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P01
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"Combatting Multiple Myeloma by CTL specific for Plasma cell-associated transcription factors"
S. Abdel Mutallib, C. Lotz, U. Liewer, C. Huber, M. Theobald
Department of Hematology and Oncology, Johannes Gutenberg-University, Mainz
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Growing clinical and experimental evidence indicates that multiple myeloma (MM) is susceptible to cytotoxic T-lymphocyte (CTL)-based immune interventions. These CTL recognize peptides derived from the endogenous processing of cellular proteins and presented by MHC class I molecules. Transcription factors being indispensable for the terminal differentiation of mature B-lymphocytes into malignant and non-malignant plasma cells provide abundant and universal MM-associated target molecules. HLA-A*0201 (A2.1)-transgenic (tg) mice were used to identify A2.1-presented CTL epitopes derived from the plasma cell-associated transcription factors XBP-1 and PRDI-BF1. CTL with specificity for identified peptides were able to efficiently kill various MM cell lines. However, recognition by CTL was not limited to malignant plasma cells. In case PRDI-BF1, substantial protein levels were detected in melanomas, osteosarcomas, breast cancer, and a hepatocellular carcinoma. The broader recognition patttern of PRDI-BF1- and XBP-1-specific CTL suggested that the self-A2.1-restricted human T cell repertoire with specificity for both transcription factors is affected by self-tolerance. To study this hypothesis, naive CD8+ human T-lymphocytes underwent in vitro stimulation with A2.1+ autologous DC pulsed with the relevant PRDI-BF1 and XBP-1 peptides. Resultant effector cells specifically produced IFN-gamma in response to peptide-pulsed target cells and MM cells as well, but were only able to kill peptide-pulsed targets. Our results indicate that plasma cell-associated transcription factors can serve as MM-associated and, to some extent, as broader tumor antigens. The transduction of appropriate T cell receptors selected from A2.1-tg mice into human T-lymphocytes offers a possibility to turn self-A2.1-restricted human T cells into efficient MM-reactive CTL.
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P02
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"NemodDC: a fully functional human DC line for therapy"
H. Baumeister*, U. Hilbert*, H. Tandara*, K. Daemen*, P. Trinder*, R. Scheper# and S. Goletz*
*NEMOD Immuntherapie AG, Berlin, Germany. #Free University of Amsterdam, Netherlands.
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NemodDC represents a CD34+/CD14+ cell line of leukemic origin that can be differentiated and matured to fully functional immature (iDC) and mature dendritic cells (mDC) as well as Langerhans cells. To demonstrate the potential of NemodDC in cancer immunotherapy we used Nemod-mDC to prime tumor specific, cytotoxic CD8+ T-cells. Nemod-iDC were loaded with tumor cell lysates and further matured to Nemod-mDC, or mature Nemod-mDC were loaded with peptides derived from tumor-associated antigens. IFN-gamma-ELISPOT analyses revealed that both procedures resulted in mDC that efficiently and antigen-specifically stimulated CD8+ T-cells. Nemod-mDC primed CD8+ T-cells were able to specifically lyse antigen-loaded T2 cells and tumor cells carrying the respective antigen. The results show, that Nemod-iDC are able to take up tumor antigens from lysates which are processed and presented during maturation by Nemod-mDC.
Based on these data, the HLA-A2+ Nemod-DC line is being developed as an off-the-shelf semi-allogeneic mDC vaccine in order to combine the induction of a specific immune response via syngeneic antigen presenting HLA with a strong bystander non-specific T cell helper responses via allogeneic HLA. Additional therapeutic approaches to be applied with Nemod-DC include adoptive T-cell transfer and exosome based therapies. In summary, NemodDC has great potential as a novel immunotherapeutic for treatment of cancer and infectious diseases.
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P03
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"Specifically activated CD45R0+ central and effector memory but not CD45RA+ naïve T cells from bone marrow of cancer patients selectively home to and reject xenotransplanted autologous tumors"
P. Beckhove, M. Feuerer, M. Dolenc, C, Choi, N. Sommerfeldt, J. Schwendemann
Deutsches Krebsforschungszentrum Heidelberg
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Bone marrow (BM)of breast cancer patients contains CD8+ T cells specific for breast cancer antigens MUC1 and Her-2/neu.These cells had central (CM) or effector memory (EM)phenotype (CD45RA-CD62L+ or CD45RA-CD62L-). To test their in vivo function, BM derived CD45RA+ naïve or CD45RA- memory T cells were separated, stimulated with autologous DCs pulsed with tumor lysate and transferred into NOD/SCID mice transplanted with autologous breast tumors and normal skin. Transferred memory but not naïve T cells infiltrated autologous tumor but not skin tissues. Tumor infiltrating cells had either CM or EM phenotpye, produced perforin, expressed the P selectin ligand PSGL1 and were found around P-selectin+ tumor endothelium. Tumor infiltration included cluster formation in situ by memory T cells together with co-transferred DCs. It was associated with tumor cell apoptosis and tumor reduction. We suggest that tumor infiltration and rejection is based on the recognition of TAAs on tumor cells and DCs by specifically activated CM and EM T cells and that PSGL1/P-selectin interactions facilitate tumor selective homing.
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P04
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"Clonal RNA as an antigenic format for the peptide-independent monitoring of antigen-specific T cell responses"
C.M. Britten, R.G. Meyer, C. Huber, T. Wölfel
III. Department of Medicine, Hematology and Oncology, University of Mainz
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The monitoring of T cell responses is widely applied in malignant and infectious diseases and in immunotherapy trials. Its quality depends very much on the availability of well-defined pure antigens that can be consistently produced with an appropriate effort. RNA as an antigen format meets these requests in many respects. In addition the use of RNA allows to monitor antigen-specific T cell responses independently of known peptides and patients´ HLA phenotype. Herein we compared RNA-transfected and peptide-loaded antigen-presenting cells (APCs) in their ability to detect antigen-specific T cells.
Model antigens were CMV pp65 and human tyrosinase. Effector cells were non-stimulated ex vivo T lymphocytes and T cell populations expanded in vitro from the peripheral blood of healthy donors. These effectors were tested in IFN-gamma ELISPOT assays for their reactivity towards autologous dendritic cells (DCs) either electroporated with RNA or loaded with CMV pp65- or tyrosinase-derived peptides. Both types of stimulators induced equivalent spot numbers both in non-stimulated and in pre-stimulated CD8+ and CD4+ lymphocyte subsets reactive with tyrosinase or CMV pp65. RNA electroporation did not induce background cytokine secretion.
With autologous DCs electroporated with clonal RNA we detected the overall antigen-specific T cell response in individual donors. The subsequent use of RNA-electroporated K562 cells stably transfected with single HLA class I alleles allowed to dissect complete CD8+ T cell responses into partial responses restricted by single HLA class I molecules.
Our observations provide evidence that RNA is a preferable antigen source in immunomonitoring. In addition its use might facilitate the identification of new T cell epitopes.
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P05
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"A recombinant bispecific single-chain Fv antibody against HLA class II and FcgammaRIII (CD16) triggers effective lysis of lymphoma cells"
J. Brünke, B. Fischer, K. Barbin, M. Gramatzki, R. Repp, R. Valerius, H.G. Fey
Universität Erlangen, Lehrstuhl für Genetik
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Bispecific antibodies offer the possibility to improve effector-cell recruitment for antibody therapy. Therefore, a recombinant bispecific single-chain Fv antibody (bsscFv) directed against FcgRIII (CD16) and HLA class II was constructed here and tested in functional assays. RNA from the hybridomas 3G8 and F3.3, reacting with CD16 and HLA class II, respectively, was used to generate phage display libraries. From these libraries, reactive phages were isolated and the bsscFv was constructed by connecting both scFv components through a 20 amino acid flexible linker. After expression in SF21 insect cells and chromatographic purification, the bsscFv bound specifically and simultaneously to both antigens. The affinities of the anti-CD16 and the anti-HLA class II scFv components of the bsscFv were 8.6 x 10-8 M and 13.7 x 10-8 M, respectively, which was approximately 7-fold lower than the F(ab) fragments of the parental antibodies. In antibody-dependent cellular cytotoxicity (ADCC) experiments with human mononuclear cells (MNCs) as effectors, the bsscFv mediated specific lysis of both HLA class II-positive, malignant human B-lymphoid cell lines and patient-derived primary B-CLL cells. Optimal lysis was obtained at bsscFv concentrations of approx. 400 ng/ml, similar to the concentration required for maximum lysis by the corresponding chemically linked bispecific antibody (bsAb). Thus, this recombinant bispecific scFv-antibody is an efficient molecule for effector-cell mediated lysis of malignant human B-lymphoid cells.
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P06
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"mRNA-based Vaccine- Characterization and Optimisation"
J.-P. Carralot, J. Probst, S. Aulwurm, B. Scheel, R. Teufel, H.-G. Rammensee; S. Pascolo
Curevac GmbH
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We showed that direct injection in mice of non-replicative in vitro transcribed messenger RNA (mRNA) leads to an immune response against the antigen it encodes (Hoerr et al., Eur. J. immunol., 2000). Such a genetic vaccination strategy is safer than DNA-based vaccination (where the injected DNA might persist in the organism and eventually integrate in the genome) or replicative RNA-based vaccination (where the replicative RNA that codes for a virus-derived replicase might recombine with other RNA viruses).
We studied in more detail the mRNA-based vaccination. We show that at the site of injection (ear pinna) many cells are able to take-up the mRNA and translate it into proteins. We demonstrate that a TH2 type of response is triggered when the mRNA-based strategy is used to induce a ßgal specific immune response. However, when we inject recombinant Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) and mRNA, we observe a switch toward a TH1 type anti-ßgal immune response. This result is of interest in the context of anti-viral or anti-tumor immune response.
We are currently testing such a mRNA plus GM-CSF vaccine using viral (HBV, Influenza) and tumor antigens. Concerning anti-tumor vaccination, the utilization of autologous total tumor-derived RNA for the preparation of the mRNA vaccine is being evaluated in a mouse model. The efficiency of this treatment will be presented and discussed.
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P07
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"ITGO: ImmunoTherapy in Gynecologic Oncology A nationwide network of institutions working in gynecologic oncology towards application of immunotherapy with emphasis on dendritic cell therapy"
G. Bastert / M. Lindner / A. Marmé (Heidelberg); A. Schneider / A. M. Kaufmann (Jena); J. Pfisterer / F. Hilpert / A. Heiser (Kiel); K. Diedrich / M. Löning (Lübeck); U. Wagner / S. Reinarzt (Marburg); D. Wallwiener / B. Gückel / J. Huober (Tübingen); M. Suetterlin / U. Kämmerer (Würzburg)
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Gynecologic tumors are known to frequently (over)express well characterized tumor associated antigens (TAA) such as CEA, MUC-1, Her-2/neu, members of the cancer/testis-antigen family (e.g. NY-ESO-1, SSX-2, MAGE-1), as well as virus-related antigens such as human papillomavirus (HPV). Several of these TAA have been shown to be recognized by antibodies (Abs) and/or cellular effectors of the immune system, rendering the tumor cells susceptible to specific immunotherapy. Approaches using therapeutical Abs, cellular vaccines (tumor-cell based or immune-cell-based), and defined peptide vaccines have been tested in preclinical and clinical studies. Induction of immune responses has been shown in vitro or/and in clinical phase I trials, also by members of this network. Clinical trial quality depends on factors including validated approaches, well defined patient's characteristics and efficient recruitment, GMP/GCP procedures, quality control and monitoring, support by public authorities and sufficient funding, and last not least the unrestricted communication of results. The ITGO network of gynecologic clinics/laboratories cooperates in clinical trial organization and conduction. Shared methods/competence for immunomonitoring procedures as well as shared efforts in contacting authorities and funding agencies will stimulate and facilitate trial conduction. Ongoing research and clinical trials using dendritic cells are performed in breast (HD, KI, TÜ, Wü), in cervix (J), and in ovarian cancer (HD, KI, Lü, Wü). To date, early clinical trials have been successfully performed locally in e.g. in J (cervix: DC/HPV E7), or are ongoing in Tü (breast: DC/multiple HLA-A2 restricted peptides), HD (breast: DC/multiple HLA-A2 restricted peptides). Anti-idiotypic and bispecific Abs are also investigated for ovarian cancer (CA125 anti-idiotype Abs (Ma), biAbs HEA125xOKT3 (HD), L1 mAb (HD)). We will present data of these early trials. This network will serve as a nation wide platform for immunotherapy in gynecologic cancers and will represent our participants in the european community.
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P08
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"Reduction of lung metastasis by local adjuvant immunotherapy using the Macrophage-Activating Lipopeptide -2 (MALP-2)"
Y. Hama, C. Kruschinski, A. Luhrmann, T. Tschernig, S. von Horsten, R. Pabst
Functional and Applied Anatomy, Medical School of Hannover
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Primary surgery of tumors may trigger metastasis to organs such as the lungs and perioperative local stimulation of host defense mechanisms may represent a novel approach to minimize such risk. In our previous studies we were able to demonstrate that local intratracheal (i.t.) instillation of MALP-2 as a bacterial-derived immunomodulator of cellular host defense responses reduces the number of lung tumor colonies induced by intravenous (i.v.) inoculation of the F344 rat syngeneic adenocarcinoma MADB106. The present studies aimed to identify the optimal time point for such local immunostimulation and to test whether repeated treatment may further potentiate the protective-like effect of i.t. applied MALP-2. In the first experiment MALP-2 was instilled i.t. 3 days, 1 day and 1 h before injection of MADB106 cells. In the second experiment repeated MALP-2 treatment was conducted 1 h before and immediately after tumor cells. Lung surface tumor colonies were quantified 16 days later. Results demonstrate that local i.t. pre-treatment of lungs with MALP-2 at 1 h before but not earlier reduces significantly the number of lung tumor colonies to about 25% of control levels. Repeated local i.t. treatment at 1 h before and directly after inoculation of tumor cells did not result in a further potentiation of this protective-like effect. The present findings provide strong experimental evidence that local MALP-2 treatment is effective in suppressing MADB106 tumor growth in the lungs when applied in a time frame of 1 h before or parallel to the metastatic process. In general these studies strengthen the concept of local adjuvant immunotherapy for prevention of organ-specific metastasis as apparent in the context of primary tumor surgery.
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P09
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"Phase I trial of patients with advanced gynaecologic malignancies using autologous tumour antigen-pulsed dendritic cells"
J.J. Hernando, T.-W. Park, K. Kübler, H. Schlebusch
Universitäts-Frauenklinik, University of Bonn
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Purpose: DC-based immunotherapy has proven to be effective in patients with malignant lymphoma, melanoma, renal and prostate carcinoma. To evaluate the clinical usefulness of DC vaccination in patients with advanced gynaecologic malignancies, we have initiated a phase I trial using DC pulsed with keyhole limpet hemocyanin (KLH) and unfractionated tumour-associated antigens.
Methods: Monocyte-derived, mature DC were generated under serum-free conditions with GM-CSF, IL-4 and TNF-a, and for delivery of TH and CTL epitopes, loaded with endotoxin-free KLH and autologous tumour-derived antigens. FACS analysis were done to quantify the number of CD83+/CD86+ cells. T-cell priming to KLH or tumour antigens was analysed in vivo by DTH skin testing and in vitro by determining PBMC proliferation, as well as secretion of IFN-g; in an ELISPOT assay upon antigen stimulation.
Results: 2 patients with uterine sarcoma and 6 with ovarian carcinoma received 3 to 23 intradermal injections of antigen-pulsed DC at 10-day or 4-week intervals. 3 patients showed stable disease lasting 25 weeks to 16 months, 5 had tumour progression within the first 14 weeks. KLH- and tumour antigens-specific DTH reactions were observed in 6 and 1 patient, respectively. Lymphoproliferative responses to KLH stimulation were recorded in 6 patients and to tumour lysate in 2. Tumour antigen-directed IFN-g; secretion by PBMC of 1 patient with ovarian carcinoma who showed disease stabilization lasting 16 months was consistent with a TH type 1 cytokine bias. Treatment was safe, well tolerated, immunologically and clinically active and devoid of adverse effects.
Conclusions: Our results show that patients with advanced gynaecologic malignancies can be effectively vaccinated with DC pulsed with KLH and tumour-derived antigens.
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P10
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"A batch-release test for hybrid cells generated by electrofusion of dendritic cells and ovarian carcinoma cells"
F. Hilpert, A. Heiser, C. Krüger, A. Rosenau, N. Arnold, W. Jonat, J. Pfisterer
Klinik für Gynäkologie und Geburtshilfe, Universitätsklinikum Schleswig-Holstein Campus Kiel
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Electrofusion of tumor cells (TC) and dendritic cells (DC) is an elegant method to generate a polyvalent anticancer vaccine. Considering preclinical and clinical evaluation, standardized methods for the detection and quantification of cell-cell hybrids are essential. DC and TC were transferred into low conductive medium and fused with a single electric pulse under varying electrical conditions. DC and TC were labeled with dyes of distinct emission wavelength with either unspecific cell trackers prior to electrofusion or immunocytochemically after electrofusion. Fusion results were analyzed with dual-fluorescent flow cytometry and fluorescence microscopy.
Fluorescence microscopy confirmed formation of double-fluorescent polynuclear hybrids but was ineligible to quantify fusion efficacy due to aggregation and fragmentation, irrespective of the staining method. Transfer of cells into low conductive medium reduced viability and staining intensity of cell trackers. Flow cytometry and fluorescence microscopy indicated leakage and transposition of cell trackers during electrofusion. Unspecific staining prior to electrofusion is ineligible if further evaluation of an unstained vaccine is recommended. Immunofluorescence was feasible as a batch-release test of fusion products and showed no significant alteration of antigen expression and/or staining intensity in flow cytometry. Opposing mAb-labeled DC, TC and double positive cells could be clearly separated with flow cytometry. Overall, fusion efficacy expressed in percentage of double fluorescent cells ranged between 2-23 % and varied with 2-7% within several experiments under the same fusion conditions. Electrofusion efficacy results with DC as a fusion partner tend to vary in a large proportion, enforcing necessity of batch-release testing with immunofluorescence.
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P11
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"Killer T Cells - A Promising New Tool For Adoptive Immunotherapy"
C. Jursik, M. Prchal, R. Voglauer, J. Grillari, H. Jungfer, H. Katinger
Institute of Applied Microbiology
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Cell-mediated cytotoxicity is a very important mechanism involved in several basic immunological processes in vivo. Previously we have established a method for enrichment of a special kind of human killer T cells (KTCs) from peripheral blood mononuclear cells, which kill different tumor cell lines, but leave the normal counterparts unattacked. KTC´s are derived from a patient´s peripheral blood mononuclear cells (PBMC) and expanded in serum-free medium supplemented with growth stimulants to cell numbers needed for re-infusion into the patients. The activation occurs by incubation with an antibody-producing cell line that mediates proliferation of KTCs and their cytotoxicity against tumor cells of different origin. To measure and characterize the killing mechanism of KTCs on different human tumor cell lines conventional test systems like 51Cr release assay or DNA staining methods cannot be applied due to the characteristics of KTC killing, which is a process lasting several days. In the first approach green fluorescence protein (GFP) expressing tumor targets were incubated with KTCs, resulting in the release of GFP into the culture supernatant, which then was analysed with a highly sensitive ELISA. Since this method requires the establishment of different GFP expressing targets and lacks appropriate negative control cells, we established another cytotoxicity assay. First several adherent target cells were labelled with PKH-26 for distinction of target and effector cells. Thereafter targets were exposed to KTCs at various E:T ratios for up to 24 hours, followed by the flow cytometric analysis of apoptotic and necrotic cells using annexin-V-FITC and propidium iodide. Using this highly sensitive and reproducible in-vitro assay we were able to characterize the cell-mediated cytotoxicity of KTCs on various targets.
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P12
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"Targeting of a human Natural Killer cell line by chimeric immunoglobulin T cell receptor gene transfer leads to tumor growth inhibition in vitro and in vivo"
G. Pecher, T. Schirrmann
Medizinische Klinik m.S. Onkologie u. Hämatologie, Charité Campus Mitte, Humboldt-Universität zu Berlin
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The gene transfer of tumor-specific chimeric immunoglobulin T cell receptors (cIgTCR) combining antibody-like specificity with the effector cell function could be an attractive tool in immunotherapy. In this study, we directed the human Natural Killer (NK) cell line YT to tumor cells by gene transfer of a cIgTCR with specificity against the carcinoembryonic antigen (CEA). The cIgTCR was constructed of a CEA-specific humanized single-chain Fv antibody fragment fused to the IgG1 Fc domain and the CD3 ? chain. YT cells were transfected with the cIgTCR gene by electroporation and cIgTCR expressing cells were enriched by immunoaffinity purification. cIgTCR expressing YT cells specifically lysed CEA+ colon carcinoma cell lines which were resistant to the parental YT cell line. The lysis was not inhibited in the presence of soluble CEA. cIgTCR gene modified YT cells retained their specific cytolytic activity after ?-irradiation in vitro and inhibited the tumor growth in vivo after adoptive transfer into Nod/SCID mice. cIgTCR gene modified YT cells specifically directed to CEA expressing tumor cells and available in unlimited source might be used for an immunotherapy of CEA+ cancer.
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P13
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"Generation and expansion of tumor-reactive cytotoxic T lymphocyte (CTL) clones for adoptive immunotherapy of cancer patients"
(1) Schmidt, B., (1), Fischer, G., (2), Busch, D. H., (1) Huster, K., (2) Wagner, H., (1) Peschel, C. and (1) Bernhard, H.
(1) Department of Hematology/Oncology and (2) Department of Microbiology/ Immunology Klinikum rechts der Isar, Technical University of Munich, Germany
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Patients with advanced cancer have an impaired cellular immune system. The rationale of adoptive T cell therapy is based on the attempt to circumvent pre-existing tolerance mechanisms by stimulating anergic, potentially tumor-reactive T cells ex vivo. Attempts to eradicate cancer by adoptive T cell transfer have been limited due to the difficulty of generating T cells with defined antigen specificity. The current study focuses on the isolation of HLA class I-restricted tumor-reactive CTL clones with defined specificity against peptides derived from the oncogene HER2, the cancer-testis antigen NY-ESO-1, and the differentiation antigens Melan-A, tyrosinase and gp100.
Peptide-pulsed DCs were used as antigen presenting cells for stimulating autologous peripheral blood lymphocytes from HLA-A2+ healthy donors and cancer patients. After several simulations the frequency of peptide-specific T cells increased to numbers that allow visualization by fluorochrome-labeled HLA-A2/peptide multimers. Multimer+ T cells were sorted and cloned by limiting dilution. We succeeded in expanding HLA-A2-restricted CTL clones targeting the tumor-associated antigens HER2, NY-ESO-1, Melan-A, tyrosinase or gp100 to numbers sufficient for adoptive T cell therapy.
Current studies evaluate the feasibility, safety, in vivo persistence and efficacy of adoptively transferred HER2-specific T cells for the treatment of patients with HER2-overexpressing breast cancer. So far, autologous HLA-A2+ T-cell clones specific for HER2p369-377 were transferred to a 47-year-old patient with metastatic breast cancer. Repetitive transfer of HER2-specific T cell clones was feasible without major side effects. Trafficking of transferred T cells was evaluated by HLA-A2/peptide multimer analyses and by T cell labeling with Indium-111 oxine.
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P14
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"Autologous dendritic cell (DC) vaccination of elderly patients with acute myeloid leukemia (AML) can elicit a cytotoxic T cell response"
M. Schmitt, L. Li, P. Reinhardt, A. Schmitt, J. Greiner, M. Ringhoffer, M. Wiesneth, H. Döhner
University of Ulm, 3rd Dept. of Internal Medicine
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Recently, several leukemia-associated antigens (LAA), e.g. WT-1, PRAME and RHAMM have been defined in patients with AML. The immunogenicity of these LAA has been proven in vitro. Myeloid leukemias (AML/CML) are the only diseases where DC can be generated from the malignant cell, thus constitutively presenting LAA. DC vaccination might result in an enhanced T cell response to AML blasts. Therefore, we inaugurated a phase I trial for the vaccination of elderly AML patients with autologous DC. DC could be generated from 22/29 (79 %) patients with refractory AML, testing positive for HLA-ABC, -DR, CD40, CD80, CD83 and CD86, thus fulfilling the prerequisites for antigen presentation. At least one of the LAA mentioned above was detectable by RT-PCR in all DC. For three patients, DC were generated under GMP conditions. 5x10E6 DC were injected s.c. in the vicinity of inguinal lymph nodes four times at a biweekly interval. No serious adverse events were observed after DC vaccination. As two patients died of intracranial hemorrhage due to thrombocytopenia in the further course of the leukemia, they received only a single DC vaccination. An allo-sensitization against platelets or HLA could not be detected. The third patient required blood transfusions and remained in stable condition for several months, but died from pneumonia 13 months after the DC vaccinations. After completion of DC vaccination, ELISPOT analysis revealed a twofold increase of CD8+ T cells recognizing leukemic blast lysate as well as LAA peptides, when compared to the initial T cell frequency. In summary, DC could be generated from AML blasts and preserved LAA expression. DC vaccination was well tolerated and resulted in an enhanced and specific response of cytotoxic T cells.
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P15
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"Inhibition of tumor growth and metastatic lung tumors I mice by adenovirus-mediated Inteferon-b gene therapy"
R. Wiewrodt (1), (2), M. Odaka (3), P.A. DeLong (2), T. Tanaka (3), V. Kapoor (2), L. R. Kaiser (3), S. M. Albelda (2),
(1) Pulmonary Division, Mainz University, Germany, (2) Pulmonary Divison and (3) Cardiothoracic Surgery, University of Pennsylvania, Philadelphia, PA 19104
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RATIONALE: For non-resectable patients, treatment of lung cancer (LC) is ineffective. In other tumors immune-modulatory therapies using various interferons (IFN) delivered via viral vector, have shown some promise. We thus evaluated the use of a replication-deficient adenoviral (Ad) vector encoding for the murine IFN-ß gene (Ad.muIFN-ß) in mouse LC models.
METHODS: L1C2 (a mouse LC cell line syngeneic in immune competent BALB/c) was injected via four routes: intrapleural (i.pl.), intratracheal (i.t.), intravenous (i.v.) and intraperitoneal (i.p.). Established tumors were treated with a single dose (109 pfu) of Ad.muIFN-ß, a control Ad vector (Ad.HSVtk), or saline given via various routes.
RESULTS: 80% of animals with intraperitoneal tumors were cured after a single i.p. dose of Ad.IFN-b. A single i.t. dose of Ad.muIFN-ß significantly prolonged the survival of BALB/c mice after i.t. tumor (90% survival at 90 days), whereas all controls had a median survival of less then 50 days. In the more aggressive i.pl. model, Ad.muIFN-ß doubled median survival compared with Ad.HSVtk. Intraperitoneal treatment of animals injected with tumors both i.p. and i.v. led to marked inhibition of the metastatic lung tumors. The special role of lung and pleural immunity with regard of initiation of a cellular immune response was evaluated.
CONCLUSION: Local (intratracheal, intrapleural, and intraperitoneal) delivery of the murine interferon-bets gene via an adenoviral vector provided effective local and distant treatment for lung cancers established at various sites, as well as protective acquired immunity.
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