| ANTIBODY THERAPY |
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01 Thursday, May 6, 2004 at 10:30
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"Bispecific costimulatory molecules for activation of tumor-killing lymphocytes"
Robert Weth, Markus Biburger, Winfried Wels
Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, Frankfurt am Main, Germany
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The receptor tyrosine kinase ErbB2 (HER2) is overexpressed in multiple human tumors of epithelial origin. High ErbB2 expression is functionally involved in tumorigenesis and correlates with poor clinical prognosis. For immunotherapy of ErbB2 expressing tumors, we developed a strategy to supply the tumor cells with costimulatory activity. A bispecific fusion protein was constructed (BIg5), containing the IgV-like domain of huCD86, the CH2/CH3 domain of huIgG1 and the ErbB2-specific single chain antibody fragment scFv(FRP5). A similar fusion protein lacking the CD86 domain (Ig5) was used as a control. Upon binding of BIg5 to ErbB2 on tumor cells, these cells display CD86 on their surface and thus can deliver costimulatory signals for T-cell activation. In addition, NK cells could be activated by CD86 binding to CD28. BIg5 is secreted by eukaryotic cells as a homodimer with increased stability compared to monomers and possibly enhanced costimulatory activity due to crosslinking of CD28 on effector cells. By FACS analysis, specific binding of the scFv(FRP5) domain to ErbB2 as well as CD86 IgV binding to CTLA-4 could be demonstrated. Together with anti-CD3 antibody, BIg5 stimulates proliferation of human CD2-purified lymphocytes in vitro. After binding to ErbB2 on murine Renca-lacZ/ErbB2 tumor cells, about 50% of initially bound BIg5 is still present on the cell surface after 4 hours. For delivery of chimeric fusion proteins in vivo, we used syngeneic, stably transfected HC11 mammary epithelial cells continuously secreting the proteins. Inoculation of these bystander cells close to subcutaneously growing Renca-lacZ/ErbB2 tumors should provide a long-lasting source to achieve high local concentrations of BIg5 at the tumor site. In vivo HC11-BIg5 cells proved to be non-tumorigenic and secreted BIg5 for several weeks, causing a strong anti-BIg5 antibody response. Treatment of established Renca-lacZ/ErbB2 or ErbB2-negative Renca-lacZ tumors by peritumoral inoculation of either HC11-BIg5 or HC11-Ig5 cells led to rejection of all Renca-lacZ/ErbB2, but none of the Renca-lacZ tumors. HC11neo control cells had no effect on tumor growth. Rejection of ErbB2+ tumors led to long-term protection also against subsequent challenge with intravenously injected ErbB2- tumor cells. Intraperitoneal injection of bystander cells secreting the fusion proteins did not lead to tumor regression suggesting that high local concentrations at the tumor site are necessary to target ErbB2 on tumor cells and to overcome elimination of BIg5 or Ig5 by neutralizing antibodies. The CD86 IgV domain of BIg5 did not play a major role in the observed antitumoral immune response suggesting NK-cell mediated ADCC as the initial effector mechanism followed by activation of tumor specific T cells. Targeting of ErbB2 on tumor cells with antibody fusion proteins that interact specifically with the host immune system could be an efficient and specific approach for therapy of solid ErbB2+ tumors.
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02 Thursday, May 6, 2004 at 10:45
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Antibody-based immunoreceptors: the impact of signalling domain, binding affinity and costimulation.
Andreas Hombach, Claudia Heuser, Hinrich Abken
Tumorgenetik, Klinik I für Innere Medizin, Klinikum der Universität zu Köln
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The immunoreceptor strategy is based on grafting of T cells with recombinant T cell receptor molecules that consist extracellularily of a scFv domain for MHC-independent antigen binding and intracellularily of the CD3 z- or FceRI g-signalling domain for cellular activation. Upon binding to antigen-positive cells, grafted T cells are activated, secrete IFN-g, and lyse specifically antigen-positive, but not antigen-negative target cells. During the last years, we generated a panel of immunoreceptors with specficity for "tumor associated antigens" as targets for use in adoptive immunotherapy of malignant diseases: CEA, CA72-4, CA19-9 for gastrointestinal carcinomas, CD30 for Hodgkin´s lymphoma and cutaneous T cell lymphoma, HMW-MAA and melanotransferrin for melanoma, and ErbB2 for a variety of carcinomas (1, 2). T cells taken from the peripheral blood of tumor patients and grafted with the appropriate immunoreceptor mediate a highly efficient immune response towards autologous, antigen expressing target cells in vitro (3).
One of the major advantages of the immunoreceptor strategy lies in the modular compositon of the receptor molecule. However, little is known about the impact of the individual receptor modules on cellular activation in a complex immunological context. We have identified several items that affect the efficacy of receptor mediated cellular activation including
- the signalling domain that affects the stability of immunoreceptor expression and function in T cells (4);
- the affinity of the scFv domain that affects the efficacy of T cell activation;
- B7-1 and B7-2 costimulation that affects the quality of T cell activation (5, 6, 7).
The high complexity of the recognition and signalling process makes it unlikely that a universal configuration of the immunoreceptor exists. The design of the receptor molecule, however, has major impact on the stability and function in T cells and thereby on the efficacy of adoptive immunotherapy.
(1) Hombach et al., Curr. Gene Ther 2, 211-226 (2002); (2) Abken et al., Curr. Pharm. Des. 9, 639 - 652 (2003); (3) Hombach et al., Gene Ther. 8, 81-895 (2001); (4) Heuser et al., Gene Ther. 10, 1408-1419 (2003); (5) Hombach et al., Cancer Res. 61, 1976-1982 (2001); (6) Abken et al., Trends Immunol. 23, 240-245 (2002); (7) Hombach et al., J. Immunol. 167, 6123-6131 (2001)
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03 Thursday, May 6, 2004 at 11:00
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"Tolerability and efficacy of the trifunctional antibody removab® (anti-EpCAM x anti-CD3) in patients with malignant ascites due to ovarian cancer: Results of a phase I/II study"
Pauline Wimberger1, Alexander Burges2, Vera Gorbounova3, Harald Sommer4, Barbara Schmalfeldt5, Jacobus Pfisterer6, Michail Lichinitser3, Anatoliy Makhson7, Michael Ströhlein8, Wolfgang Eiermann9, Michail Biakhov10, Vladimir Moiseenko11, Andreas du Bois12, Rainer Kimmig1
1Department of Obstetrics and Gynecology, University Hospital, Essen, Germany
2Department of Obstetrics and Gynecology, University Hospital Munich/Grosshadern, Munich, Germany
3Research Center for Oncology, Moscow, Russia
4Department of Obstetrics and Gynecology, University Hospital Munich-City, Munich
5Department of Gynecology, University Hospital Technical University, Munich, Germany
6Department of Obstetrics and Gynecology, University Hospital, Kiel, Germany
7Moscow Oncology Clinical Hospital, Moscow, Russia
8Department of Surgery, University Hospital Munich/Grosshadern, Munich, Germany
9Frauenklinik vom Roten Kreuz, Munich, Germany
10Central Clinical Hospital of Ministry, Moscow, Russia
11St. Petersburg Scientific Oncology Institute, St. Petersburg, Russia
12Department of Gynecology and gynecological Oncology, Dr. Horst-Schmidt-Kliniken, Wiesbaden, Germany
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Introduction: Malignant ascites in patients with gynecological malignancies is associated with poor prognosis and poor quality of life. The bispecific trifunctional antibody removab® (anti-EpCAM x anti-CD3) belongs to a new class of intact antibodies that has been developed for targeted therapy of epithelial tumors. The two binding sites of removab® are directed against epithelial tumor cells (EpCAM+) and T-cells (CD3+) thus recruiting T-cells in the direct environment of tumor cells. Simultaneously, mediated by the intact Fc-fragment removab® binds to FcgI/IIIR+ accessory cells (e.g. macrophages, natural killer cells, dendritic cells) that are mandatory for the induction of a tumor-specific immune response.
Patients and methods: In an open-label multicenter phase I/II-dose escalating study, a total of 23 patients with ovarian cancer and symptomatic ascites at FIGO stage III-IV were treated intraperitoneally with removab®. The patients had received a median of 3 (1-8) previous therapies. Their mean age was 62 (42-80) years. The treatment consisted of up to 5 intraperitoneal applications of the antibody within 13 days using increasing dosages.
Results: The intraperitoneal treatment with removab® was able to stop the production of ascites in 22 of 23 patients. These patients were ascites-free at the end of the study (day 37). Immunocytochemical quantification of tumor cells in the ascites fluid showed a dramatic reduction of EpCAM+ cells (> log 5). In addition, clinically significant improvement of the quality of life was observed. The majority of adverse events was mild to moderate. The most common side effects observed in the study were fever (82.6%), nausea (60.9%), vomiting (56.5%), and abdominal pain (30.4%). The MDT (maximal tolerated dose) was reached at the increasing dosages of 10mg-20mg-50mg-200mg-200mg.
Conclusion: In conclusion, intraperitoneal treatment with removab® was safe, well tolerated and showed encouraging efficacy in patients with malignant ascites due to ovarian cancer. Thus, the new concept of the anti-EpCAM x anti-CD3 antibody might offer a promising treatment option for patients with epithelial tumors.
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04 Thursday, May 6, 2004 at 11:15
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"HLA DR-directed bispecific single-chain Fv antibodies for lymphoma therapy"
Y.Wachter, J.Brünke, S.Schuster, K.Barbin, M.Peipp, T.Valerius, M. Gramatzki, G.Fey, R.Repp
Medizinische Klinik III mit Poliklinik der Universität Erlangen,Germany
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Fc receptors are important for the clinical efficacy of therapeutic antibodies. Bispecific antibodies (BsAb) are immunoglobulin-conjugates with two different binding specifities, targeting tumor antigens and effector cell trigger molecules. BsAb, produced by chemical coupling of one antibody against a tumor cell surface antigen with another against a Fc receptor, mediate effective interactions between effector and target cells.
Here, genetically coupled bispecific single chain Fv (bsscFv) were produced - as they easily enable further modifications of the molecule - directed against one of the effector cell antigens FcaRI(CD89)or FcgRIII(CD16) and against HLA class II or Lym-2. Lym-2 represents a variant form of the HLA-DR antigen and is highly expressed on the surface of malignant B cells, but only at low levels on normal cells. HLA class II and Lym-2 are both known as effective targets for effector cell-mediated lysis of malignant human B-lymphoid cells. CD89 is an interesting trigger molecule for BsAb therapy, as it recruits neutrophils as effector cells, which have tumor cytolytic potential against a broad spectrum of tumor cells and are the most abundant circulating blood leukocytes. Antibodies against CD16 have already shown biological activity in vitro and in tumor patients by recruting NK cells. The two component scFv were fused via a flexible 20aa linker. ScFv fragments were generated by producing phage display libraries from corresponding hybridomas, and screening the libraries with antigen-positive cells. Recombinant scFv against HLA class II, Lym-2, CD89 and CD16 were thus obtained from the hybridomas F3.3, Lym-2, A77 and 3G8 respectively. Functional bsscFv were expressed and secreted by insect cells and were purified via Nickel chelate chromatography. Purified BsAb reacted with HLA class II or Lym-2-positive target cells and one of the effector cell antigens, CD89 or CD16, respectivly. In ADCC experiments all constructs mediated specific lysis of HLA class II or Lym-2-positive malignant human B-lymphoid cell lines with human MNC or PMN as effector cells. The [CD89 x HLA class II] and the [CD16 x HLA class II ] bsscFv also mediated significant lysis of primary cells from patients with B-cell chronic lymphocytic leukaemia (B-CLL). In conclusion, these recombinant bsscFv may allow the specific recruitment of effector cells for an improved therapy in B-lymphoid malignancies.
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| IMMUNE ESCAPE / NK CELLS |
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05 Thursday, May 6, 2004 at 15:00
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"Increased frequency of CTLA4+ TGFb+ CD4+ CD25+ T cells in peripheral blood of patients with chronic lymphatic leukemia and multiple myeloma"
Marc Beyer, Matthias Kochanek, Michael von Bergwelt-Baildon, Alexey Popov, Jürgen Wolf, Joachim L. Schultze
Molecular Tumor Biology and Tumor Immunology, Center for Internal Medicine, University of Cologne, Joseph-Stelzmann-Str. 9, 50924 Cologne, Germany
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A globally suppressed T cell function has been described for cancer patients including patients with chronic lymphatic leukemia (CLL) or multiple myeloma (MM). This has been mainly associated with inhibitory factors released by the tumor cells, while the role of recently characterized regulatory immune cells is not understood. Several different regulatory T cells such as CD4+ CD25+ T cells (Treg), TGFb producing TH3 or IL-10 producing TR1 cells have been described in murine models, and Treg cells have also been implicated in the control of graft versus host disease after allogeneic transplantation in humans. In contrast, little is known about frequencies and function of regulatory cells in leukemias and lymphomas. To address this issue we have analyzed 82 peripheral blood samples from 24 CLL patients, 18 MM patients and 26 healthy individuals. By assessing CD4+ CD25+ T cells we established a strongly significant increase of this subpopulation in both CLL (13±7% mean ± SD, p<0.01) and MM (13± 9%, p<0.01) when compared to healthy individuals (3.5±1.5%). While CDC4+ CD25+ T cells could also comprise previously activated T cells, the expression of CTLA4 has been associated with Treg cells. In fact, 80±14% respectively 67±26% of CD4+ CD25+ T cells in CLL resp. MM were also CTLA4+, while only 30±22% were found to be positive in healthy individuals strongly suggesting that these cells are mainly Treg cells. In contrast, using the BDCA-4 specific antibody for Neuropilin-1, we were unable to detect this molecule recently described on murine Treg cells on any of our samples. Interestingly, a significant proportion of CD4+ CD25+ T cells in CLL 28±13% and MM 22±12% also expressed intracellular TGFb, which was only found in 6±4% of these T cells in healthy individuals. Whether TGFb production reflects a particular activation status of Treg cells or whether these cells are a defined subpopulation requires further investigation. By analyzing co-expression of CCR7 and CD45RA we established that the fraction of CD4+ CD25+ T cells was particularly increased in the naïve and the central memory pool in peripheral blood of CLL and MM patients. Next we assessed T cell activation as a function of Treg cells. As expected, there was already a significantly decreased proliferative response of CD4+ T cells in many CLL patients even when CD25+ cells were depleted. These findings are most likely explained by chronic exposure to inhibitory cytokines as well as Treg cells. However, even under these conditions, coculture experiments of CD4+ CD25- and CD4+ CD25+ Treg cells supported the inhibitory role of Treg cells in CLL. We therefore propose that immunotherapy in any malignancy including CLL and MM characterized by an increase of regulatory factors and cells will significantly benefit from strategies inhibiting immune repression.
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06 Thursday, May 6, 2004 at 15:15
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"MMP-7 inhibits CTL - tumor cell interaction"
Sebastian Gregor1, Vijay Alla1, Jürgen Kuball2, Matthias Theobald2, Peter R. Galle1 , Dennis Strand1 and Susanne Strand1
1I. Medizinische Klinik und Poliklinik, 2 III. Medizinische Klinik und Poliklinik, Johannes Gutenberg-Universität Mainz,Germany
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MMP-7, the smallest member of the MMP-family, is a zinc-dependent metalloproteinase and is overexpressed in colon cancer and many other human cancers. Along with its prometastatic function, a fundamental role for MMP-7 has also been established in early tumor development, but the mechanism by which MMP-7 contributes to this, is still unknown.
Tumor specific cytotoxic T cells (CTLs) play a critical role in the control of tumor growth. They can induce apoptosis by CD95 as well as perforin/granzyme mediated pathways. Loss of CD95 may contribute to apoptosis resistance and immune escape of tumor cells leading to successful tumor outgrowth.
In our project we analyzed MMP-7 for its influence on CD95 mediated apoptosis and the cytolytic effector functions of CTLs. Furthermore we investigated the influence of MMP-7 cleavage activity on the adhesion and deadhesion of peptide specific CTL´s to tumor targets. In a cleavage assay with recombinant CD95 protein, we could show that MMP-7, cleaved approximately 2-3 kDa from the extracellular N-terminal end of CD95. In coculture experiments with 51Cr-labeled HepG2-cells, we found a significant decrease of cytotoxic action of peptide specific CTLs in the presence of MMP-7. In addition MMP-7 leads to a higher adhesion of CTLs and inhibits their deadhesion from HepG2 cells. Considering that CTL´s are serial killers, alteration in adhesion/deadhesion functions can be detrimental for tumor specific CTL killing.
Our results show, that MMP-7 can contribute to the apoptosis resistance of tumor cells by different mechanisms. These activities may explain the contribution of MMP-7 to early tumor growth.
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07 Thursday, May 6, 2004 at 16:00
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"Genetically modified natural killer cells specifically recognizing the tumor-associated antigens ErbB2/HER2 and EpCAM"
Christoph Uherek1, Tina Müller1, Torsten Tonn1,2, Barbara Uherek1 Hans-Georg Klingemann3, and Winfried S. Wels1
1Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, Frankfurt/Main, Germany
2Institute for Transfusion Medicine and Immunohematology RCBDS, Frankfurt/Main, Germany;
3Section of Bone Marrow Transplant and Cell Therapy, RUSH Medical Center, Chicago, IL, USA
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The continuously growing natural killer (NK) cell line NK-92 is highly cytotoxic against malignant cells of various origin without affecting normal human cells. Based on this selectivity, the potential of NK-92 cells for adoptive therapy is currently being investigated in phase I clinical studies. To further enhance the antitumoral activity of NK-92 cells and expand the range of tumor entities suitable for NK-92-based therapies, here by transduction with retroviral vectors we have generated genetically modified NK-92 cells expressing chimeric antigen receptors specific either for the tumor-associated ErbB2 (HER2/neu) antigen or the human Epithelial Cell Adhesion Molecule (Ep-CAM). Both antigens are overexpressed by many tumors of epithelial origin. The chimeric antigen receptors consist of either the ErbB2 specific scFv(FRP5) antibody fragment or the Ep-CAM specific scFv(MOC31), a flexible hinge region derived from CD8, and transmembrane and intracellular regions of the CD3 zeta chain.
Transduced NK-92-scFv(FRP5)-zeta or NK-92-scFv(MOC31)-zeta cells express high levels of the fusion proteins on the cell surface as determined by FACS analysis. In europium release assays no difference in cytotoxic activity of NK-92 and transduced NK-92 cells towards ErbB2 or Ep-CAM negative targets was found. However, even at low effector to target ratios transduced NK-92 cells specifically and efficiently lysed established ErbB2 or Ep-CAM expressing tumor cells that were completely resistant to cytolytic activity of parental NK-92 cells. Similarly, ErbB2-positive primary breast cancer cells isolated from pleural effusions of patients with recurrent disease were selectively killed by NK-92-scFv(FRP5)-zeta. In an in vivo model in immunodeficient mice treatment with retargeted NK-92-scFv(FRP5)-zeta, but not parental NK-92 cells resulted in markedly delayed growth of ErbB2 transformed cancer cells.
These results demonstrate that efficient retargeting of NK-92 cytotoxicity can be achieved, and might allow the generation of potent cell-based therapeutics for the treatment of ErbB2 and Ep-CAM expressing malignancies. This therapeutic approach might be applicable for a large variety of different cancers where suitable cell surface antigens have been identified.
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08 Thursday, May 6, 2004 at 16:15
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"Rapid functional exhaustion and deletion of cytotoxic T lymphocytes following immunization with recombinant adenovirus"
Elke Scandella1, Philippe Krebs1,2, Bernhard Odermatt3, and Burkhard Ludewig1
1Research Department, Kantonal Hospital St. Gallen, 9007 St. Gallen, Switzerland
2Institute of Experimental Immunology , University Hospital Zürich, 8091 Zürich, Switzerland
3Department of Pathology, University Hospital Zürich, 8091 Zürich, Switzerland
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Replication-deficient adenoviruses (rec-AdV) expressing different transgenes are widely employed vectors for gene therapy and vaccination. We examined here the generation of b-galactosidase (bgal)-specific CTL following administration of bgal-recombinant adenovirus (Ad-LacZ). Using MHC class I tetramers to track bgal-specific CTL in different organs, we found that a significant expansion of bgal-specific CTL could only be achieved in a very narrow dose range (2 ´ 108 - 2 ´ 109 pfu). Functional analysis revealed that adenovirus-induced bgal-specific CTL produced only very low amounts of effector cytokines and were unable to lyse bgal peptide-pulsed target cells. Injection of optimal doses of Ad-LacZ into transgenic mice which express bgal exclusively in non-lymphoid organs, led to physical deletion of bgal-specific CTL. Our results indicate first that CTL deletion in the course of adenoviral vaccination is preceded by their functional impairment and second, that the outcome of rec-AdV vaccination depends critically on the antigen load in peripheral tissues. The presented findings thus impinge on the rationale to use adenoviral vectors in clinical vaccination.
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09 Thursday, May 6, 2004 at 16:30
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"High frequency of functionally active Melan-A specific T cells in a patient with progressive immunoproteasome-deficient melanoma"
Norbert Meidenbauer1, Alfred Zippelius2, Mikaël J. Pittet2, Monika Laumer1, Sandra Vogl1, Jana Heymann1, Michael Rehli1, Barbara Seliger3, Stephan Schwarz4, Frederique-A. Le Gal5, Pierre Y. Dietrich5, Reinhard Andreesen1, Pedro Romero2, Andreas Mackensen1.
1 Dept. of Hematology/Oncology, University of Regensburg, Regensburg, Germany
2 Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, University Hospital (CHUV), Lausanne, Switzerland
3 Johannes Gutenberg University, III. Dept. of Internal Medicine, Mainz, Germany
4 Dept. of Pathology, University of Regensburg, Regensburg, Germany
5 Lab. of Tumor Immunology, Division of Oncology, University Hospital of Geneva, Geneva, Switzerland.
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Tumor-reactive T cells play an important role in cancer immunosurveillance. Applying the multimer technology, we report here an unexpected high frequency of Melan-A-specific CTL in a melanoma patient with progressive lymph node (LN) metastases, consisting of 18% and 12.8% of total peripheral blood and tumor-infiltrating CD8+ T cells, respectively. Melan-A-specific CTL revealed a high cytotoxic activity against allogeneic Melan-A-expressing target cells but failed to kill the autologous tumor cells. Loading of the tumor cells with Melan-A peptide reversed the resistance to killing, suggesting impaired function of the MHC class I Ag processing and presentation pathway. Mutations and/or down-regulation of the MHC class I heavy chain, the antigenic peptide TAP, and tapasin could be excluded. However, RT-PCR and immunohistochemical analysis revealed a deficiency of the immunoproteasomes low molecular weight protein (LMP)2 and LMP7 in the primary tumor cells, that affects the quantity and quality of generated T cell epitopes and might explain the resistance to killing. Overall, this is the first report of an extremely high frequency of tumor-specific CTL that exhibit competent T cell effector functions, but fail to lyse the autologous tumor cells. Immunotherapeutic approaches should not only focus on the induction of a robust anti-tumor immune response, but also have to target tumor immune escape mechanisms.
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| ADOPTIVE T CELL TRANSFER |
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10 Friday, May 7, 2004 at 10:15
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"Adoptive T cell therapy using antigen-specific CD8+ T cells for the treatment of patients with metastatic melanoma: a phase I clinical study"
Norbert Meidenbauer, Sandra Vogl, Monika Laumer, Jana Heymann, Reinhard Andreesen, Andreas Mackensen
Department of Hematology/Oncology, University of Regensburg, D-93042 Regensburg, Germany
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The adoptive transfer of in vitro induced and expanded tumor antigen-specific cytotoxic T lymphocytes (CTL) provides a promising approach to the immunotherpy of cancer. We have previously shown that Melan-A-specific CTL can be generated from HLA-A2.1+ melanoma patients by 4 rounds of in vitro stimulation of purified CD8+ T cells with autologous dendritic cells pulsed with a mutated HLA-A2 binding Melan-A (ELAGIGILTV) peptide. Based on these results we have initiated a pilot study of adoptive T cell therapy in advanced melanoma patients demonstrating that in vitro generated Melan-A specific CTL survive intact in vivo for several weeks and localize preferentially to tumor (Meidenbauer et al., J. Immunol. 170: 2161, 2003). Here we report on the clinical results of a phase I study of 12 HLA-A2+ melanoma patients that received at least three i.v. infusions of Melan-A-specific CTL i.v. at 2-week intervals. Each T cell infusion was accompanied by a 6-day course of s.c. IL-2 (3x106 IU daily). A total of 51 T-cell infusions were administered, averaging 1.48x108 Melan-A multimer+ T cells per infusion, with a range from 0.11 - 6.58x108 Melan-A-specific T cells per infusion. Clinical side effects were mild and consisted of chills and low-grade fever (WHO grade I-II) in 8 out of 12 patients that typically occurred within 6 to 8 h post infusion. Hematological effects, observed after T cell transfer, consisted of an increase in eosinophils up to 30% in 7 out of 12 patients, peaking 24h post transfer. Clinical and immunological responses consisted of antitumor responses in 3 out of 12 patients (2 PR, 1 mixed response), an elevated frequency of circulating Melan-A multimer+ T cells up to 2% of total CD8+ T cells up to 14 days post transfer, suggesting long-term survival and/or proliferation of transferred CTL, and a complete loss of Melan-A expression in lymph node metastases of 2 patients after T cell transfer. Our data indicate that the adoptive transfer of antigen-specific T cells in melanoma patients is capable of inducing clinical and systemic tumor-specific immune responses without provoking major side effects.
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11 Friday, May 7, 2004 at 10:30
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"Large Scale In Vitro Expansion of Polyclonal Human CD4+CD25high Regulatory T Cells"
Petra Hoffmann1, Rüdiger Eder1, Leoni A. Kunz-Schughart2, Reinhard Andreesen1, Matthias Edinger1
1Department of Hematology & Oncology, University Hospital Regensburg, 93053 Regensburg, Germany
2Institute of Pathology, University HospitalRegensburg, 93053 Regensburg, Germany
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CD4+CD25+ regulatory T (Treg) cells are pivotal for the maintenance of self-tolerance and their adoptive transfer protects from autoimmune diseases and pathogenic alloresponses after solid organ or bone marrow transplantation in murine model systems. In vitro, human CD4+CD25+ Treg cells display similar phenotypic and functional characteristics as murine CD4+CD25+ Treg cells, namely hyporesponsiveness to TCR stimulation and suppression of CD25- T cells. Thus far, the detailed characterization and potential clinical application of human CD4+CD25+ Treg cells was hampered by their paucity in peripheral blood and the lack of appropriate expansion protocols. Here we describe the up to 40,000-fold expansion of highly purified human CD4+CD25high T cells in vitro through the use of artificial APC for repeated stimulation via CD3 and CD28 in the presence of high dose IL-2. Expanded CD4+CD25high T cells were polyclonal, maintained their phenotype, exceeded the suppressive activity of freshly isolated CD4+CD25high T cells and showed characteristics of central memory T cells. The ability to rapidly expand human CD4+CD25high Treg cells large scale will not only facilitate their further exploration but also accelerate their potential clinical application in T cell-mediated diseases and transplantation medicine.
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12 Friday, May 7, 2004 at 10:45
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"Reversible HLA multimers (streptamers) for isolation of human cytotoxic T lymphocytes functionally active against tumor- and virus-derived antigens"
Julia Neudorfer1, Burkhard Schmidt1, Katharina Huster2, Matthias Schiemann2, Thomas Schmidt3, Hermann Wagner2, Christian Peschel1, Dirk H. Busch2 and Helga Bernhard1
1Department of Hematology/Oncology and 2Department of Microbiology/Immunology Klinikum rechts der Isar, Technical University of Munich, Germany. 3IBA GmbH, Göttingen, Germany.
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Attempts to treat patients with tumor-reactive or viral-specific cytotoxic T lymphocytes (CTLs) have been limited due to the difficulty of isolating and expanding functionally active T cells present in low numbers in the peripheral blood. Recently developed MHC/peptide multimers mimic T cell receptor (TCR) ligands and, therefore, allow visualization and isolation of antigen-specific CTLs. However, the persistence of multimers leads to prolonged TCR signaling and subsequently to overstimulation and cell death. We have generated a new type of MHC/peptide multimers, termed streptamers, which can be dissociated from the TCR. In the mouse model, the dissociation of streptamers from the TCR, prevents T cells from multimer-induced cell death (Knabel et al. Nature Med. 8:631, 2002). In this study, we investigate the efficacy of reversible HLA/peptide multimers for isolation of human antigen-specific T cells. Melan-A and CMV have been chosen as representative tumor-associated and viral antigen (Ag), respectively. Specificity and reversibility of A2/CMV and A2/Melan-A streptamers was documented by staining of Ag-specific T cell clones and loss of staining after streptamer removal. Streptamer-stained Ag-specific T cells remained functionally active following dissociation, whereas lytic function of T cells was impaired in the presence of non-reversible multimers (tetramers). Furthermore, CMVpp65(495-503)-specific T cells were streptamer- or tetramer-sorted from HLA-A2-positive, CMV-seropositive donors either directly out of the blood or following repetitive peptide stimulations in vitro. Both attempts successfully led to the isolation of CMV-specific CTLs that were cloned by limiting dilution. Clonal proliferation was superior for CMV-specific streptamer-sorted T cells compared to tetramer-sorted T cells. CMV-specific T cell clones isolated with streptamers and tetramers displayed a similar TCR repertoire and avidity. Growing CTL clones were capable of lysing CMVpp65(495-503)-pulsed as well as CMVpp65-transfected HLA-A2-positive target cells. For isolation of melanoma-reactive CTLs, the modified decapeptide Melan-A(26-35)A27L was chosen to construct streptamers respective tetramers. Again, streptamer-sorted Melan-A-specific CTL clones proliferated better than tetramer-sorted CTL clones. The isolated Melan-A-specific CTL clones displayed different TCR motifs, which can be explained by the broad repertoire of Melan-A-specific T cells physiologically present in vivo. All Melan-A(26-35)A27L-specific CTL clones crossreacted with the naturally processed peptide Melan-A(27-35), but only some CTL clones lysed HLA-A2-matched, Melan-A-expressing melanoma cells. Of note, tumor recognition by some streptamer-sorted CTL clones was superior to tumor lysis by tetramer-sorted CTL clones. Our current experiments focus on the isolation of T cells using reversible multimers coupled with microbeads allowing us to sort antigen-specific T cells under the guidelines of good manufacturing practice. Clinical goal is the adoptive transfer of antigen-specific T lymphocytes for treatment of patients with cancer or infectious diseases.
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13 Friday, May 7, 2004 at 11:00
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"Therapeutic efficacy of RAGE-1 and MAGE-9 peptide specific cytotoxic T cell clones in renal cell carcinoma"
Nicole Oehlrich1, Michael Linnebacher2, Stefan Stevanovic3, Margot Zoeller1
1 Department of Tumor Progression and Tumor Defense, German Cancer Research Center, Heidelberg, 2 Department of Molecular Pathology, University of Heidelberg, Heidelberg, 3 Department of Immunology, Institute for Cell Biology, University of Tübingen, Tübingen, Germany
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Renal cell carcinoma (RCC) are supposed to be immunogenic and several clinical trials of immunotherapy using tumor-lysate pulsed dendritic cells have been performed. We here report on the generation and therapeutic efficacy of RAGE-1 and MAGE-9 peptide specific cytotoxic T cell clones.
RAGE-1 and MAGE-9 are expressed on 61% and 40% of RCC. Six MAGE-9 and 14 RAGE-1 derived peptides were found to be immunogenic in the context of the HLA-A2.1 MHC complex. CTL were generated by co-culture with peptide pulsed dendritic cells (DC) or peptide pulsed CD40-activated B cells. The latter are as efficient in antigen presentation as DC, but have the advantage of being easier to generate in great quantitities and to display functional activity for a prolonged period of time. Therefore, they are well suited for the generation of CTL clones to be used in adoptive transfer. Three MAGE-9 and RAGE-1 specific CTL clones were generated. The clones were strictly peptide-specific and displayed high cytotoxic activity not only against peptide-loaded T2 cells, but also against HLA-A2.1-positive RCC lines that naturally expressed MAGE-9, RAGE-1 or both. The in vivo efficacy of these MAGE-9 and RAGE-1 peptide specific CTL clones is currently being evaluated in human RCC-bearing SCID, which received after intraperitoneal RCC injection repeated applications of the CTL clones at weekly intervals. So far, the majority of control mice became moribund, but only one mouse receiving a MAGE-9 and a RAGE-1 specific CTL developed a tumor, that had retained MHC class I as well as MAGE-9 and RAGE-1 expression. CTL recovered from these mice at 2 and 3 weeks after the last T cell transfer revealed that CTL had retained their specificity and cytotoxic activity was only slightly reduced.
Thus, B cells appear well suited as antigen presenting cells for the generation of large quantities tumor peptide specific CTL as required for adoptive transfer and cancer testis antigens may well provide suitable target for immunotherapy of RCC.
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14 Friday, May 7, 2004 at 11:15
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"The role of glycosylation as a modulator for signalling strength in T cells"
Beate Hauptrock, Jürgen Kuball, Ralf-Holger Voss, Magdalena Brkic, Christoph Huber and Matthias Theobald
Department of Hematology and Oncology, Johannes Gutenberg-University, 55101 Mainz, Germany
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Adoptive immunotherapy has proven efficiency in patients suffering from malignancies. However, the quality of in vitro generated tumor antigen-specific T cells which can be used for such therapies is limited. Therefore, methods have to be developed in order to increase potency of these tumor antigen-specific T lymphocytes. One possibility arises from the modification of glycoproteins such as the T cell receptor (TCR) or CD8. By their size and charge, sugar chains of these molecules play a role in the interaction of a T cell with the major histocompatibility complex (MHC). It is also reported that glycosylation decreases mobility of cell surface molecules by cross linking to carbohydrate binding proteins (so called lectins, e.g. Galectin-3). The resulting molecular lattice constrains TCR and CD8 clustering. Deglycosylation of the TCR and CD8 might be a possibility in order to increase mobility of the TCR or CD8 and to augment signalling strength. Unspecific global O- and N-deglycosylation were compared as well as a specific removal of selective glycosylation motives in defined proteins as the TCR.
For exogenous deglycosylation, T cells were treated with neuraminidase, which removes sialic acids at the end of sugar chains. This resulted in a better communication of the O-glycosylated CD8 with the MHC I molecule which was demonstrated by an increased specific tetramer-binding. Surprisingly, the ability to kill tumor cells was not increased. To inhibit the interaction of N-linked sugars of the TCR with Galectin-3 and so to increase mobility of the TCR, T cells were treated with lactose, which competes with the Galectin-binding sugars for Galectin-3. However, lactose-treatment did also not ameliorate target cell lysis. Thus, T cell deglycosylation could increase tetramer-binding, but this was not translated into a better T cell activity. We assumed, that other surface molecules which are important for signalling were negatively affected by these treatments. As consequence, we silenced N-glycosylation motives in the TCR molecule itself by point mutation. Wild-type and mutant TCRs were transduced into human T cells or the murine T cell hybridoma 58a-b- by retroviral gene transfer. Efficacy of TCR deglycosylation was ascertained by western blot analysis. Removing one or two defined sugar chains increased antigen-specific cytokine secretion. Deletion of three or more glycosylation sites impaired T cell function.
In summary, T cell deglycosylation is a sensitive mechanism. Deletion of defined glycosylation motives within a TCR is an efficient tool to increase signalling strength of a T cell.
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| THERAPEUTIC VACCINATION |
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15 Friday, May 7, 2004 at 14:45
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"Migration of dendritic cells to regional lymph nodes in a vaccination trial"
Angela Riccobon1, Ruggero Ridolfi1, Riccardo Galassi2, Massimiliano Petrini1, Monica Stefanelli1, Laura Fiammenghi3, Gianluigi Giorgetti4, Andrea Moretti2, Laura Ridolfi1 and Giuseppe Fiorentini2.
1Department of Medical Oncology, Pierantoni Hospital, Forlì, Italy;
2Department of Nuclear Medicine, Morgagni Hospital, Forlì, Italy;
3Istituto Oncologico Romagnolo, Forlì, Italy;
4Department of Health Physics, Pierantoni Hospital, Forlì, Italy.
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Dendritic Cell (DC) vaccination is a very promising therapeutic strategy in cancer patients. The immunizing ability of DC is critically influenced by their migration activity to lymphatic tissue, where they prime naïve T cells. In the present study DC were differentiated from PBMC obtained by leukapheresis (7-day culture of adherent cells with IL-4 and GM-CSF). On day 7 the DC were defined as immature DC (iDC). After a transient (3 h) and continuous (48 h) stimulation with a cocktail of cytokines (IL-1b, IL-6, TNF-a, PGE2), the iDC were defined as transientlyDC (tsDC) and mature DC (mDC), respectively. During a phase I-II clinical vaccination trial for patients with advanced melanoma and renal carcinoma, we evaluated the migration ability of iDC, tsDC and mDC, and compared intradermal (id) and subcutaneous (sc) administration. DC were labelled with 99Tc-HMPAO or 111In- Oxine and the presence of labelled DC was detected in regional lymph nodes up to a maximum of 72 h after inoculation.
It was verified that id administration resulted in about a threefold higher migration to lymph nodes than sc administration. iDC and mDC migration was compared in 8 patients. mDC showed, on average, a six-to eightfold higher migration than iDC. The first DC were detected in lymph nodes after 20-60 min and the maximum concentration was reached 8 h-72 h after vaccination. In two patients a similar migration activity of mDC and tsDC was observed up to 36 h after inoculation.
These data confirm the validity of therapeutic vaccination with mDC administered intradermally. Further investigation is also needed to determine whether tsDC could be used in clinical vaccination trials to have a longer T cell activation and a more potent stimulation capacity.
Partially financed by Ministry of Health Research Project 2002, n.234.
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16 Friday, May 7, 2004 at 15:00
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"TRANSITORY RESPONSE TO VACCINATION WITH PSCA-/PSA-PEPTIDE LOADED, AUTOLOGOUS DENDRITIC CELLS IN PATIENTS WITH METASTATIC, HORMONE-REFRACTORY PROSTATE CANCER"
A.K. Kaskel, R. Zeiser, R. Jochim, W. Schultze-Seemann*, C.F. Waller and H. Veelken
Department of Hematology/Oncology and *Department of Urology, Freiburg University Medical Center, D-79106 Freiburg, Germany
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Clinical trials employing vaccination with prostate-specific membrane antigen (PSMA) loaded dendritic cells (DC) in patients (pts) with advanced prostate cancer yielded response rates of >30%. We decided for HLA-A2 restricted peptides derived from prostate stem cell antigen (PSCA) and prostate specific antigen (PSA 1-3) because both antigens are overexpressed in >85% of prostate cancer and were demonstrated to induce antigen specific T-cell responses in vitro. Cell penetrating peptide (CPP) is a peptide derived from the HIV-tat protein that prolongs antigen presentation in vitro leading to enhanced antitumor immunity in vivo. Therefore, half of the pts were assigned to receive CPP-PSCA loaded DCs. The purpose of this study is to assess feasibility, patient safety, PSCA-/PSA-specific T-cell responses and tumor regression in vivo. Pts were vaccinated s.c. in 14d intervals. Response was assessed two weeks after the 4th vaccination. Delayed-type hypersensitivity (DTH) was tested d4+d46. Immune competence was monitored by HBV vaccination d1+d43. So far, 9/12 planned pts completed vaccination. No toxicities were observed. 5/9 pts achieved SD (3 with <50% decrease, 2 with <50% increase in PSA), and 3 of these but no non-responders developed a positive DTH after the 4th vaccination. The addition of CPP did not correlate with outcome. Responding pts received at mean two further vaccinations 4 weeks apart. At a median follow-up of 8.6 months, 70% of pts are alive compared to a reported OS of 59% and 28% at a follow-up of 6 and 11 months, respectively for pts with metastatic, hormone-refractory disease (1-3). No peptide specific T-cells were detected in two pts evaluated so far, however, the correlation of clinical and DTH responses suggests a tumor specific immunity. Our data indicate that vaccination with PSA/PSCA-peptide-loaded, autologous DCs is safe and well tolerated. Vaccination of more patients, long-term follow-up and completion of immune monitoring are needed to evaluate anti tumor effects.
1. Fossa SD et al. Br J Urol 1992; 69: 175-179
2. Kelly WK et al. J Clin Oncol 1993; 11: 607-615
3. Smaletz O et al. J Clin Oncol 2002; 20: 3972-3982
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17 Friday, May 7, 2004 at 15:35
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"Vaccination with WT1 induces high frequency memory and effector T cells in peripheral blood and bone marrow associated with complete remission of recurrent AML"
Anne Letsch, Carmen Scheibenbogen, Anne Marie Asemissen, Volker Mailänder, Eckhard Thiel, Ulrich Keilholz.
Medizinische Klinik III, Hämatologie, Onkologie und Transfusionsmedizin, Charité, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany
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The transcription factor Wilms tumor protein 1 (WT1) is an interesting antigen for use in vaccination and T cell therapy in myeloid leukemias. WT1 is strongly expressed in the majority of myeloid leukemic blasts and is a key molecule for blast proliferation. A phase I/II study has been initiated in our institution in patients with acute myeloid leukemia to analyze the immunogenicity and toxicity of WT1 126-134 peptide vaccination ín combination with the adjuvants GM-CSF and keyhole limpet hemocyanin. We report here on the first 4 patients who have completed vaccination and have received a total of 15, 12, 5, and 5 vaccination cycles, respectively. While, following chemotherapy, patients 1 and 2 both had 5-10% blasts in bone marrow when vaccination was initiated, patient 3 and 4 had 90% and 70% blasts, respectively. Induction of high frequency T cell responses was detected in 3 of 4 patients with up to 0.92%, 0.43%, and 0.42% in peripheral blood and up to 0.8% in bone marrow by tetramer analysis. Detailed phenotypical analysis in patient 1 showed that WT1-specific peripheral blood T cells were almost exclusively CD45RA+CCR7-granzymeB+, and directly produced IFNg in response to WT1 peptide, resembling cytotoxic effector T cells, while in the bone marrow both WT1-specific effector and CD45RA-CCR7- effector memory T cells were found. Patient 3 and 4 had progressive disease after 5 vaccinations. Patient 1 who had progressed during the first 4 weeks of vaccination with an increase of blasts to 30% in bone marrow was induced into complete remission after 6 vaccinations, which lasted for 12 months. Patient 2 is in continuous remission for currently 20 months. WT1 transcripts in bone marrow and peripheral blood were quantitated by PCR to monitor residual disease. In accordance with the clinical course in patients 1 and 2 we observed an approximately 100-fold, and 50-fold reduction, respectively, of WT1 transcripts following vaccination. No side effects as those typically seen with GM-CSF were noted. Taken together, these findings underline the efficacy of the vaccine, inducing high frequency WT1-specific effector and memory T cells in peripheral blood and bone marrow associated with complete remission of leukemia in the absence of hematological or renal toxicity.
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18 Friday, May 7, 2004 at 15:50
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"LUD00-014: Phase I Study of recombinant vaccinia-NY-ESO-1 (rV-NY-ESO-1) and recombinant fowlpox-NY-ESO-1 (rF-NY-ESO-1) in patients with NY-ESO-1 or LAGE positive cancers"
Armin Bender, Julia Karbach, Antje Neumann, Melina Biskamp, Dirk Jäger, Sacha Gnjatic, Eric Hoffman, Lloyd J. Old, Alexander Knuth,Elke Jäger
II. Medizinische Klinik, Krankenhaus Nordwest, Frankfurt, Germany; Ludwig Institute for Cancer Research, New York, USA
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Objectives: To determine the toxicity and NY-ESO-1-specific immune responses induced by immunization with rV-NY-ESO-1 or rF-NY-ESO-1. Study design: Eligible patients (pts) for the first part of the study were HLA-A2+ with advanced NY-ESO-1/LAGE+ tumors assessed by RT-PCR or immunohistochemistry. For the second part, pts were enrolled irrespective of the HLA type. Four injections of rV-NY-ESO-1, 3.1x107pfu i.d., or rF-NY-ESO-1, 7.41x107 pfu s.c., were administered 4x at 4 week intervals in part 1. Pts enrolled to part 2 received 2 injections of rV-NY-ESO-1, 3.1x107pfu i.d., followed by multiple injections of rF-NY-ESO-1, 7.41x107 at monthly intervals in case there was no evidence for progressive disease during vaccination. Toxicity was evaluated every 2 weeks. Delayed-type hypersensitivity (DTH) against HLA-A2 binding NY-ESO-1 peptides was assessed at baseline and after the last vaccination in HLA-A2+ pts. Immunogenicity was assessed by ELISPOT and tetramer analysis against HLA-A2 binding NY-ESO-1 peptides. Results: 29 pts with different types of cancer were enrolled, 9 pts are ongoing, 11 completed at least 4 cycles of treatment, 9 were withdrawn for disease progression or at their own discretion. No remarkable toxicities occured. Positive post-vaccination DTH reactions against 2 HLA-A2 binding NY-ESO-1 peptides occurred in 6/6 pts tested. At baseline all HLA-A2+ pts of part 1 were NY-ESO-1 serum antibody- and CD8+ T-cell negative. In 2 pts a conversion of NY-ESO-1 serum antibody was observed after 3 vaccinations. In 9 pts for whom immune assay data are available, NY-ESO-1 specific CD8+ T-cell responses were induced during vaccination. After 4 injections maximum ELISPOTS ranged between 160-800 per 50,000 CD8+ T-cells in the 2 vaccinia pts and 100-500 in the 2 fowlpox pts as confirmed by tetramer assays. At 4 months 14 pts were found to have stable disease. 1 pt experienced a partial remission of subcutaneous and peritoneal melanoma metastases after 3 months of immunization. Assessments are underway for NY-ESO-1 specific T cell responses restricted by non-HLA-A2 and HLA class II alleles. Conclusion: Immunization with recombinant vaccinia- and fowlpox-NY-ESO-1 is safe and induces NY-ESO-1 specific immune responses.
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19 Friday, May 7, 2004 at 16:05
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"Tumor vaccination after allogeneic bone marrow cell reconstitution of the non-myeloablatively conditioned tumor-bearing murine host"
Margot Zöller
Department of Tumor Progression and Tumor Defense, German Cancer Research Center, D-69120 Heidelberg, Germany
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Allogeneic bone marrow cell (BMC) reconstitution of the non-myeloablatively conditioned host is supposed to provide an optimized platform for tumor vaccination. We recently showed that an allogeneic T cell-depleted graft was well accepted if the tumor-bearing host was NK-depleted. Based on this finding a vaccination protocol in tumor-bearing, non-myeloablatively conditioned, allogeneically reconstituted mice was elaborated. Vaccination was most efficient when allogeneically reconstituted, tumor-bearing mice received tumor-primed, donor T cells, which had matured in the allogeneic host together with host-derived tumor lysate-pulsed dendritic cells. High frequencies of tumor-specific proliferating and cytotoxic T cells were recorded, the survival time of tumor-bearing mice was significantly prolonged and in over 50% of mice the tumor was completely rejected. Notably, GvH was not aggravated after vaccination with tumor-primed, donor-derived T cells that had matured in the host, i.e. T cells were tolerant towards the host, but not towards the tumor. The finding convincingly demonstrates the feasibility and efficacy of tumor vaccination of the allogeneically reconstituted, non-myeloablatively conditioned host after establishment of thymic tolerance and ceasing of initial GvH reactivities. Furthermore, they support the working hypothesis that reconstitution protocols favoring tolerance induction, which are time consuming, rather than a rapid establishment of full donor chimersim may allow more efficient tumor vaccination without potentially refreshing GvH reactivites.
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