy donors and patients with acute myeloid leukemia (AML) elicited an antigen-specific and HLA-A2– or HLA-A3–restricted cytolytic activity against tumor cells endogenously expressing the RGS5 protein, including RCCs, breast cancer, melanoma, multiple myeloma cells, ovarian carcinoma, and primary autologous blasts from an AML patient. Materials andMethods Tumor cell lines. MCF-7 (breast cancer, RGS5, HLA-A2); A498, MZ1774, and MZ1257 (RCC cell lines, RGS5, HLA-A2; kindly provided by Prof. A. Knuth, Zürich, Switzerland); U266 (multiple myeloma, RGS5, HLA-A2); HCT116 (colon cancer, RGS5, HLA-A2); Mel1479 (malignant melanoma, RGS5, HLA-A3; kindly provided by Prof. G. Pawelec, Tübingen, Germany); SKOV-3 (ovarian cell line, RGS5, HLA-A3; kindly provided by O.J. Finn, Pittsburgh, PA); Caki-1 (RCC, RGS5); CROFT (EBV-immortalized B-cell line, HLA-A2; kindly provided by O.J. Finn); SD-1 (human peripheral blood B lymphoblastoid cells, RGS5); THP-1 (RGS5 , human acute monocytic leukemia); NT-2 (NTERA-2, pleuripotent, embryonal carcinoma cells, RGS5); KASUMI-1 (AML, RGS5); WERI-RB-1 (human retinoblastoma, RGS5); HL-60 (human AML, RGS5). Generation of dendritic cells from adherent peripheral blood mononuclear cells. Dendritic cells were generated from peripheral blood monocytes as described previously (19, 20). In brief, peripheral blood mononuclear cells (PBMC) were isolated by Ficoll/Paque (Biochrom) density gradient centrifugation of blood obtained from buffy coats of healthy volunteers from the blood bank of the University of Tübingen. Cells were seeded (1 10 per 3 mL per well) into six-well plates (Corning) in serum-free X-VIVO 20 medium (Bio Whittaker). After 2 h of incubation at 37jC/5% CO2, non-adherent cells were removed and cryopreserved at -80jC to be used later for cell isolation or restimulations. Human recombinant granulocyte macrophage-colony stimulating factor (100 ng/mL; Leukine Liquid Sargramostim, Berlex) and interleukin-4 (20 ng/mL; R&D Systems) were added every 2nd day starting at the 1st day of culture to generate immature dendritic cells. Maturation was induced at day 6 of culture by adding tumor necrosis factor-a (R&D Systems; 10 ng/mL) for 24 h. Synthetic peptides. RGS5 A*02: LAALPHSCL, RGS5 A*03: GLASFKSFLK (1), HER-2/neu (E75) A*02: KIFGSLAFL, BCR-ABL A*02: SSKALQRPV, and BCR-ABL A*03/A*11: KQSSKALQR (21) were synthesized in an automated peptide synthesizer EPS221 (Abimed) following the fluoren-9-ylmethoxycarbonyl/t-butyl strategy and analyzed by high-performance liquid chromatography (Varian star, Zinsser Analytics) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (future, GSG). Gene expression analysis by high-density oligonucleotide microarrays. Frozen fragments of tumors RCC0044, RCC0068, RCC0070, RCC0073, RCC0075, RCC0098, and RCC0103 were homogenized with mortar and pestle under liquid nitrogen. Total RNA was prepared from these samples using TRIzol (Invitrogen) according to the manufacturer’s protocol, followed by a cleanup with RNeasy (Qiagen). Quality and quantity were assessed on an Agilent 2100 Bioanalyzer (Agilent) using the RNA 6000 Pico LabChip kit (Agilent). Gene expression analysis of RNA samples from RCC0044, RCC0068, RCC0070, RCC0073, RCC0075, RCC0098, and RCC0103 was done by Affymetrix Human Genome U133A oligonucleotide microarrays (Affymetrix). For all other samples, HG-U133 Plus 2.0 was used. The same normal kidney sample was hybridized to both array types to achieve comparability (data not shown). All steps were carried out according to the Affymetrix manual. Briefly, double-stranded cDNA was synthesized from 5 to 8 Ag total RNA, using SuperScript RTII (Invitrogen) and the oligo-dT-T7 primer (MWG Biot