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Identification of C-Met Oncogene As a Broadly Expressed Tumor- Associated Antigen Recognized by Cytotoxic T-Lymphocytes

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Identification of C-Met Oncogene As a Broadly Expressed Tumor- Associated Antigen Recognized by Cytotoxic T-Lymphocytes 3658 Vol. 10, 3658–3666, June 1, 2004 Clinical Cancer Research Featured Article Identification of C-Met Oncogene as a Broadly Expressed Tumor- Associated Antigen Recognized by Cytotoxic T-Lymphocytes Kerstin Schag,1 Susanne M. Schmidt,1 INTRODUCTION Martin R. Mu¨ller,1 Toni Weinschenk,2 C-Met encodes a heterodimeric transmembranous receptor 1 1 with tyrosine kinase activity that is composed of an ␣ chain that Silke Appel, Markus M. Weck, ␤ 1 2 is disulfide-linked to a subunit (1, 2). Both subunits are Frank Gru¨nebach, Stefan Stevanovic, expressed on the surface; the heavy ␤ subunit is responsible for 2 1 Hans-Georg Rammensee, and Peter Brossart the binding of the ligand, hepatocyte growth factor (HGF), and 1Department of Hematology, Oncology and Immunology, University the ␣ subunit contains an intracellular domain that mediates the of Tu¨bingen, and 2Department of Immunology, Institute for Cell activation of different signal transduction pathways. Met signal- Biology, Tu¨bingen, Germany ing is involved in organ regeneration, as demonstrated for liver and kidney, embryogenesis, hematopoiesis, muscle develop- ment, and in the regulation of migration and adhesion of nor- ABSTRACT mally activated B cells and monocytes. Furthermore, numerous Purpose: C-Met proto-oncogene is a receptor tyrosine studies indicated the involvement of c-Met overexpression in kinase that mediates the oncogenic activities of the hepato- malignant transformation and invasiveness of malignant cells cyte growth factor. Using a DNA chip analysis of tumor (3–12). samples from patients with renal cell carcinoma and se- Using an integrated functional genomics approach that quencing of peptides bound to the HLA-A*0201 molecules combines gene expression profiling with analysis of MHC li- on tumor cells a peptide derived from the c-Met protein was gands by mass spectrometry to identify genes and corresponding identified recently. MHC ligands that are selectively expressed or overexpressed in Experimental Design: We used this novel HLA-A*0201 malignant tissues an HLA-A*0201-presented peptide derived peptide for the induction of specific CTLs to analyze the from the c-Met proto-oncogene could be identified (13). In the presentation of this epitope by malignant cells. present study we analyzed the possible function of this peptide Results: The induced CTL efficiently lysed target cells as a T-cell epitope and its presentation by malignant cells using pulsed with the cognate peptide, as well as HLA-A*0201- antigen-specific CTLs that were generated by in vitro priming matched tumor cell lines in an antigen-specific and HLA- with monocyte-derived dendritic cells (DCs) as antigen present- restricted manner. Furthermore, the induced c-Met-specific ing cells. We show here that the CTLs generated from several CTLs recognized autologous dendritic cells (DCs) pulsed healthy donors elicited an antigen-specific and HLA-A*0201- with the peptide or transfected with whole-tumor mRNA restricted cytolytic activity against tumor cells endogenously purified from c-Met-expressing cell lines. We next induced expressing the c-Met protein including renal cell carcinomas, c-Met-specific CTLs using peripheral blood mononuclear breast cancer, colon cancer, melanoma, and multiple myeloma cells and DC from an HLA-A*0201-positive patient with cells. Furthermore, they lysed autologous DCs pulsed with the plasma cell leukemia to determine the recognition of pri- antigenic peptide or electroporated with RNA isolated from c-Met-expressing tumor cell lines and primary autologous ma- mary autologous malignant cells. These CTLs lysed malig- lignant plasma cells while sparing nonmalignant B- and T- nant plasma cells while sparing nonmalignant B- and T- lymphocytes, monocytes, DCs, and bone marrow-derived or lymphocytes, monocytes, and DCs. mobilized CD34ϩ hematopoietic cells. Our results demonstrate Conclusion: Our results demonstrate that c-Met onco- that c-Met oncogene is a tumor rejection antigen recognized by gene is a novel tumor rejection antigen recognized by CTL CTLs and expressed on a broad variety of epithelial and hema- and expressed on a broad variety of epithelial and hemato- topoietic malignancies. poietic malignant cells. MATERIALS AND METHODS Tumor Cell Lines. Tumor cell lines used in the experi- ments were grown in RP10 medium (RPMI 1640 supplemented Received 11/25/03; revised 2/11/04; accepted 2/16/04. with 10% heat inactivated FCS and antibiotics). The following Grant support: Deutsche Forschungsgemeinschaft (SFB 510, Projekt c-Met-expressing tumor cell lines were used in experiments: B2) and Deutsche Krebshilfe. MCF-7 (breast cancer, HLA-A*0201ϩ, purchased from Amer- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ican Type Culture Collection), A498 (renal cell carcinoma, advertisement in accordance with 18 U.S.C. Section 1734 solely to HLA-A*0201ϩ) and MZ1257 (renal cell carcinoma, HLA- indicate this fact. A*0201ϩ, kindly provided by Prof. Alexander Knuth, Frank- Note: K. Schag and S. Schmidt contributed equally to the manuscript. furt, Germany), U266 (multiple myeloma, HLA-A*0201ϩ), Requests for reprints: Peter Brossart, University of Tu¨bingen, Depart- ϩ ment of Hematology, Oncology and Immunology, Otfried-Mu¨ller- HCT116 (colon cancer, HLA-A*0201 ), Mel1479 (malignant Strasse-10, D-72076 Tu¨bingen, Germany. Phone: 49-7071-2982726; melanoma, HLA-A*0201ϩ, kindly provided by Prof. Graham Fax: 49-7071-295709; E-mail: [email protected] Pawelec, University of Tu¨bin, Tu¨bingen, Germany), and SK- Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2004 American Association for Cancer Research. Clinical Cancer Research 3659 OV-3 (ovarian cell line, HLA-A*03ϩ, kindly provided by O. J. (ILKEPVHGV, pol HIV-1 reverse transcriptase peptide, amino Finn, Pittsburgh, PA). The c-Met-negative cell lines T2 (HLA- acids 476–484) were synthesized using standard F-moc chem- A*0201ϩ, TAP-deficient) and Croft (EBV-immortalized B-cell istry on a peptide synthesizer (432A; Applied Biosystems, Weit- line, kindly donated by O. J. Finn, Pittsburgh, PA, HLA- erstadt, Germany) and analyzed by reversed-phase high-perfor- A*0201ϩ) were used as controls. K562 cells were used to mance liquid chromatography and mass spectrometry. determine the natural killer-cell activity. For CTL induction, 5 ϫ 105 DCs were pulsed with 50 Cell Isolation and Generation of DC from Adherent ␮g/ml of the synthetic c-Met peptide for 2 h, washed, and Peripheral Blood Mononuclear Cells (PBMNC). Genera- incubated with 2.5 ϫ 106 autologous PBMNC in RP10 medium. tion of DCs from peripheral blood monocytes was performed as After 7 days of culture, cells were restimulated with autologous described previously (14). In brief, PBMNCs were isolated by peptide-pulsed PBMNC, and 2 ng/ml human recombinant inter- Ficoll/Paque (Biochrom, Berlin, Germany) density gradient cen- leukin 2 (R&D Systems) was added on days 1, 3, and 5. The trifugation of blood obtained from buffy coat preparations of cytolytic activity of induced CTL was analyzed on day 5 after healthy donors from the blood bank of the University of Tu¨b- the last restimulation in a standard 51Cr-release assay. ingen. Cells were seeded (1 ϫ 107 cells/3 ml/well) into six-well CTL Assay. The standard 51Cr release assay was per- plates (BD Falcon, Heidelberg, Germany) in RP10 medium. formed as described (14). Target cells were pulsed with 50 ␮ 51 After2hofincubation at 37°C and 5% CO2, nonadherent cells g/ml peptide for 2 h and labeled with [ Cr]sodium chromate were removed, and the adherent blood monocytes were cultured in RP10 for1hat37°C. Cells (104) were transferred to a well in RP10 medium supplemented with the following cytokines: of a round-bottomed 96-well plate. Varying numbers of CTLs human recombinant granulocyte macrophage colony-stimulat- were added to give a final volume of 200 ␮l and incubated for ing factor (Leukomax, Novartis; 100 ng/ml), interleukin 4 4hat37°C. At the end of the assay supernatants (50 ␮l/well) (R&D, Wiesbaden, Germany; 20 ng/ml), and tumor necrosis were harvested and counted in a ␤-plate counter. The percentage factor ␣ (R&D; 10 ng/ml). The phenotype of DCs was analyzed of specific lysis was calculated as: 100 ϫ (experimental re- by flow cytometry after 7 days of culture (data not shown). lease Ϫ spontaneous release/maximal release Ϫ spontaneous Mobilized CD34ϩ hematopoietic progenitor cells, B- and release). Spontaneous and maximal release were determined in T-lymphocytes, as well as monocytes were isolated from pe- the presence of either medium or 2% Triton X-100, respectively. ripheral blood using magnetic cell sorting technology. Activa- Antigen specificity of tumor cell lysis was additionally tion of B and T cells was performed as described recently (15). determined in a cold target inhibition assay (14) by analyzing Monocytes were grown in RP10 medium with granulocyte the capacity of peptide-pulsed unlabeled T2 cells to block lysis macrophage colony-stimulating factor (Leukomax; Novartis; of tumor cells at a ratio of 20:1 (inhibitor:target ratio). 100 ng/ml) overnight before being used as targets. Bone marrow IFN-␥ Enzyme-Linked Immunospot Assay. C-Met- cells and CD34ϩ progenitor cells were incubated for 24–48 h specific CTLs were generated in vitro using autologous DCs with stem cell factor (SCF; R&D; 100 ng/ml). pulsed with the c-Met peptide. These CTLs were incubated at a Reverse Transcription-PCR (RT-PCR). RT-PCR was concentration of 2 ϫ 105 cells/well in a 96-well plate coated performed with some modifications as described recently (14). with antihuman IFN-␥ antibody (mAb 1-D1K, 10 ␮g/ml; Total RNA was isolated from cell lysates using Qiagen RNeasy Mabtech AB, Hamburg, Germany) together with autologous “Mini” anion-exchange spin columns (Qiagen GmbH, Hilden, PBMNCs pulsed for 1 h with the HLA-A2 binding peptides Germany) according to the instructions of the manufacturer and derived from the tumor antigens c-Met or adipophilin.
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