Abstracts

General Information

Abstracts that have been selected for poster presentation have been divided into four sessions:

Session 1: Modulation of Immune Responses
Session 2: Vaccination A
Session 3: Vaccination B
Session 4: Cellular Therapy

Session	Session 1	Session 2	Session 3	Session 4
Title	Modulation of Immune Responses	Vaccination A	Vaccination B	Cellular Therapy
Date	May 12	May 12	May 13	May 13
Time	13.30 - 14.30	13.30 - 14.30	13.20 - 14.20	13.20 - 14.20
Chaired by:	T. Wölfel	M. Schmitt	E. Kaempgen	M. Nishimura
	Mainz	Ulm	Erlangen	Chicago
	P1 P2 P3 P4 P6 P7 P25 P28 P31 P33	P5 P8 P9 P10 P11 P13 P15 P16 P19	P20 P21 P22 P23 P24 P26 P27 P30 P32	P12 P14 P17 P18 P29

List of poster presentations

P01	γ/δ T cell cytotoxicity is diminished by soluble MIC in patients with pancreatic carcinoma and can be restored by treatment with IFN-α" Angela Märten, Marie von Lilienfeld-Toal, Jan Schmidt, Markus W. Büchler,Helmut Salih Abstract

P02	"In vitro Analysis of the role of Interferon-alpha in combined Chemoradioimmunotherapy in the adjuvant treatment of pancreatic adenocarcinoma (CapRI)" Jan Schmidt, Emilia Patrut, Markus W. Büchler, Angela Märten Abstract

P03	"Phase III Trial CapRI (adjuvant ChemoRadioImmunotherapy of pancreatic adenocarcinoma) versus 5-FU alone (ISRCTN 62866759)" Jan Schmidt, Angela Märten, Katrin Hoffmann, Katja Lindel K, Stefan Fritz, Thomas Herrmann, Hartmut Goldschmidt, Ulrich Mansmann, Jürgen Debus, Volker Diehl, Reiner Kunz, Lorenzo Capussotti, Markus Büchler Abstract

P04	"Immunomonitoring of the CapRI Trial: First Results" Jan Schmidt, Markus Büchler, Angela Märten Abstract

P05	"Induction of antitumor immune responses by DNA vaccines encoding APC-targeted ErbB2 and NY-ESO-1 fusion proteins" Arjen Sloots, Florian Rohrbach, Robert Weth, Elke Jäger, Winfried Wels Abstract

P06	"A supra-agonistic, bispecific single-chain antibody purified from the serum of cloned, transgenic cows induces CD28 mediated killing of glioblastoma cells in vitro and in vivo" Ludger Grosse-Hovest, Wolfgang Wick, Rosa Minoia, Michael Weller, Hans-Georg Rammensee, Gottfried Brem, Gundram Jung Abstract

P07	"Selective Stimulation of the Fas/Apo-1/CD95-Death Receptor on Human Glioblastoma Cells with Bispecific Antibodies" Tanja Herrmann, Ludger Grosse-Hovest, Hans-Georg Rammensee, Gundram Jung Abstract

P08	"The differentiation antigen NY-BR-1 is overexpressed in mammary tumors and encodes a novel membrane protein - a potential target for immunotherapy" Inka Seil, Silke Deckert, Claudia Frei, Kurt Zatloukal, Holger Sültmann, Elke Jäger, Alexander Knuth, Dirk Jäger, Roland H. Stauber Abstract

P09	"A bi-specific linker molecule employs NK cell help to generate T cell action against tumors" Markus Jensen, Carolin Hoyer, Martin Klehr, Deanna Berger, William W. Kwok, Marc Beyer, Joachim Schultze, Frank Berthold Abstract

P10	"Identification of tumor-associated T-cell epitopes, Part I: Lessons to be learned from primary renal cell carcinoma"Oliver Schoor, Margret Müller, Anna Missiou, Tobias Krüger, Claudia Lemmel, Björn Krämer, Christian Reichle, Jörn Dengje1, Toni Weinschenk, Jörg Hennenlotter, Arnulf Stenzl, Hans-Georg Rammensee, Stefan Stevanović Abstract

P11	"A new polyclonal antibody reveals GAGE protein expression in malignant melanoma"Nicole Wiedemann, Alexandr V. Bazhin, Dirk Schadendorf, Stefan B. Eichmüller Abstract

P12	"Signaling via chimeric receptors containing the costimulatory domain of CD28 fails to induce tumor-specific T cell proliferation in Epstein-Barr virus specific T cells" Bianca Altvater, Sibylle Pscherer, Martin Pule, Heribert Jürgens, Claudia Rössig Abstract

P13	"CTL Priming by Transcutaneous Peptide Immunization with R-837" Tobias Warger & Gerd Rechtsteiner, Philipp Osterloh, Hansjörg Schild, Markus P. Radsak Abstract

P14	"Varicella–Zoster-virus-specific T cells as mediators of chmimeric-receptor redirected tumor-specific Immunity" Silke Landmeier, Bodo Eing, Verena Niggemeier, Cliona Rooney, Heribert Jürgens, Claudia Rössig Abstract

P15	"Evaluation of genetic melanoma vaccines in cdk4-mutant mice provides evidence for immunological tolerance against authochthonous melanomas in the skin" Julia Steitz, Julia Lenz, Stefanie Büchs, Christoph Huber, Thomas Wölfel, Mariano Barbacid, Marcos Malumbres, and Thomas Tüting Abstract

P16	"Stabilized RNA activates several human blood cell types through a TLR signalling pathway" Birgit Scheel, Regina Teufel, Jean-Philippe Carralot, Jochen Probst, Markus Radsak, Hans-Georg Rammensee, Ingmar Hoerr, Steve Pascolo Abstract

P17	"T cell immunity in non-myeloablative allogeneic stem cell transplanted renal cell and breast carcinoma patients" Els Verdegaal, Renée Barge, Conny Hoogstraten, Jeanne van Steijn, Jan Ouwerkerk, Willem Fibbe, Fred Falkenburg, Susanne Osanto Abstract

P18	"CTL transfer in metastatic melanoma patients; a phase I/II clinical trial" Tamara Ramwadhdoebé, Carolien van der Minne, Conny Hoogstraten, Jeanne van Steijn, Els Verdegaa, Susanne Osanto Abstract

P19	"Electroporation of human blood cells with messenger RNA provides target cells able to stimulate memory-cytotoxic T cells in vitro" Regina Teufel, Birgit Scheel, Jochen Probst, Jean-Philippe Carralot, Steffen Walter, Hans-Georg Rammensee, Steve Pascolo Abstract

P20	"Reduced frequencies and suppressive function of CD4+ CD25high regulatory T cells in patients with chronic lymphocytic leukemia after therapy with fludarabine" Marc Beyer, Matthias Kochanek, Kamruz Darabi, Alexey Popov1, Markus Jensen, Elmar Endl, Percy A. Knolle, Michael von Bergwelt-Baildon, Svenja Debey, Joachim L. Schultze Abstract

P21	"Immunotherapy with autologous PSA-peptide loaded dendritic cells of hormone refractory prostate carcinoma after prestimulation with Interferon-gamma – A pilot study" Bernd Hildenbrand, Barbara Sauer, Anja Langenkamp, Christoph Stoll, Oana Kalis, Marina A. Freudenberg, Chris Galanos, Gabi Niedermann, Brigitte Häring, Regina Leo ,Clemens Unger, Marc Azemar Abstract

P22	"Expression of RHAMM/CD168, fibromodulin, survivin, OFAiLRP and hTERT as potential immunotherapeutical targets in patients with B-cell chronic lymphocytic leukemia" K. Giannopoulos, L. Li, J. Rolinski, A. Dmoszynska, J.Tabarkiewicz, K. Wojas, I. Hus, J. Greiner, H. Döhner, M. Schmitt Abstract

P23	"Improvement of the production of IL-12p70 by human monocyte-derived dendritic cells maturated with various highly purified LPS-derivatives from Salmonella minesota and INF-α under serum-free conditions" B. Hildenbrand, C. Kalis, M. Azemar, B. Sauer, G. Niedermann, O. Kalis, C. Unger, C. Galanos, M.A. Freudenberg Abstract

P24	"RHAMM/CD168 is a novel target of immunotherapies for patients with acute myeloid leukemia (AML)" M. Schmitt, L. Li, M. Ringhoffer, K. Giannopoulos, H. Döhner, J. Greiner Abstract

P25	"Interaction of TLR2 and TLR4 ligands with Gp96 amplifies innate and adaptive immune responses" T. Warger, G. Rechtsteiner, N. Hilf, P. von Landenberg, H. Jonuleit, H.-G. Rammensee, M. P. Radsak, H. Schild Abstract

P26	"Phenotypic and funtional characterization of in vitro maturated dendritic cells: differential influence of microbial extract conditioned media on in vitro dendritic cell maturation" Kang-Hun Lee, Mareen Reichert, Benjamin Günther, Carmen Scheibenbogen, Eckhard Thiel Abstract

P27	"Definition of potential T cell epitopes for the immunotherapy of cutaneous T cell lymphoma" J. Müller-Berghaus, H. Schönhaber, X.D. Nguyen, H. Klüter, S. Eichmüller, D. Schadendorf Abstract

P28	"Structural imbalance of chromosome 15 contributes to the irreversible loss of HLA class I expression on melanoma cells" A. Sucker, N. Arens, S. Striegel, R. Hildenbrand, M. Maio, D. Schadendorf, A. Paschen Abstract

P29	"T-cell depleted haematopoietic stem-cell transplantation followed by preemptive CD8-depleted DLI" R.G. Meyer, C.M. Britten, A. Konur, K. Bender, D. Wehler, U. Hartwig, T.C. Wehler, C. Huber, K. Kolbe, W. Herr Abstract

P30	"Development of a Clinical-Grade Protocol to mature moncyte-derived dendritic cells in TH1-direction" B. Hildenbrand, B. Sauer, M. Freudenberg, C. Galanos, C. Kalis, G. Niedermann, C. Stoll, C. Unger, M. Azemar Abstract

P31	"The role of MMP-7 in cancer-mediated immunotolerance in vivo" S. Gregor, V. Alla, J. Kuball, M. Theobald, P.R. Galle, D. Strand, S. Strand Abstract

P32	"The idiotype vaccination program for B cell lymphoma at Freiburg University - Current status" K. Mikesch, C. Bertinetti, H. Veelken Abstract

P33	Bispecific single-chain antibodies induce target-cell restricted, supra–agonistic CD28 stimulation and T-cell mediated killing of lymphoma cells T. Otz, L. Große-Hovest, H.-GT. Rammensee and G. Jung Abstract

Abstracts Poster Presentations

P01

γ/δ T cell cytotoxicity is diminished by soluble MIC in patients with pancreatic carcinoma and can be restored by treatment with IFN-α

Angela Märten¹, Marie von Lilienfeld-Toal², Jan Schmidt¹, Markus W. Büchler¹, Helmut Salih³

¹ Department of Surgery, University of Heidelberg, 69120 Heidelberg, Germany
² Department of Internal Medicine, University of Tübingen, 72016 Tübingen, Germany
³ Department of Internal Medicine I, University of Bonn, 53105 Bonn, Germany

Release of ligands of the activating immunoreceptor NKG2D from the cell surface in a soluble form is considered to be an immune escape mechanism of tumor cells. However, up to now the direct effect of soluble MIC (sMIC) on cellular cytotoxicity of NK cells and Gamma/Delta T cells is unknown. We determined the presence of sMIC in sera of patients with pancreatic carcinoma by ELISA. The effect of sMIC content in patient serum on cellular cytotoxicity of NK and Gamma/Delta T cells was investigated by cytotoxicity assays. Furthermore, we evaluated the expression of NKG2DL on the surface of tumor cells and tried to increase them by treatment with IFN-alpha. Subsequently the effect of IFN-alpha induced increase of NKG2DL expression for recognition by Gamma/Delta T cells was investigated. Pancreatic carcinoma cells express NKG2DL to various extents, and analysis of patient sera revealed elevated sMIC levels that correlate with tumor stage, grade and differentiation. Furthermore, sMIC in patient sera inhibits cellular cytotoxicity of NK cells and Gamma/Delta T cells, and this can be reversed by sMIC depletion and preincubation with MIC specific antibodies. Immunotherapy of pancreatic carcinoma with IFN-alpha induces expression of NKG2DL without induction of sMIC resulting in enhanced lysis of tumor cells. Overall, our results indicate that determination of sMIC serum levels may be used as prognostic indicator and implicate an important role for NKG2D mediated immunity in pancreatic carcinoma. Furthermore, presence of sMIC directly inhibits cellular cytotoxicity of NK cells and Gamma/delta T cells. Since IFN-alpha treatment induces NKG2DL expression without increasing sMIC levels our results indicate that the beneficial effects of IFN-alpha immunotherapy might be mediated by modulation of NKG2D mediated immunesurveilance.

P02

In vitro Analysis of the role of Interferon-alpha in combined Chemoradioimmunotherapy in the adjuvant treatment of pancreatic adenocarcinoma (CapRI)

Jan Schmidt¹, Emilia Patrut¹, Markus W. Büchler¹, Angela Märten¹

¹Department of Surgery, University of Heidelberg, 69120 Heidelberg, Germany

Adjuvant treatment of pancreatic adenocarcinoma has failed so far to produce long-lasting benefits. Results from a phase II trial, where chemoradiotherapy was combined with IFN-alpha (CapRI scheme), are very encouraging. We hypothesize that IFN-alpha is the agent that turns a slightly effective treatment (radiochemotherapy) into a potent therapy. Therefore, the role of IFN-alpha regarding a) direct inhibitory effects; b) radio- and chemosensitizing effects; c) anti-angiogenic properties and d) enhancement of immunogenicity was investigated. Eight human ductal pancreatic carcinoma cell lines were treated with the CapRI scheme (5-Fluorouracil, Cisplatin, IFN-α and radiation). Apoptosis and proliferation rate were investigated as well as surface expression pattern and VEGF secretion. For immunomodulatory studies, NK and T cells were preincubated with 1,000 U/ml IFN-α over 24 hours and tested in cytotoxicity assays against these cell lines and the mechanism of cell lysis was investigated. The induction of the immunoproteasome in tumor cells after IFN α stimulation was analyzed by immunoblot and RT PCR. Our results show that IFN-α has direct inhibitory properties and some synergistic influence as determined by AnnexinV/PI stain and cell count. IFN-α is also able to prevent the increase in proliferation rate and VEGF secretion of CDDP resistant cells. The most interesting results were found with regard to immunomodulatory effects. We observed an increase in cytotoxic activity of peripheral blood cells after IFN α treatment from 12.5% to 34.3% (p<0.05). This cytotoxicity was NK cell mediated as shown after depletion of T cells (T cells 4% lysis, NK cells 42.7% lysis) and was mediated as well by Fas-induced apoptosis as by perforin release. Pretreatment of tumor cells with 5-FU and combinations showed a significant increase in the susceptibility of tumor cells against NK cells (untreated tumor cells 34.3%, CapRI scheme 69.1%). Treatment of tumor cells with IFN-α induced a switch to the immunoproteasome and enhanced their vulnerability to T cells. This is the first description of this phenomenon in pancreatic carcinoma cells with implications for their immunogenicity In conclusion, IFN-alpha has direct cytotoxic effects, acts as a radiosensitizer and circumvents tumor cell-regrowth after CDDP treatment. However, the most important effects are the activation of NK cells against pancreatic carcinoma cells by IFN-α and the observation that 5 FU treatment makes tumor cells more susceptible. Furthermore, IFN-α induces the immunoproteasome with impact on the immunogenicity of pancreatic carcinoma cells. These mechanisms may be responsible for the improved clinical outcome of CapRI.

P03

Phase III Trial CapRI (adjuvant ChemoRadioImmunotherapy of pancreatic adenocarcinoma) versus 5-FU alone (ISRCTN 62866759)

Jan Schmidt¹, Angela Märten^1;5, Katrin Hoffmann¹, Katja Lindel K^2;5, Stefan Fritz^1;5, Thomas Herrmann³, Hartmut Goldschmidt^3:5, Ulrich Mansmann⁴, Jürgen Debus^2;5, Volker Diehl⁵, Reiner Kunz⁶, Lorenzo Capussotti⁷, Markus Büchler^1;5

¹ University of Heidelberg, Department of Surgery, 69120 Heidelberg, Germany
² University of Heidelberg, Department of Radiology, 69120 Heidelberg, Germany
³ University of Heidelberg, Department of Medicine, 69120 Heidelberg, Germany
⁴ LMU München, IBE Medical School, 81377 München, Germany
⁵ National Centre for Tumor Diseases, 69120 Heidelberg, Germany
⁶ St. Josef Hospital, 12101 Berlin, Germany
⁷ Istituto per la Ricerca e la Cura del Cancro, Department of Surgical Oncology, Italy

Carcinoma of the exocrine pancreas has an especially poor prognosis. The five-year survival for all stages is <1% with a median survival of 4-6 months. Even after surgical intervention with a curative intention the two-year survival is only 25%. Investigators from the Virginia Mason Clinic have recently published data from a phase II trial of postoperative cisplatin (CDDP), 5-fluorouracil (5-FU), interferon alpha-2b (IFN-α), and external-beam radiation (RT) administered following pancreatico-duodenectomy. We termed this regimen CapRI for adjuvant treatment of pancreatic adenocarcinoma with ChemoRadioImmunotherapy. They have treated 43 patients with mainly stage III tumors. 84% had positive lymph nodes and 19% had positive cut margins. After a mean follow-up of 31.9 months, 67% of the patients were still alive. Actuarial overall survival for the 1-, 2-, and 5-year survival rate were 95%, 64%, and 55%, respectively. The treatment was quite toxic and 42% patients were hospitalized during treatment, but there were no treatment-related deaths. We decided to compare in this phase III trial CapRI with 5-FU plus folinic acid, i.e. the best arm of the first large European randomised ESPAC-1 trial. Patients in study arm A are treated with 200 mg/m²/day 5-Fluorouracil by continuous intravenous infusion on days 1-38; 30 mg/m² Cisplatin IV over 60 minutes (with pre- and posthydration) on days 1, 8, 15, 22, 29, 36 (6 doses) and 3 million units SQ Interferon alpha-2b d1-38 (17 total doses). External beam radiation is given concurrently with chemotherapy with a total dose of 50.4 Gy in 28 fractions over 5.5 weeks (1.8 Gy/day). In study arm A the patients receive post-chemoradiation 5-FU Infusions of 200/mg/m² /day by continuous intravenous infusion on days 64-101 and 120-161. Patients in study arm B are treated with 20 mg/m2 intravenous bolus injection of Folinic Acid, D-L form, followed by 425 mg/m²/day intravenous bolus injection of 5-FU given on 5 consecutive days every 28 days for 6 cycles, i.e. 24 weeks. Primary objective is the overall survival in both arms. Secondary objective is to evaluate the role and the mechanism of interferon alpha 2b in patients chemoradiation regimen, to assess the toxicity, the disease-free interval, the quality of life and to test different factors for their potential role as predictive marker. A total of 110 patients with R0 or R1 resected pancreatic adenocarcinoma will be enrolled. Recruiting phase started in August 2004 and should be finished after 2 years. With a follow-up of two years a total running-time of approx. 4 years should be expected. One year after inclusion of the first patient an interim analysis will be done. If already at this time point there is a significant difference between both groups favoring the Virginia Mason scheme (p <0.001) group B (5 FU/Folinic Acid) will be stopped and replaced by group C (Radiochemotherapy without Interferon alpha).

P04

Immunomonitoring of the CapRI Trial: First Results

Jan Schmidt¹, Markus Büchler^1;2, Angela Märten^1;2

¹ University of Heidelberg, Department of Surgery, 69120 Heidelberg, Germany
² National Centre for Tumor Diseases, 69120 Heidelberg, Germany

In August 2004 we have started a phase III trial, where we compare combined chemoradiotherapy plus IFN-alpha (arm A) with 5-FU bolus infusion plus folinic acid (arm B) in patients with pancreatic adenocarcinoma in the adjuvant setting. We hypothesize that IFN-alpha is the agent that turns a slightly effective treatment (radiochemotherapy) into a potent therapy. Since our in vitro data indicate that the main mechanism of IFN-α in this regimen is via immunomodulation, we perform a broad immunomonitoring. Patients in study arm A at the CapRI trial receive a single low-dose injection of 3 million units IFN-α (LDI) prior to therapy. Accompanying immunological analysis should help to define predictive response markers. During therapy patients blood samples from patients of both arms are drawn at several time-points for immunological studies. The panel includes immunophenotyping, detection of cytokine mRNA in lymphocytes, determination of cytokine patterns in peripheral blood, cellular cytotoxicity assays, proliferation assays and immunohistochemical analysis of tumor samples (IFN-receptors, MHC expression, leukocyte infiltration, EGF-R). At this time-point we have analyzed blood samples from 24 patients for cellular cytotoxicity, proliferation, granzyme B secretion, serum cytokine levels and by flow-cytometry. Interestingly, we observed with regard to NK cell mediated cytotoxicity an increase in cytotoxicity for PBL against K562 cells from patients in arm A during treatment. Cytotoxicity increased from 13% before first administration of IFN-α (pre LDI) to 45% after 24hrs, dropped then but remained increased during treatment. The mean cytotoxicity of about 20% at an effector to target cell ratio of 80:1 in arm B did not change. Specific granzyme B secretion was analyzed in ELISpot after MUC-1 and/or CA19.9 stimulation. Again, there was no change in arm B patient’s PBL and no specific secretion (compared with medium control). Arm A patients showed an increase in CA 19.9 and MUC-1 specific granzyme B secretion after the second IFN-α dosage, i.e. one day after treatment start. With regard to the proliferation rate after PHA stimulation we observed no change in any group. Furthermore, we analyzed serum levels of various cytokines. IL-6, IFN-gamma and VEGF were below the detection level. TNF-alpha, IL-2 and TGF-beta did not change in group A or B during treatment. Only with regard to IL-12 we observed elevated serum levels in group with peaks one day after IFN-α injection. Flow-cytometric analysis of peripheral blood showed a significant increase in monocytes, peripheral dendritic cells, CD80, CD40 and IFN-αR expression on monocytes and central and effector memory T cells in arm A patients. Percentage of PI+ lymphocytes decreased in arm A during treatment as well as naïve lymphocytes and expression of IFN-αR on lymphocytes. Percentage of NK cells and regulatory T cells remained unchanged in both groups. The effects were most pronounced after the first administration of IFN-α. To summarize, we observed so far an increase in NK and T cell cytotoxicity, IL-12 secretion and an activation of monocytes in group A patients with a peak one day after the first administration of IFN α. At the end of the trial we will correlate these analyses with the clinical outcome.

P05

Induction of antitumor immune responses by DNA vaccines encoding APC-targeted ErbB2 and NY-ESO-1 fusion proteins

Arjen Sloots¹, Florian Rohrbach¹, Robert Weth¹, Elke Jäger² and Winfried Wels¹

¹Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus and
²Krankenhaus Nordwest, II. Medizinische Klinik, Onkologie/Hämatologie, Frankfurt am Main, Germany

DNA vaccination constitutes a powerful approach to elicit or modulate specific immune responses. DNA vaccines offer a number of possible advantages, such as relatively easy and cost-effective development, production and storage. In addition, molecular cloning techniques allow rapid manipulation of plasmid DNA in order to alter the quantitative and qualitative aspects of the immune response to be elicited, for example by optimizing codon usage, adding immunostimulatory CpG motifs or incorporating genes encoding cytokines or costimulatory molecules. Furthermore, DNA vaccines can encode multiple immunogenic epitopes and have been shown to trigger both humoral and cell-mediated immune responses.
To target the human ErbB2 (HER2, neu) tumor antigen to antigen presenting cells (APC), we have previously generated chimeric protein vaccines that contain the extracellular domain of CTLA-4 for specific binding to B7 molecules on the APC surface, fused to amino acid residues 1-222 of human ErbB2 as an antigenic determinant. Vaccination of mice with recombinantly produced CTLA-4-ErbB2 fusion proteins resulted in the induction of ErbB2-specific CTLs and CTL-dependent protection from subsequent challenge with ErbB2 expressing murine renal carcinoma cells (Renca-lacZ/ErbB2). Here we have introduced the sequence encoding a CTLA-4-ErbB2 fusion protein into the mammalian expression vector pSecTag2B, for direct expression and subsequent secretion of these protein vaccines in vivo following intramuscular injection of plasmid DNA. To investigate the general applicability of such CTLA-4 fusion vaccines, a similar construct was designed, which carries the unrelated cancer-testis antigen NY-ESO-1.
Following transfection of mammalian cells with CTLA-4-ErbB2 or CTLA-4-NY-ESO-1 DNA constructs in vitro, expression of the respective fusion proteins could be detected in culture supernatants. Specific binding of these secreted proteins to B7 molecules on the surface of cells could be demonstrated by FACS analysis. Balb/c mice vaccinated with the CTLA-4-ErbB2 DNA construct were protected against subsequent challenge with Renca-lacZ/ErbB2, but not against ErbB2-negative Renca-lacZ cells. Vaccination resulted in activation of ErbB2-specific T cells, indicated by increased numbers of IFN-gamma producing CD8+ cells upon ex vivo restimulation of splenocytes with a synthetic ErbB2 peptide. In addition, ErbB2-specific antibodies were detected in immunized mice. For analysis of antitumoral activity of the CTLA-4-NY-ESO-1 DNA vaccine a similar animal model was established based on Renca-lacZ cells stably transfected with human NY-ESO-1 cDNA (Renca-lacZ/NY-ESO-1). Also in this case, vaccination of Balb/c mice with the DNA vaccine induced protective effects against subsequent challenge with Renca-lacZ/NY-ESO-1 cells.
Our results indicate that both, the ErbB2- and NY-ESO-1-based DNA vaccines can induce specific immune responses in vivo against tumor cells expressing the respective antigens. Thereby inclusion of the CTLA-4 domain markedly improved efficacy of the DNA vaccines.

P06

A "supra-agonistic", bispecific single-chain antibody purified from the serum of cloned, transgenic cows induces CD28 mediated killing of glioblastoma cells in vitro and in vivo

Ludger Grosse-Hovest¹, Wolfgang Wick², Rosa Minoia³, Michael Weller², Hans-Georg Rammensee¹, Gottfried Brem⁴, Gundram Jung¹

1 Institute for Cell Biology, Department of Immunology, Eberhard Karls University Tübingen, Germany
² Department of Neurology, Eberhard Karls University Tübingen, Germany
³ Department of Animal Production, University of Bari, Italy
⁴ Ludwig Boltzmann Institute for Immuno-, Cyto- and Molecular Genetic Research,
University of Veterinary Medicine, Vienna, Austria

ABSTRACT
Endowing tumor cells with costimulatory signals for T cell activation has emerged as promising strategy for tumor immunotherapy. Costimulatory molecules were either transfected into tumor cells to generate vaccines or were fused, e.g. to antibodies against tumor-associated antigens, to achieve targeted T cell-costimulation in vivo.
Here we further characterize the anti-tumor activity of a recombinant bispecific single-chain antibody (bi-scFV) triggering T-cell costimulation in an exceedingly efficient way. The antibody, termed r28M, isolated from the serum of cloned transgenic cows, is directed to a melanoma-associated-proteoglycan, also expressed on glioblastoma cells, and to human CD28. Bound to tumor cells, r28M induced "supra-agonistic" T-cell activation via the CD28 molecule without an additional stimulus via the TCR/CD3 complex. In vitro, T-cells and NK-cells, contributed to tumor cell killing after r28M-mediated activation of PBMC. However, NK-activity depended on T-cell derived cytokines. In vivo, r28M markedly inhibited the growth of human glioblastoma cells in nude mice. The serum half-life of the protein after i.v.-injection was approximately 6 hrs. Thus, r28M is unique not only in inducing "supra-agonistic", CD28 mediated T-cell activation against tumor cells in vitro and in vivo. It also meets two additional requirements which are critical for clinical application: a relatively long serum half-life and the possibility of obtaining large amounts of active material from cloned, transgenic livestock.

P07

Selective Stimulation of the Fas/Apo-1/CD95-Death Receptor on Human Glioblastoma Cells with Bispecific Antibodies

Tanja Herrmann, Ludger Grosse-Hovest, Hans-Georg Rammensee, and Gundram Jung

Institute for Cell Biology, Dept. of Immunology, University of Tübingen, Auf der Morgenstelle 15,
Tübingen, Germany.

We reported previously that bispecific antibodies with CD20xCD95 specificity can selectively induce apoptosis in CD20 positive CD95 sensitive lymphoma cells.
Here, we extended this approach using chemically hybridized bispecific F(ab’)2 fragments directed to two different target antigens expressed on human glioblastoma cells: a melanoma associated proteoglycan (MAPG) and the EGF-receptor (EGFR). We found that MAPGxCD95 can kill Fas sensitive glioblastoma cells, whereas the EGFRxCD95 construct was only marginally effective. This is most likely not only due to the mere antigen expression but to the fact that in some cases bispecific antibodies bind in a „uni-cellular" fashion thus preventing „bi-cellular" binding, which is necessary to induce an effective mutual crosslinking of the CD95 molecule. This hypothesis is supported by the finding that both bispecific antibodies can lead to comparable lysis of CD95 positive bystander cells which do not express the relevant target antigens.
We also constructed a recombinant bispecific single chain antibody (scFv) with MAPGxCD95 specificity and found that its activity was comparable to that of the chemically hybridized molecule with identical specificities.
These results show that bispecific antibodies directed against the Death Receptor CD95 may be used to activate this receptor in a tumor cell restricted manner. This may not only allow the selective induction of apoptosis in sensitive tumor cells but also the initiation of immunostimulatory events via the CD95 molecule.
Taken together, our findings may support the development of antibody-based immuno-therapy in patients.

P08

The differentiation antigen NY-BR-1 is overexpressed in mammary tumors and encodes a novel membrane protein - a potential target for immunotherapy

Inka Seil, Silke Deckert, Claudia Frei*, Kurt Zatloukal³, Holger Sültmann¹, Elke Jäger², Alexander Knuth*, Dirk Jäger* and Roland H. Stauber

Georg-Speyer-Haus, Institute for Biomedical Research, Paul-Ehrlich-Str. 42–44, D-60596 Frankfurt, Germany
* Klinik und Poliklinik für Onkologie, Universitätsspital Zürich, Rämistrasse 100, 8091 Zürich, Switzerland
¹ Division of Molecular Genetics, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280,
D-69120 Heidelberg, Germany.
² Krankenhaus Nordwest, II. Medizinische Klinik, Steinbacher Hohl 2-26, 60488 Frankfurt
³ University of Graz, Department of Pathology, Division of Experimental Cell Research and Oncology,
A-8036 Graz, Austria

ABSTRACT
Breast cancer is one of the most common malignancies and a leading cause of death among women. Although early stage breast cancer can be effectively treated, there are limited numbers of treatment options available for patients with advanced and metastatic forms of the disease. Thus, there is a clear need to define new targets for the development of novel therapeutic approaches as well as tumor markers for monitoring both progression and treatment of the disease. Tumor-associated antigens are immunogenic proteins expressed predominantly in cancer cells and are therefore considered promising target molecules for immunotherapy approaches.
We report for the first time the cloning, molecular characterization and expression analysis of the differentiation antigen NY-BR-1, we previously identified by SEREX (1). NY-BR-1 was confirmed as a tumor antigen since we could detect NY-BR-1 specific immune responses in several breast cancer patients. Analysis of NY-BR-1 mRNA and protein expression levels in a systematic multicenter study demonstrated that NY-BR-1 was overexpressed in breast cancer patients. Cloning of the full length NY-BR-1 cDNA predicted an open reading frame of 4.2 kbp, encoding a protein of 1397 amino acids. Using a monoclonal antibody, NY-BR-1 was detectable as a 160 kDa protein in the membrane fraction of breast cancer tumor specimens. Transient expression studies confirmed that NY-BR-1 localized to the plasma membrane and the cytoplasm. Biochemical labeling and cell sorting experiments further confirmed that NY-BR-1 encodes a novel membrane protein. NY-BR-1 might not only be functionally involved in breast cancer development but also represents a highly attractive target molecule for immunotherapy approaches in breast cancer patients.
References
(1) Jäger et al. (2001) Cancer Res 61: 2055-2061

P09

"A bi-specific linker molecule employs NK cell help to generate T cell action against tumors"

Markus Jensen^1-3, Carolin Hoyer^1-3, Martin Klehr^1-3, Deanna Berger⁴, William W. Kwok⁴, Marc Beyer³, Joachim Schultze³, Frank Berthold^1-2

¹ Department of Paediatric Oncology and -Hematology, University of Cologne, Germany
² Center for Molecular Medicine Cologne (CMMC), University of Cologne, Germany
³ Molecular Tumor Biology and Tumour Immunology (MTBTI) at the Department I of Internal Medicine,
University of Cologne, Germany
⁴ Benaroya Research Institute, Seattle, USA

Natural killer cells (NK cells) can spontaneously attack pathogens, virus-infected or malignant cells. NK cells are also known to enhance T- and B cell responses. We constructed a CD3xCD56 bi-specific linker molecule connecting CD56+ natural killer cells with CD3+ T cells to stimulate T cells and investigated the role of NK cells for T cell activation in this model.
CD3 binding provided by the bi-specific linker induced strong T cell activation. Cytotoxic T cell activity against CD56-expressing malignant target cells (neuroblastoma, small cell lung cancer) was also induced by this bi-specific linker.
Depletion experiments of CD56+ blood derived cells revealed that T cell activation during incubation of peripheral blood mononuclear cells (PBMC) was dependent on the presence of CD56+ cells. Add-back experiments of purified NK cells to CD56 depleted PBMC revealed that indeed NK cells were the cells enhancing T cell activation after stimulation by the CD3xCD56 bi-specific linker. More important, T cells pre-activated in the presence of the linker molecule and NK cells showed significantly augmented cytotoxicity against CD56+ malignant neuroblastoma cells.
We next addressed the question whether antigen-specific T cell responses might also be enhanced by pre-activation with the linker molecule. To this end, we have stimulated T cells with specific antigen using tetanus toxoid as a model and have been able to demonstrate that the number of IFNγ secreting T cells after stimulation by the specific antigen can be further enhanced in the presence of the bi-specific linker molecule. Using MHC class II tetramers against the DR*1501 restricted epitope TT23 derived from tetanus toxoid, we could demonstrate that the number of tetramer binding T cells is also increased in the presence of the CD3xCD56 linker molecule, suggesting that antigen-specific T cells are expanded under these conditions.
From these data we conclude that the CD3xCD56 bi-specific linker molecule might be used not only to redirect T cells toward CD56+ tumor targets but also as an amplifier of antigen-specific T cell responses e.g. in context of cancer vaccine strategies.

P10

Identification of tumor-associated T-cell epitopes, Part I: Lessons to be learned from primary renal cell carcinoma

Oliver Schoor¹, Margret Müller¹, Anna Missiou¹, Tobias Krüger¹, Claudia Lemmel¹, Björn Krämer¹, Christian Reichle¹, Jörn Dengjel¹, Toni Weinschenk¹, Jörg Hennenlotter², Arnulf Stenzl², Hans-Georg Rammensee¹, Stefan Stevanović¹

¹ Institute for Cell Biology - Department of Immunology, University of Tübingen, 72076 Tübingen, Germany.
² Department of Urology, University of Tübingen, 72076 Tübingen, Germany

The lack of sufficient well-defined tumor-associated antigens is still a drawback on the way to a cytotoxic T lymphocyte-based immunotherapy of renal cell carcinoma (RCC). We are defining larger numbers of such targets by a combined approach involving HLA ligand characterization by mass spectrometry and gene expression profiling by oligonucleotide microarrays. Here we present the results of a large-scale analysis of several RCC specimens. We were able to identify more than 700 peptides, mostly from self proteins without any evident tumor association. However, some HLA ligands derived from previously known tumor antigens in RCC. In addition, gene expression profiling of tumors and a set of healthy tissues revealed novel candidate RCC-associated antigens. For several of them we were able to characterize HLA ligands after extraction from tumor tissue. Apart from universal RCC antigens, some proteins seem to be appropriate candidates in individual patients only. This underlines the advantage of a personalized therapeutic approach. Further analyses will contribute additional HLA ligands to this repertoire of universal as well as patient-individual tumor antigens.

P11

A new polyclonal antibody reveals GAGE protein expression in malignant melanoma

Nicole Wiedemann, Alexandr V. Bazhin, Dirk Schadendorf, Stefan B. Eichmüller

Skin Cancer Unit, German Cancer Research Center, Heidelberg, Germany

The GAGE family of tumor-associated antigens is present in a wide spectrum of human tumors but its expression in normal tissues is restricted to testis. So far, it has been reported that GAGEs expression on mRNA level implies poor prognosis in malignant melanoma. We have generated a rabbit polyclonal antibody for characterization of GAGE-7b protein expression.
GAGE-7b protein was cleaved from recombinant GST-fusion protein by thrombin and purified by anion exchange chromatography using a HPLC-operated MonoQ column. Purified GAGE-7b was used for rabbit immunization and subsequent purification of the monospecific antibody from the rabbit hyperimmune serum. The antibody was used in both Western blotting and immunocytochemistry. 50% of all melanoma cell lines tested (n=14) showed expression of GAGE-7b in Western blotting. GAGE-7b immunostaining could be abolished by preincubation of the antibody with antigen. Moreover, 6% of melanoma patients (n=72) showed detectable autoantibodies against the recombinantly expressed GAGE-7b protein in comparison to 0% recognition in controls (n=36). In conclusion, we describe for the first time the generation of a rabbit polyclonal antibody against GAGE-7b which can be used in Western blotting and immunocytochemistry. Most likely, the antibody recognizes not only GAGE-7b but all 8 different GAGE proteins since they mainly differ by single amino acid substitutions. Using our GAGE-specific antibody we can now investigate functional aspects of GAGE expression in malignant melanoma.

P12

Signaling via chimeric receptors containing the costimulatory domain of CD28 fails to induce tumor-specific T cell proliferation in Epstein-Barr virus specific T cells

Bianca Altvater¹, Sibylle Pscherer¹, Martin Pule², Heribert Jürgens¹, Claudia Rössig¹

¹Department of Pediatric Hematology and Oncology, University of Münster, Germany
²Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, USA

Genetic modification of cytotoxic T cells with tumor antigen-specific chimeric receptors (chRec) specifically redirects their effector functions towards tumor cells. Integration of the signaling domains of the costimulatory molecule CD28 into chRec enhances antigen-specific proliferation of polyclonal human T cell populations. While CD28 plays an essential role in the priming of naïve CD4+ T cells, its contribution to effector memory T cell responses is controversial. We compared the function of optimized CD28ζ chRec with native T cell receptor signaling in in vitro expanded T cells with native specificity for Epstein-Barr virus (EBV).
Chimeric T cell receptors containing an extracellular single-chain antibody domain directed against the tumor ganglioside antigen GD2 or the B cell antigen CD19 fused to the intracellular signaling domains of both the CD28 and the T cell receptor (TCR) ζ chain or TCR ζ alone, were cloned and expressed in EBV-specific cytotoxic T cell (CTL) lines from two seropositive donors by retroviral gene transfer. The transduced CTL maintained their capacity to specifically lyse autologous EBV targets in 4 hr 51Cr release assays and to proliferate after stimulation with autologous EBV targets. Furthermore, tumor targets were lysed in a comparable and antigen-specific manner. However, while antigen engagement by CD28ζ chRec efficiently induces expansion of transduced peripheral blood lymphocytes (PBL), CD28ζ signaling fails to mediate tumor antigen-specific proliferation in transduced EBV-CTL.
Thus, the costimulatory requirement for the highly efficient activation response of EBV-specific CTL to viral antigen can not be mimicked by combined CD28 and ζ signaling alone. Exploring the mechanisms of effector memory T cell reactivation by native T cell receptors may help to design more effective strategies for adoptive immunotherapy of cancer.

P13

CTL Priming by Transcutaneous Peptide Immunization with R-837

Tobias Warger¹* & Gerd Rechtsteiner¹*, Philipp Osterloh^1,2, Hansjörg Schild¹, Markus P. Radsak¹

¹ Institute for Immunology, Johannes Gutenberg-University, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany
² Institute for Cell Biology, Department of Immunology, Eberhard-Karls-University,
Auf der Morgenstelle 15, D-72076 Tübingen, Germany
* contributed equally

Cytotoxic T lymphocytes (CTL) are important in combating cancer and viruses due to their high efficiency in specifically eliminating virus infected and malignant cells and their ability to mediate long term protection after formation of memory cells. New vaccination approaches targeting the activation of CTL are needed because this will not only improve prophylaxis of tumor or virus related diseases, but also open opportunities for effective therapeutic immunizations.
Utilizing a novel transcutaneous immunization (TCI) protocol, we show that TCI by epicutaneous application of a creme containing a CTL epitope in association with the synthetic Toll-like receptor 7 ligand imiquimod (R-837) is highly effective in activating naïve T-cell receptor transgenic CTL. These CTLs mount a full-blown immune response against the target epitope characterized by proliferation, cytolytic activity and the production of IFN-gamma. Beyond this we demonstrate a similar efficacy for CTL priming against the ovalbumine derived CTL epitope SIINFEKL in Wild- Type mice.
In conclusion, we developed a new transcutaneous immunization protocol which allows the robust induction of a CTL response against and restricted to a defined CTL epitope. These results might be useful in developing new vaccines against cancer or virus associated diseases.

P14

Varicella–Zoster-virus-specific T cells as mediators of chmimeric-receptor redirected tumor-specific Immunity

Silke Landmeier¹, Bodo Eing², Verena Niggemeier¹, Cliona Rooney³, Heribert Jürgens¹, Claudia Rössig¹

¹ Department of Pediatric Hematology and Oncology, University Children's Hospital Münster, Münster, Germany
² Department of Virology, University of Münster, Münster, Germany
³ Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, USA

Adoptive transfer of gene-engineered T cells expressing tumor antigen-specific chimeric receptors is a promising tool in cancer immunotherapy. A major limitation is the failure of chimeric receptors to induce T cell proliferation and reexpansion, resulting in a rapid loss of function. We propose to use cytotoxic T cells with native specificity for Varicella-Zoster-Virus (VZV) antigens as mediators of chimeric receptor directed anti-tumor immunity. Booster doses of a VZV vaccine may allow long-term in vivo survival and reactivation of gene-modified dual specific CTL following adoptive transfer.
To investigate the potential of cytotoxic T cells specific for VZV to serve as chimeric receptor-mediated antitumor effector cells, we generated VZV-specific cytotoxic T cell lines (VZV-CTL) from four seropositive donors by culturing peripheral blood-derived T cells with lysates extracted from VZV-infected fibroblasts. By repeated stimulation with VZV lysate and autologous PBMCs, VZV-CTL could be expanded to large numbers and maintained in culture for more than 12 weeks.
Transduction with recombinant retrovirus encoding the GD2-specific chimeric receptor 14.G2a-ζ resulted in surface expression on up to 70% of cells. Gene-modified VZV-CTL efficiently recognized GD2+ neuroblastoma targets, as demonstrated by antigen-specific secretion of IFN-γ in response to coincubation with GD2-expressing tumor targets. Furthermore, 14.G2a-ζ-transduced VZV-CTL performed potent and antigen-specific tumor cytolysis in a MHC-independent manner in standard 51Cr release assays. Antibody blocking experiments revealed that tumor cells were lysed in a granulysin-dependent manner.
Chimeric receptor-transduced VZV-CTL may provide a source of highly potent tumor-reactive cells for adoptive immunotherapy cancer. Their responsiveness to a well tolerated vaccine may allow selective reexpansion of adoptively transferred T cells in vivo, permitting long lasting anti-tumor immune control.

P15

Evaluation of genetic melanoma vaccines in cdk4-mutant mice provides evidence for immunological tolerance against authochthonous melanomas in the skin

Julia Steitz¹ , Julia Lenz¹, Stefanie Büchs¹, Christoph Huber², Thomas Wölfel², Mariano Barbacid³, Marcos Malumbres³, and Thomas Tüting¹

¹ Laboratory of Experimental Dermatology, Department of Dermatology, University of Bonn, Germany;
² Department of Internal Medicine III, University of Mainz, Germany;
³ National Cancer Research Center, Madrid, Spain;

We evaluated the efficacy of a candidate melanoma vaccine approach in mice genetically prone to develop melanoma due to the introduction of an oncogenic mutation (R24C) in the germline sequence of the cyclin dependent kinase 4 (cdk4), a protein critically involved in cell cycle regulation. Melanomas were induced in cdk4-mutant mice by chemical carcinogenesis. A genetic prime-boost strategy targeting the clinically relevant differentiation antigen tyrosinase-related protein 2 (TRP2) was performed which was able to stimulate a melanocyte-specific cellular immune response associated with localized autoimmune vitiligo-like depigmentation. However, significant destruction of carcinogen-induced autochtnonous melanocytic neoplasms in the skin was not observed following immunization. We provide evidence that autochthonous melanomas expressed TRP2 but not the MHC molecule H2-Kb and are immunologically tolerated in the skin. Our results highlight the importance of assessing melanoma vaccines in genetic mouse models which more adequately represent the expected clinical situation in order to identify strategies, which eventually may be of benefit for melanoma patients.

P16

Stabilized RNA activates several human blood cell types through a TLR signalling pathway

Birgit Scheel^†,*, Regina Teufel^†,*, Jean-Philippe Carralot^*,†, Jochen Probst^†, Markus Radsak^†, Hans-Georg Rammensee^†, Ingmar Hoerr* and Steve Pascolo*,^†.

^† Institute for Cell Biology, Department of Immunology; Auf der Morgenstelle 15; 72076 Tübingen, Germany
* CureVac GmbH, Paul Ehrlich Str. 15, 72076 Tübingen, Germany

We reported that stabilized RNA is a danger signal that activates mouse cells through an MyD88-dependent pathway. Now, we show that this danger signal stimulates several human blood cell types. Stabilized RNA strongly activates human DCs and monocytes leading to secretion of TNF-α and interferon-α, for instance. In addition, it directly activates B-cells, NK-cells and granulocytes, as shown by upregulation of CD69, CD86 and cytokine production. The detailed analysis of the activated cell types, the characterization of cytokines released by PBMCs cultured in the presence of stabilized RNA and recently published data suggest that TLR-7 and TLR-8 are involved in the recognition of this new danger signal. It can be assumed that stabilized RNA is a Pathogen Associated Molecular Pattern (PAMP) that mimicks infections with viruses such as retroviruses. Our results show that stabilized RNA activates more cell types than other known TLR ligands (CpG DNA for example) without triggering side effects like splenomegaly. In addition, our recent pre-clinical data indicate that due to its immunostimulatory potency and short half-life, stabilized RNA can be of interest for immunotherapies.

P17

T cell immunity in non-myeloablative allogeneic stem cell transplanted renal cell and breast carcinoma patients

Els Verdegaal¹, Renée Barge², Conny Hoogstraten¹, Jeanne van Steijn¹, Jan Ouwerkerk¹, Willem Fibbe², Fred Falkenburg² and Susanne Osanto¹.

¹ Department of Clinical Oncology, Leiden University Medical Center, Leiden, The Netherlands
² Department of Experimental Hematology, Leiden University Medical Center, Leiden, The Netherlands

T cell responses observed against renal cell (RCC) and breast carcinoma encouraged the exploration of allogeneic stem cell transplantation as a novel approach in the treatment of solid tumors. This approach aims at induction of T cell mediated graft-versus-tumor (GVT) responses similar to the powerful responses that have been observed in hematological malignancies. Non-myeloablative allogeneic stem cell transplantation (NM-alloSCT) has been used as an alternative strategy to conventional high dose transplant regimens thus facilitating stem cell engraftment with limited regimen related toxicity. Graft-versus-Host-Disease (GVHD) remains a significant cause of mortality or morbidity following NM-alloSCT that prevents the subsequent application of cellular immunotherapy in many patients. We investigated a novel in-vitro T-cell depleted non-myeloablative transplantation protocol followed by donor lymphocyte infusion (DLI).
Conditioning consisted of fludarabine 30 mg/m2 6 days, ATG 10 mg/m2 4 days, busulfan i.v. 3.2 mg/m2 2 days and cyclophosphamide 750 mg/m2 2 days. High numbers of G-CSF mobilized blood stem cells from HLA-identical siblings were collected (median 15 x 106 CD34+ cells/kg). The graft was T-cell depleted by incubation with 20 mg Campath-1H. No post-transplant GVHD prophylaxis was administered.
Currently, 2 breast cancer patients and 6 RCC patients underwent NM-alloSCT according to this regimen. The transplant period was very well tolerated. No graft failures were observed. All patients had sustained engraftment of donor cells with a median of 96% donor cells at 3 months after NM-alloSCT. No acute GVHD>grade 1 was observed following this T-cell depleted NM-alloSCT approach. At 3-6 months after NM-alloSCT donor lymphocyte infusion (107 mononuclear cells/kg) was administered in 7/8 patients to initiate GVT effects. This resulted in GVHD in 7/7 patients at a median of 1.5 month after DLI. A durable regression (>1284 days) was obtained in one RCC patient with progressive lung metastases before treatment. Stable disease was observed in 2 patients for more than 158 and 523 days, respectively. Three other patients showed initial stabilization of disease after NM-alloSCT followed by disease progression, whereas two other patients had progressive disease only (2 of these 5 patients developed brain metastases). Reactivation of CMV and herpes simplex occurred in a high number of patients. One patient with GVHD died from unknown infection. With a median follow up of 390 days, 3 patients (all RCC) are still alive.
We conclude that in-vitro T-cell depleted alloSCT following reduced intensity conditioning leads to durable donor engraftment without GVHD. The high level of donor chimerism allows the subsequent DLI to initiate GVT effects. Studies to unravel whether T cell mediated GVT responses occur in these patients in the presence or absence of GVHD are initiated.

P18

CTL transfer in metastatic melanoma patients; a phase I/II clinical trial

Tamara Ramwadhdoebé¹, Carolien van der Minne¹, Conny Hoogstraten¹, Jeanne van Steijn¹, Els Verdegaal¹ and Susanne Osanto¹

¹ Department of Clinical Oncology, Leiden University Medical Centre, The Netherlands

The successful activation and ex vivo expansion of anti-melanoma cytotoxic T lymfocytes (CTL) are preliminary conditions for the approach of adoptive immunotherapy in patients with metastatic melanoma. In this study the possibility to generate and expand CTL for adoptive transfer in melanoma patients is evaluated using both autologous and HLA-class I matched melanoma cell lines as stimulator cells. Previous studies have shown that specific CTL transfer in combination with high dose rhIL-2 can provide clinical responses. Since the use of high dose rhIL-2 is dose limiting, we designed an alternative protocol using IFN-α s.c. to maintain activation status of the transferred CTL in vivo. Importantly, IFN-α may also mediate additional beneficial effects by increasing HLA class I expression on tumor cells and growth inhibition of tumor cells in vivo. Three cohorts of patients will receive escalating doses of CTL infusions in combination with IFN-α s.c. and will be monitored for toxicity, tumor responses and immunological anti-melanoma responses. Melanoma cell lines were generated in our GMP facility from surgical specimens. We successfully cultured 11 melanoma cell lines (41 percent success rate). All established cell lines were sub-typed for HLA class I antigens and studied for expression of melanoma antigens MART, MAGE, tyrosinase, gp100, NY-Eso, Prame and Camel using FACS analysis and RT-PCR. The panel of cell lines covers more than 90% of the possible HLA types and can thus be used for inductions of anti-melanoma CTL utilizing HLA class I-matched allogeneic tumor as stimulator cells in the majority of melanoma patients if autologous melanoma tumors are not available.
Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats and stimulated with irradiated autologous or allogeneic HLA-class I-matched melanoma cell lines as stimulator cells to generate tumor specific cytotoxic T lymphocytes (CTL). Every two weeks cells were tested for specific lytic activity using the 51Chromium release assay and phenotyped by FACS analysis. After 4-8 weeks of culturing, autologous CTLs can be released for administraton to the patient if specific lytic activity against stimulator melanoma cells is demonstrated, reactivity against non-malignant autologous cells (autologous EBV-transformed B cells) is absent and viral and bacteriologic culture tests are negative.
Presently we generated two CTL lines in an autologous setting and one CTL line after stimulation using an allogeneic HLA class I-matched melanoma cell line. All three CTL line batches consist of more than 70% CD8+ T cells, show tumor specific lysis but no reactivity against normal cells of the patient. The first metastatic melanoma patient received CTLs in combination with IFN-α s.c. and no major toxicity was observed. So far, we have demonstrated the feasibility to generate and expand large numbers of antitumor CTL in our GMP facility for adoptive immunotherapy.

P19

Electroporation of human blood cells with messenger RNA provides target cells able to stimulate memory-cytotoxic T cells in vitro

Regina Teufel¹, Birgit Scheel^1,2, Jochen Probst², Jean-Philippe Carralot^1,2, Steffen Walter², Hans-Georg Rammensee² and Steve Pascolo^1,2.

¹ CureVac GmbH, Paul Ehrlich Str. 15, 72076 Tübingen, Germany
² Institute for Cell Biology, Department of Immunology; Auf der Morgenstelle 15; 72076 Tübingen, Germany

The efficacy of immunotherapies is currently evaluated by in vitro measurement of lymphocyte´s activity. Specifically, in the context of innovative anti-tumor vaccination protocols, the triggered adaptive immune response specific for tumor antigens has to be analysed. Usually, for these assays autologous target cells expressing the vaccine antigen(s) are needed. We show that Peripheral Blood Mononuclear Cells (PBMCs) can be directly transfected with mRNA by electroporation and that such cells are as efficient as mRNA-transfected Monocyte-derived Dendritic Cells (Mo-DCs) for the activation of specific memory T-cells in vitro. The production of PBMCs-targets cells requires 10 times less blood from the patients than the production of Mo-DCs and does not need intensive in vitro culture. Frozen PBMCs and fresh PBMCs can be used. They do not prime in vitro naïve lymphocytes whereby guarantying that only re-call responses are monitored. Thus, mRNA-transfected PBMCs are an adequate and convenient replacement of mRNA-transfected MoDCs for the in vitro monitoring of natural or vaccine-induced immune responses.

P20

Reduced frequencies and suppressive function of CD4+ CD25high regulatory T cells in patients with chronic lymphocytic leukemia after therapy with fludarabine

Marc Beyer¹, Matthias Kochanek¹, Kamruz Darabi¹, Alexey Popov¹, Markus Jensen¹, Elmar Endl², Percy A. Knolle², Michael von Bergwelt-Baildon¹, Svenja Debey¹, Joachim L. Schultze¹

¹ Molecular Tumor Biology and Tumor Immunology, Clinic I for Internal Medicine, University of Cologne, Germany
² Institute for Molecular Medicine and Experimental Immunology, University of Bonn, Germany

Globally suppressed T cell function has been described in many cancer patients to be a major obstacle for the development of clinically efficient cancer immunotherapy. Inhibition of anti-tumor immune responses has been mainly linked to inhibitory factors present in cancer patients. More recently, increased frequencies of CD4+ CD25high regulatory T cells (Treg cells) have been described as an additional mechanism reducing immunity. Assessing 73 B cell chronic lymphocytic leukemia (CLL) patients and 42 healthy controls we demonstrate significantly increased frequencies of Treg cells in CLL patients (pts). Analysis of 26 previously untreated CLL pts revealed a correlation between Treg cell frequency and stage of disease with highest numbers of Treg cells in pts with advaced disease (Binet C) followed by pts with Binet B and with lowest, but still increased numbers in Binet A stage. Although a truly unique marker for Treg cells is still not available, several molecules have been associated with these cells including Cytotoxic T Lymphocyte-Associated Protein 4 (CTLA4), Glucocorticoid-Induced TNFR-Related Protein (GITR), Forkhead Box P3 (FOXP3), and L-selectin (CD62L). Further characterization of the increased fraction of Treg cells in CLL showed higher numbers of Treg cells in CLL pts expressing CTLA4+ FOXP3+ GITR+ CD62L+ TGFβ1+ IL-10+ as compared to healthy controls. In 18 CLL pts sufficient numbers of highly purified CD4+ CD25high T cells were isolated to analyze their inhibitory function in comparison to Treg cells from 16 healthy controls. Regulatory function of Treg cells was assessed using an allogeneic MLR of CD4+ CD25- T cells and allogeneic irradiated PBMC as stimulators. In controls as well as CLL pts never treated with fludarabine a significant inhibition of allogeneic CD4+ CD25- T cells was observed. Surprisingly, in the majority of CLL pts treated with fludarabine containing therapy regimens the inhibitory function of Treg cells was decreased or even abrogated. Assessment of IFNγ production by the CD4+ CD25- T cells confirmed the proliferation results. In addition, frequencies of Treg cells were significantly decreased after therapy with fludarabine. Serial analysis of 8 CLL pts treated with fludarabine, of whom we were able to obtain samples at two different time points at least 6 months apart, showed reduced Treg cell frequencies after fludarabine therapy. First in vitro experiments demonstrated a preferential induction of apoptosis in CD4+ CD25+ T cells after incubation with fludarabine. Inhibition of Treg cells by drugs such as fludarabine and cyclophosphamide might not only be exploited prior to adoptive T cell transfer and non-myeloablative stem cell transplantation but also prior to cancer vaccination to enhance vaccine efficacy by reducing regulatory T cell function.

P21

Immunotherapy with autologous PSA-peptide loaded dendritic cells of hormone refractory prostate carcinoma after prestimulation with Interferon-gamma – A pilot study

Bernd Hildenbrand, Barbara Sauer, Anja Langenkamp, Christoph Stoll, Oana Kalis, Marina A. Freudenberg**, Chris Galanos**, Gabi Niedermann**, Brigitte Häring, Regina Leo*, Clemens Unger, Marc Azemar

Tumor Biology Center, Breisacher Str. 117, 79106 Freiburg;
* Kitaro Biotec GmbH, Feodor-Lynen-Str. 23; 30625 Hannover;
** Max-Planck-Institute für Immunbiologie, Stübeweg 51, 79108 Freiburg

Purpose: We conducted a pilot trial to evaluate the feasibility and tolerability of a prime/boost vaccine strategy using autologous dendritic cells (DC) loaded with PSA-peptides after prestimulation with Interferone-gamma (INF-γ) for patients with hormone refractory prostate carcinoma. The induction of PSA-specific immunity and clinical response was also evaluated.
Patients and Methods: Fifeteen HLA-A2 positive patients with hormone-refractory, metastatic prostate cancer were vaccinated for four times with intracutaneously applicated PSA-peptide loaded DC after prestimulation with INF-γ (50 µg/m² KO). Primary end points were response rate (NPCP), development of PSA and clinical benefit (pain intensity). Secondary end points were quality of life (EORTC QLQ-C30), safety and immunological parameters.
Results: The vaccination was well tolerated without any vaccination associated adverse events. Twelve of fifeteen patients could be fully evaluated. One patient dropped out due to early progress, one patient with tumor far in progress died tumor related and one patient was not evaluable because of radiotherapy during vaccination. Of the 12 eligible patients two showed a PSA-decline, three a decrease in PSA-velocity, one no change and six an increase of PSA. In regard to the clinical response four patients showed stable disease, one a mixed response and one a partial response. No complete response was seen. DTH-response to PSA-loaded DC was strongly positive for nine of the vaccinated patients. The statistical evaluation of ELISPOT and Tetramer testing is still in progress. First results show some hints for correlation between PSA-specific T-cell activation and PSA-velocity.
Conclusion: Therapy with PSA-Peptide loaded dendritic cells after pre-stimulation with INF-γ was feasible and associated with minimal toxicity. No evidence for the induction of autoimmunity could be detected. The biochemical and clinical response rate implicate a potential antitumor activity. Key Words: Prostate carcinoma, dendritic cells, PSA, Immunotherapy, clinical trail

P22

Expression of RHAMM/CD168, fibromodulin, survivin, OFAiLRP and hTERT as potential immunotherapeutical targets in patients with B-cell chronic lymphocytic leukemia

K. Giannopoulos^1,3, L. Li¹, J. Rolinski³, A. Dmoszynska², J.Tabarkiewicz³, K. Wojas³, I. Hus², J. Greiner¹, H. Döhner¹ and M. Schmitt¹

¹ 3rd Dept. of Internal Medicine, University of Ulm, 89081 Ulm, Germany
² Dept. of Hematooncology and 3Dept. of Clinical Immunology, Medical University of Lublin, 20-950 Lublin, Poland

Key words: B-cell chronic lymphocytic leukemia (B-CLL), tumor associated antigens (TAAs), leukemia associated antigens (LAAs), cytotoxic T lymphocytes (CTLs), receptor for hyaluronic acid mediated motility (RHAMM/CD168) ABSTRACT

Purpose: Antigen targeted immunotherapy might represent a novel option for the treatment of patients with B-cell chronic lymphocytic leukemia (B-CLL), especially in early stages of the disease.
Methods: The mRNA expression pattern of potential tumor associated antigens (TAAs)/leukemia associated antigens (LAAs) in B-CLL known from the literature fibromodulin, survivin, OFAiLRP, hTERT and defined earlier by SEREX analysis of patients with myeloid leukemias by our group RHAMM/CD168 were screened in 30 B-CLL and 20 healthy volunteers (HVs). Peripheral blood mononuclear cells were examined by conventional and quantitative RT-PCR. To evaluate the immunogenicity of the RHAMM/CD168, a novel LAA candidate in B-CLL, mixed lymphocyte peptide cultures and ELISPOT assays were performed for R3 (ILSLELMKL), a recently characterized highly immunogenic T-cell epitope peptide derived from RHAMM/CD168.
Results: mRNA of RHAMM/CD168, fibromodulin, was expressed in 55-90%, mRNA of OFA-iLRP in 90-100% of the B-CLL patients. No expression of hTERT and survivin was observed. Only RHAMM/CD168 and fibromodulin showed expression in B-CLL patients but not in HVs, all others antigens showed expression frequencies of 28-100% in HVs. However after stimulation with sCD40L, survivin expression appeared in B-CLL patients. The relative amount of mRNA for RHAMM/CD168 was significantly higher in B-CLL patients than in HV. No significant difference of RHAMM expression between ZAP-70 positive and negative patients was observed. Specific CD8+ T cell responses against RHAMM/CD168 in B-CLL patients were detected in vitro.
Conclusion: RHAMM/CD168 might be a novel possible target for future immunotherapies in both ZAP-70 (+) and ZAP-70 (-) B-CLL patients.

P23

Improvement of the production of IL-12p70 by human monocyte-derived dendritic cells maturated with various highly purified LPS-derivatives from Salmonella minnesota and INF-γ under serum-free conditions

Bernd Hildenbrand, Christoph Kalis*, Marc Azemar, Barbara Sauer, Gabi Niedermann*, Oana Kalis, Clemens Unger, Chris Galanos* and Marina A. Freudenberg*

Tumor Biology Center, Breisacher Str. 117, 79106 Freiburg;
* Max-Planck-Institut für Immunbiologie, Stübeweg 51 ,79108 Freiburg

Summary Purpose: To prime efficient antigen-specific T cell stimulation, dendritic cells (DC) need to skew T cell reactivity toward a TH1 pattern. However, under serum free conditions (without LBP) DC produce IL-10 in response to microbial stimuli like lipopolysaccharides (LPS) and thus prefer the initiation of a TH2 response. In this work we have tested several highly purified LPS-derivatives from Salmonella minnesota with or without INF-γ to improve the capacity of monocyte-derived DC to induce TH1-response under serum-free conditions
Methods: Monocyte-derived DC were generated in serum-free medium with GM-CSF and IL-4 for 7 days and matured for another 24 or 48 h with the LPS-derivatives ± INF-γ. The LPS-derivatives differ in the structure of their core region. The following LPS-derivatives from Salmonella Minnesota were tested: R60, R7, R5, R345, R595. Matured DC were phenotypically characterised and their elaboration of cytokines was measured by ELISA.
Results: (i) All investigated LPS-derivatives appeared able to provide the phenotypic markers characteristically for DC maturation. (ii) DC matured with LPS-derivatives resulted in high levels of IL-10 but only low levels of IL-12 p70. (iii) Addition of Interferon-γ leads to a significant higher levels of bioactive IL-12p70 concomitant with lower levels of IL-10. (iv) Addition of INF-γ resulted in more pronounced upregulation of CD83, major histocompatibility complex class I and B7 molecules by DC (v) R60 and R595 produced the highest amounts of IL-12p70 and upregulated CD83 most efficiently
Conclusion: All tested highly purified LPS-derivatives from Salmonella minnesota in combination with IFN-γ represent suitable agents for ex vivo maturation of monocyte-derived DC in serum free medium. All tested LPS-derivatives + INF-γ can be potentially used as cellular vaccines to promote a potent type 1 immune response. Most efficient immune stimulating adjuvants seem to be LPS R60 and LPS R595.

P24

RHAMM/CD168 is a novel target of immunotherapies for patients with acute myeloid leukemia (AML)

Michael Schmitt, Li Li, Mark Ringhoffer, Krzysztof Giannopoulos, Hartmut Döhner, Jochen Greiner.

3rd Dept. of Internal Medicine, University of Ulm, Robert-Koch-Str. 8, 89081 Ulm, Germany.

The majority of AML patients younger then 60 years reach a complete hematological remission (CR). However, often the CR is not durable and finally a high percentage of these patients relapse. Immunotherapies targeting leukemia-associated antigens (LAAs) might open new ways to maintain a CR or to challenge minimal residual disease. To identify such LAAs, we employed earlier the method of serological screening of a cDNA expression library (SEREX). The receptor for hyaluronic acid mediated motility (RHAMM/CD168) revealed to be the most promising LAA defined by this SEREX analysis: mRNA for RHAMM/CD168 was demonstrated by RT-PCR and Western blot to be expressed in leukemic blasts of more than 80% of the AML patients, but not in PBMN or CD34+ stem cells of healthy volunteers. Among normal tissues, only testis, placenta and thymus showed significant mRNA expression for the antigen RHAMM/CD168.
For the definition of T cell epitopes of RHAMM/CD168 towards specific immunotherapies for acute myeloid leukemia (AML), ten potential HLA-A2 binding RHAMM/CD168-peptides (R1-R10) were synthesized based on computer algorithms and screened by ELISPOT analysis using CD8+ T cells isolated from peripheral blood (PB) of AML patients and healthy donors. CD8+ cells from 7/13 (54%) AML patients presensitized with peptides R3 (ILSLELMKL) or R5 (SLEENIVIL) specifically recognized T2 cells pulsed with R3 (39%) or R5 (15%) peptide. In contrast, only in 4/21 (19%) healthy volunteers we detected CD8+ cells reactive with R3 or R5 pulsed T2 cells after presensitization. By staining with a HLA-A2/R3 peptide tetramer, the presence of R3 peptide specific CD8+CD45RA+CCR7- T cells PB of AML patients could be confirmed. In chromium-51 release assays, peptide primed CD8+ T cells from AML patients were able to lyse RHAMM/CD168 peptide pulsed T2 cells and AML blasts. Transfection of COS-7 cells with RHAMM/CD168 cDNA revealed that peptides R3 and R5 are naturally processed epitopes of RHAMM/CD168 presented in a HLA-A2 restricted manner. We therefore consider RHAMM/CD168 to be a promising target for immunotherapies in AML patients and have initiated a clinical vaccination trial with R3 peptide. As RHAMM/CD168 is also expressed in various other hematological malignancies and solid tumors, therefore vaccines targeting this antigen may have even wider application.

P25

Interaction of TLR2 and TLR4 ligands with Gp96 amplifies innate and adaptive immune responses

Tobias Warger*, Gerd Rechtsteiner*, Nobert Hilf^#, Philipp von Landenberg^%, Helmut Jonuleit⁺, Hans-Georg Rammensee^§, Markus P. Radsak* and Hansjörg Schild*

* Institute of Immunology, University of Mainz, Obere Zahlbacherstr. 67, 55131 Mainz, Germany
^# Immatics Biotechnologies, Paul-Ehrlich-Str.1, 72076 Tübingen, Germany
⁺ Department of Dermatology, University of Mainz, Langenbeckstr. 1, 55101 Mainz, Germany
^% Institute for Clinical Chemistry and Laboratory Medicine, Naunynweg, D-55101 Mainz, Germany
^$ Department of Immunology, University of Tübingen, Morgenstelle 15, 72076 Tübingen, Germany

A crucial event in the initiation of innate and adaptive immune responses is the activation of dendritic cells by agonistic Toll-like receptor ligation. Several classes of TLR ligands have been identified so far interacting with distinct members of the TLR-family, ranging from TLR1 to TLR11. Ligands activating TLR4 show a remarkable diversity including LPS derived from different gram-negative bacteria and viral proteins. Recent reports demonstrated the activation of dendritic cells by members of the heat shock protein family, including Hsp60, Hsp70 and Gp96. However, doubts were raised as to what extent this effect was due to LPS contaminations of the HSP preparations used for Dendritic Cells activation. We re-examined this phenomenon using a Gp96 purification protocol that significantly reduces the amount of endotoxin. As reported recently we do not observe Dendritic Cell activation by low endotoxin Gp96. However, pre-incubation of Gp96 with low amounts of TLR2 and TLR4 ligands, unable to activate DCs alone, results in the production of pro-inflammatory cytokines, including IL-12p70, the up-regulation of activation markers by mouse and human DCs and the amplification of T cell activation. Furthermore, human DC grown under serum free conditions and therefore unresponsive to LPS-mediated activation unless soluble CD14 and LBP are provided, now respond to the LPS stimulus in the presence of Gp96. Our results resolve the controversial issue of HSP-mediated DC activation and provide a new function of HSPs in the amplification of Dendritic Cell activation by bacterial products and induction of adaptive immune responses.

P26

Phenotypic and funtional characterization of in vitro maturated dendritic cells: differential influence of microbial extract conditioned media on in vitro dendritic cell maturation

Kang-Hun Lee, Mareen Reichert, Benjamin Günther, Carmen Scheibenbogen and Eckhard Thiel

Hämatologie, Onkologie und Transfusionsmedizin, Medizinische Klinik III,
Charité Campus Benjamin Franklin, Berlin, Germany

Dendritic cells (DCs) play a central role in the regulation of cellular immune response and in vitro maturated DCs derived from periperal blood monocytes are intensively studied for their utility in immunotherapy. To this end, diverse methods of in vitro DC generation have been published, mainly differing in the maturation step after induction of immature DCs with G-CSF and IL-4. Here, we systematically compared DCs maturated utilizing microbial extracts with DCs maturated with commonly used maturation stimuli.
Microbial extract conditioned media (CM) were obtained by incubation of peripheral blood mononuclear cells with extracts of gram-negative (Colibiogen, CoCM) or gram-positive (Symbioflor, SyCM) bacteria, a mixture of bacteria (BronchoMunal, BMCM) or viral proteins (Begrivac, BeCM) and added to immature DCs for DC maturation. Microbial extract CM maturated DCs were compared phenotypically to DCs obtained with a cytokine cocktail (CC, consisting of IL-1b, IL-6, TNF-b, and PGE2), monocyte CM (MCM) or LPS.
CC DCs showed a enhanced expression of CD40, CD80, CD83, CD86, and CD123, as compared to MCM or LPS maturated DCs. SyCM DCs displayed a similarly pronounced expression of CD86, to a lesser extent CD80, CD83 and CD123, but no upregulation of CD40. BeCM DCs, however, showed CD40 and CD86 expression similar to CC DCs, and interestingly an upregulation of CD64. When BeCM and SyCM were added together, the DCs were characterized by CD40 and CD86 expression comparable to CC DCs, weaker expression of CD80, CD83, and CD123, but stronger expression of CD64 and CD209. Thus, among the microbial extract CM DCs the BeCM+SyCM DCs showed phenotypically the closest resemblance to CC DCs, and were selected for further functional characterization.
The capacity to stimulate T cell proliferation in an MLR as well as the epitope-specific T cell stimulation were similar for BeCM+SyCM DCs and CC DCs, although there was a trend towards slightly stronger T cell stimulation with CC DCs. We next screened for the cytokines in BeCM and SyCM using cytokine array membranes including 20 cytokines. SyCM contained high levels of IL-1b, IL-6, IL-8, IL-10, TNF-a, and MCP-1, whereas BeCM showed only IL-8 and MCP-1, which were also detectable in the control medium (no microbial extract). We also analyzed the cytokine profile of the culture media after DC maturation. Comparable amounts of IL-1b, IL-6, IL-8, and MCP-1 were detected in CC DC and BeCM+SyCM DC culture media, whereas TNF-a could be demonstrated only in CC DC. IL-6, IL-8 and MCP-1 were also highly expressed in control DC cultures.
In conclusion, BeCM+SyCM generate mature DCs that are phenotypically and functionally comparable to CC DCs. Consistently, three of the 4 cytokines in the CC are present in high amounts in SyCM (PGE2 was not tested). Nonetheless, characteristic differences identify them as distinct DC populations and the practical relevance of these differences remains to be addressed in future studies. As the microbial extracts used in this study were made for application in humans, our protocol may contribute to a lower cost, large scale DC generation for clinical use.

P27

Definition of potential T cell epitopes for the immunotherapy of cutaneous T cell lymphoma

Jan Müller-Berghaus¹, Hiltrud Schönhaber¹, Xuan. Duc Nguyen², Harald Klüter², Stefan Eichmüller¹, Dirk Schadendorf¹

¹ German Cancer Research Center, Skin Cancer Unit, 69120 Heidelberg, Germany
² DRK, Institute for Transfusion Medicicne and Immunology, 68167 Mannheim, Germany

Recent evidence suggests that cutaneous T cell lymphoma (CTCL) may be amenable to immunotherapy. With the exception of peptides derived from the clonotypic T cell receptor (TCR) no T cell epitopes have been defined yet. The disadvantage of the usage of TCR-derived sequences for immunotherapy is the need for patient-specific epitopes. Using SEREX we recently described a novel antigen of the cancer-testis family that is almost exclusively expressed in CTCL, hence called cTAGE-1.
Using the "reverse immunology" approach we identified 17 potential HLA-A2 binding peptides from the cTAGE-1 sequence. Peptide-pulsed dendritic cells of normal donors were used to stimulate autologous T cells and peptide-specific lines were generated. Epitope-specificity was evaluated using interferon-γ ELISpot. Epitope-specific T cell lines could reproducibly be generated for 10 peptides. T cell lines specific for two of the peptides were capable of recognizing an allogeneic cTAGE-1 expressing CTCL cell line after transfection of the restricting HLA molecule. Further experiments concerning processing are currently ongoing and will be presented.

P28

Structural imbalance of chromosome 15 contributes to the irreversible loss of HLA class I expression on melanoma cells

Antje Sucker¹, Norbert Arens², Sandra Striegel¹, Ralf Hildenbrand², Michele Maio³, Dirk Schadendorf¹, Annette Paschen¹

¹ Skin Cancer Unit of the German Cancer Research Center (DKFZ) at the University Clinics of Mannheim, Germany
² Institute of Pathology, University Clinics of Mannheim, 68135 Mannheim, Germany
³ Department of Medical Oncology, Istituto di Ricovero e Cura a Carattere Scientifico, Aviano, Italy

HLA class I molecules presenting small peptide fragments derived from cellular proteins allow cytotoxic CD8+ T cells to monitor the protein composition of a cell and to detect its malignant transformation. Such HLA class I molecules consist of a heavy chain associated with a light chain, the beta2-microglobulin (b2m). Both chains have to assemble intracellular to achieve stable surface presentation of the complex. Tumor cells can escape from CD8+ T cell recognition by down-regulation or even total loss of HLA class I surface presentation. Mutations in the b2m gene have been described to be causative for the HLA class I deficiency of tumor cells. Consequently, the HLA class I negative phenotype of such cells must be the product of multiple mutational events affecting both copies of the b2m gene. To elucidate the complex nature of such mutations we analyzed four HLA class I-deficient melanoma cell lines (Mel 249, Mel 499, Mel 505, Mel 592) for their molecular defects.
Surface presentation of HLA class I molecules on these cell lines could not be restored by IFN-γ treatment. In contrast, transfection of tumor cells with b2m cDNA induced HLA class I expression, indicating that mutations affecting the b2m gene were indeed causative for the HLA class I-negative tumor cell phenotype. To characterise those defects, total RNA of the four cell lines was employed for b2m-specific RT-PCR. No b2m-specific cDNA could be obtained from Mel 505 and Mel 592 cells, suggesting that both copies of the b2m gene were either not transcribed or (partially) deleted. In contrast, Mel 249 and Mel 499 cells exhibited a b2m-specific PCR product: Sequence analysis of the products revealed a microdeletion of 2 base pairs in exon II for Mel 249 and different insertions of intron sequences between exon I and exon II for Mel 499 cells. Beside microdeletions/-insertions in the b2m coding sequence, macrodeletions due to chromosome 15q instability might also contribute to the loss of b2m expression. To detect such deletions, we studied the status of heterozygosity of specific microsatellite markers located on 15q and performed fluorescence in situ hybridizations employing a whole chromosome 15q painting probe. The analysis demonstrated that extensive loss of 15q material and 15q rearrangements (e.g. translocations) occurred in all cell lines tested which contribute to the loss of b2m expression. This suggests, that structural imbalance of chromosome 15 might be a characteristic feature of melanoma cells exhibiting an irreversible loss of HLA class I expression.

P29

T-cell depleted haematopoietic stem-cell transplantation followed by preemptive CD8-depleted DLI

R.G. Meyer, C.M. Britten, A. Konur, K. Bender, D. Wehler, U. Hartwig, T.C. Wehler, C. Huber, K. Kolbe, W. Herr

We initiated a phase-I allogeneic peripheral blood stem-cell transplantation (PBSCT) trial which combines a reduced-intensity in vivo T-cell depletion (TCD) regimen (Kottaridis et al., Blood 2000) with the preemptive administration of CD8-depleted donor lymphocyte infusions (DLI) in high-risk patients with hematological malignancies. After early withdrawal of cyclosporine A, patients received escalating doses of CD8-depleted DLI, if there was no evidence of active GvHD. CD8 depletion of donor leukaphereses using clinical grade magnetic CD8 beads resulted in a 2.5 to 4 log reduction of CD8 T cells. Fourteen patients (7 MM, 1 hg-NHL, 1 MCL, 1 CML, 1 OMF, 1 Ph+ ALL, 1 sAML, 1 HD) received PBSC from either HLA-identical sibling (1), HLA-matched (9), or HLA-mismatched unrelated donors (4). Four patients developed early spontaneous acute GvHD of the skin. Therefore in these patients, DLI were postponed (1) or cancelled (3). Up to now, 8 DLI were administered to 5 patients without any acute toxicity. An increase of CD4+ T cells of at least 1.5-fold was observed in peripheral blood after transfer. Four patients developed grade 1 GvHD of the skin after their DLI, and three required transient systemic (2) or topic (1) immunosuppression. One patient suffered from severe GvHD of the skin, bowel, and liver 60 days after the first DLI, which came into complete remission during immunosuppressive treatment. Donor chimerism of purified CD4, CD8, CD19, CD14, and CD15-positive peripheral blood cells was generally > 80% at day +30. A secondary drop of donor T-cell chimerism to a low of 40% in two patients between days +50 and +100 was successfully reverted by CD8-depleted DLI. In addition to blood and bone marrow cells, chimerism analysis on Langerhans cells was performed skin biopsies from some patients. Biopsies taken after day +100 showed 100% donor chimerism, whereas LC isolated from biopsies taken before day +30 were of host origin. LC obtained between days +30 and +100 were of mixed chimerism.
Our preliminary data suggest that the preemptive transfer of CD8-depleted DLI is feasible and does not induce acute toxicity related to the depletion procedure. In 3 patients it was followed by a mild and transient GvHD of the skin. The severe GvHD observed in one patient and its relatedness to the DLI is still under investigation. In a few patients, the early spontaneous onset of acute skin GvHD prevented the application of DLI. A local allo-stimulation by persisting host-LC might contribute to this phenomenon. Those patients who received CD8-depleted DLI demonstrated beneficial effects in terms of CD4 T-cell reconstitution, and engraftment.

P30

Development of a Clinical-Grade Protocol to mature moncyte-derived dendritic cells in TH1-direction

Bernd Hildenbrand, Barbara Sauer, Marina Freudenberg*, Chris Galanos*, Christoph Kalis*, Gabi Niedermann*, Christoph Stoll, Clemens Unger, Marc Azemar

Tumor Biology Center, Breisacher Str. 117, 79106 Freiburg;
*Max-Planck-Institut für Immunbiologie, Stübeweg 51, 79108 Freiburg

Summary
Purpose: Dendritic cells (DC) can initiate primary adaptive immune responses and skew T cell reactivity toward a TH1 or TH2 pattern. We improved the capacity of clinical-grade monocyte-derived dendritic cells to elaborate IL-12 with special regard of their state of maturation, different maturation stimuli and its regulation by TH1/TH2-influencing cytokines.
Methods: Monocyte-derived DC were generated in serum-free medium with GM-CSF and IL-4 for 7 days and matured for another 24 or 48 h with PGE2, TNF-α, ± IL-6 , ± IL-1 and ± Interferon-γ. Matured DC were phenotypically characterised and measured for IL-6, IL-10 and IL-12 p70 by ELISA.
Results: All investigated clinical-grade protocols for maturation of monocyte-derived DC appeared able to provide the phenotypic characteristics necessary to initiate DC maturation. DC matured with PGE2 and TNF-α only resulted in high levels of IL-10 but only low levels of IL-12 p70. Addition of IL-1 and IL-6 (Jonuleit cocktail) and Interferon-γ lead to significant higher levels of bioactive IL-12 concomitant with lower levels of IL-10. Besides addition of INF-γ resulted in more pronounced up-regulation of CD83, major histocompatibility complex class I and B7 molecules by DC
Conclusion: PGE2, TNF-α, IL-1, Il-6 and IFN-γ represent a suitable cytokine cocktail for the ex vivo maturation of monocyte-derived DC that can be used as cellular vaccines to promote a potent type 1 immune response.

P31

The role of MMP-7 in cancer-mediated immunotolerance in vivo

Sebastian Gregor¹, VijayAlla¹, Jürgen Kuball², Matthias Theobald², Peter R. Galle¹, Dennis Strand¹, Susanne Strand¹

¹ I. Department of Internal Medicine, Johannes Gutenberg-University, 55101 Mainz, Germany
² Department of Hematology and Oncology, Johannes Gutenberg-University, 55101 Mainz, Germany

Escape from immune surveillance prefigures the rapid progression of human cancer. Various immune escape mechanisms in cancer have been proposed. Cancer cells may secrete immunosuppressive factors to modify the host immune response. One of these factors is Matrix Metalloproteinase-7 (MMP-7) which is the smallest and most simple structured member of the MMP-family. MMP-7 is a zinc-dependent proteinase, is produced as an inactive proform and is activated by other proteinases. MMP-7 is expressed in many human cancers, such as colorectal-, gastric- and hepatocellular cancer, but also different hematopoeitic neopleasias like AML and multiple myeloma. Along with its prometastatic function, a fundamental role for MMP-7 in early tumor development and tumor progression has been established. In vitro MMP-7 can specifically cleave the CD95-receptor which results in reduced CD95 surface expression and decreased CD95-mediated apoptosis. Furthermore, specific cytotoxic T cell killing was reduced in the presence of MMP-7.
In the present study we established a tumor mouse model with MMP-7 expressed in Hek293 cells that are under control of an ecdysone inducible vector. We observed increased tumor growth in NOD/SCID mice under the influence of MMP-7 compared to the control. After adoptive transfer of specific CD8+ T-cells the MMP-7-producing tumors were able to escape from CTL killing. In MMP-7 negative tumors CD8+ T cells prevented tumor growth.
In conclusion, we have demonstrated that MMP-7 decreased the cytotoxicity of CTLs in vitro. We now show that MMP-7 influences the interaction between CTLs and tumor cells in vivo, by decreasing CTL killing after adoptive T-cell transfer in mice. Considering the promising findings in our present study, the results may explain the role of MMP-7 in early tumor development. We propose, that specific inhibition of MMP-7 may increase the efficiency of immunotherapy in gastrointestinal tumors and could therefore open new possibilities for increasing the effectiveness of tumor therapy.

P32

The idiotype vaccination program for B cell lymphoma at Freiburg University - Current status

Kristina Mikesch, Cristina Bertinetti, Hendrik Veelken

Department of Hematology/Oncology, Freiburg University Medical Center, D-79106 Freiburg, Germany

Immunization of B-NHL patients against the individual immunoglobulin expressed by the lymphoma (idiotype; Id) may induce tumor-specific immune responses. The Freiburg approach of Id vaccination is based on recombinant Fab fragments expressed in E.coli mixed with the adjuvant MF59 as intradermal injection plus GM-CSF s.c. The final analysis of a phase I trial in 18 patients with advanced, refractory or relapsed B-NHL of various histologies has shown feasibility with a 90% success rate of vaccine production and excellent tolerability with no grade III side effects observed. This patient group exhibited a severely impaired immune function as indicated by moderately to severely depressed counts of CD4+ T cells in 50% of all patients and undetectable anti-HBs antibodies after 2 hepatitis B vaccinations in 10/10 patients. Nevertheless, humoral immune responses to the vaccine were induced in 7 of 17 evaluable patients as assessed by ELISA, and cellular responses in 8/17 patients by IFNg ELISPOT, resulting in an immunological response rate of 65% (11/17). At a median follow-up of 30 months, median time to progression is 4.3 months, with 3 patients in ongoing progression-free survival. Complete clinical remission occurred in one FL and one DLCL patient, and a partial remission in one additional FL patient. Anti-idiotype immune responses were associated with superior progression-free survival (p=0.023). These data indicate strong immunogenicity of the vaccine formulation tested and hint to a possible clinical benefit in immune responders.
An ongoing phase II trial evaluates this vaccine (with GM-CSF escalated to 5 consecutive days) in patients with low-grade, stage III/IV B-NHL in untreated "watch and wait" (W&W) situation or in remission after systemic therapy with a partially reconstituted immune system. Patients receive 6 vaccinations in monthly intervals. As of March, 2005, 16 patients have commenced treatment, and 7 have completed 6 cycles. No patient had any evidence for spontaneous lymphoma regression prior to vaccination. One patient required dose reduction of GM-CSF due to cutaneous side effects; no other symptomatic toxicity was noted. Of 5 follicular lymphoma (FL) W&W patients, 3 achieved a partial remission (PR) after 6 vaccinations, and one had a mixed response with regression of lymph nodes (LN) but persistence of subcutaneous nodules. Immune monitoring has been performed on the 1st PR patient and demonstrated induction of Id-specific cellular and humoral immunity. One W&W patient with mantle cell lymphoma had stable disease after completing treatment. Of 2 patients vaccinated in PR after systemic chemotherapy, both had gradual further shrinking of residually enlarged LN after 2 and 6 vaccinations, respectively. Intradermal vaccination with recombinant idiotype in conjunction with GM-CSF may induce clinical remissions in untreated NHL patients at a substantially higher rate than expected spontaneously.
A second phase II trial was opened for patients with multiple myeloma in complete or "very good" partial remission after autologous stem cell transplantation that aims at inducing immune responses for remission maintenance with essentially the same vaccination schedule. Recently, the European Medical Agency (EMEA) has granted orphan drug status for the recombinant idiotype vaccine.

P33

Bispecific single-chain antibodies induce target-cell restricted, supra–agonistic CD28 stimulati