Katja Simon-Keller

Publications (5) View all

• Article: Survivin Blockade Sensitizes Rhabdomyosarcoma Cells for Lysis by Fetal Acetylcholine Receptor-Redirected T Cells.

  Katja Simon-Keller, Annette Paschen, Andreas A Hombach, Philipp Ströbel, Jean Michel Coindre, Stefan B Eichmüller, Angela Vincent, Stefan Gattenlöhner, Florian Hoppe, Ivo Leuschner, Sabine Stegmaier, Ewa Koscielniak, Martin Leverkus, Dario C Altieri, Hinrich Abken, Alexander Marx
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  ABSTRACT: Cellular immunotherapy may provide a strategy to overcome the poor prognosis of metastatic and recurrent rhabdomyosarcoma (RMS) under the current regimen of polychemotherapy. Because little is known about resistance mechanisms of RMS to cytotoxic T cells, we investigated RMS cell lines and biopsy specimens for expression and function of immune costimulatory receptors and anti-apoptotic molecules by RT-PCR, Western blot analysis, IHC, and cytotoxicity assays using siRNA or transfection-modified RMS cell lines, together with engineered RMS-directed cytotoxic T cells specific for the fetal acetylcholine receptor. We found that costimulatory CD80 and CD86 were consistently absent from all RMSs tested, whereas ICOS-L was expressed by a subset of RMSs and was inducible by tumor necrosis factor α in two of five RMS cell lines. Anti-apoptotic survivin, along with other inhibitor of apoptosis (IAP) family members (cIAP1, cIAP2, and X-linked inhibitor of apoptosis protein), was overexpressed by RMS cell lines and biopsy specimens. Down-regulation of survivin by siRNA or pharmacologically in RMS cells increased their susceptibility toward a T-cell attack, whereas induction of ICOS-L did not. Treatment of RMS-bearing Rag(-/-) mice with fetal acetylcholine receptor-specific chimeric T cells delayed xenograft growth; however, this happened without definitive tumor eradication. Combined blockade of survivin and application of chimeric T cells in vivo suppressed tumor proliferation during survivin inhibition. In conclusion, survivin blockade provides a strategy to sensitize RMS cells for T-cell-based therapy.
  American Journal Of Pathology 04/2013; · 4.89 Impact Factor
• Article: Targeting the fetal acetylcholine receptor in rhabdomyosarcoma.

  Katja Simon-Keller, Stefan Barth, Angela Vincent, Alexander Marx
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  ABSTRACT: Introduction: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. Recent efforts to enhance overall survival of patients with clinically advanced RMS have failed and there is a demand for conceptually novel treatments. Immune therapeutic options targeting the fetal nicotinic acetylcholine receptor (fnAChR), which is broadly expressed on RMS, are novel approaches to overcome the therapeutic resistance of RMS. Expression of the fnAChR is restricted to developing fetal muscles, some apparently dispensable ocular muscle fibers and thymic myoid cells. Therefore, after-birth fnAChR is a tumor-associated and almost tumor-specific antigen on RMS cells. Areas covered: This review gives an overview on nAChR function and expression pattern in RMS tumor cells, and deals with the immunological significance of fnAChR-expressing cells, including the risk of anti-nAChR autoimmunity as a potential side effect of fnAChR-directed immunotherapies. The article also addresses the advantages and disadvantages of vaccination strategies, immunotoxins and chimeric T cells targeting the fnAChR. Expert opinion: Finally, we suggest technical and biological strategies to improve the available immunotherapeutic tools including increasing the in vivo expression of the target fnAChR on RMS cells.
  Expert opinion on therapeutic targets 12/2012; · 3.72 Impact Factor
• Article: Adoptive T-Zell-Therapie des Rhabdomyosarkoms

  K. Simon-Keller, A. Paschen, S. Eichmüller, S. Gattenlöhner, S. Barth, E. Koscielniak, I. Leuschner, P. Stöbel, A. Hombach, H. Abken, A. Marx
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  ABSTRACT: ZielsetzungUm das Überleben bei fortgeschrittenen Rhabdomyosarkomen (RMS) zu verbessern, versuchen wir, Bedingungen für den adoptiven Transfer chimärer T-Zellen zu verbessern, die eine Spezifität für den fetalen Acetylcholinrezeptor, ein RMS-spezifisches Oberflächenmolekül, haben. MethodenZur Optimierung der Rezeptorzytotoxizität wurde dem ursprünglich vorhandenen chimären Rezeptor, bestehend aus einem humanen Anti-fAChR-Antikörper, einer Fc-Hinge-Region und einer humanen CD3ζ-Kette, eine CD28-Domäne zugefügt. Periphere Blutlymphozyten wurden mittels retroviraler Transduktion modifiziert und die Expression des chimären Rezeptors mittels Durchflusszytometrie verifiziert. Das zytotoxische Potenzial der modifizierten T-Zellen gegenüber RMS-Zellen wurde über MTT-Zytotoxizitätstests bestimmt. Die Expression kostimulatorischer Moleküle und antiapoptotischer Faktoren wurde durchflusszytometrisch bzw. mithilfe quantitativer PCR untersucht. ErgebnisseDie geringen Expressionslevel kostimulatorischer Moleküle auf RMS-Zellen führten zu der Überlegung, einen chimären Antigenrezeptor (CAR) mit einer CD28-CD3ζ-Signaldomäne zu generieren. Die erfolgreiche Modifikation der T-Zellen mit der „zweiten Generation“ des AChR-spezifischen CAR zeigte trotz guter Expressionsraten geringe Lyseraten gegenüber RMS, im Vergleich zu CD20-positiven Lymphom- oder CEA-exprimierenden Adenokarzinomzelllinien mit CD20- bzw. CEA-spezifischem CAR. SchlussfolgerungDie verminderten Lyseraten der RMS-Zellen durch einen fAChR-spezifischen CAR lassen eine Resistenz der RMS-Zellen gegenüber der T-Zell-Antwort vermuten. Die Inhibierung antiapoptotischer Stoffwechselwege könnte die Sensitivität dieser Zellen gegenüber konventioneller und auch gegenüber T-Zell-basierten Therapien verbessern. AimsTo improve survival of patients with advanced rhabdomyosarcomas (RMS), we aimed to adoptively transfer T-cells with redirected specificity for the fetal acetylcholine receptor (AChR), an RMS-specific cell surface antigen. MethodsA “second generation” chimeric antigen receptor (CAR) with a combined CD28-CD3ζ signaling domain was derived from our previously described chimeric antigen receptor composed of an extracellular human anti-fAChR antibody fragment, an Fc hinge region, and the intracellular T-cell receptor zeta chain. Lymphocytes from the peripheral blood were modified by retroviral transduction and monitored by FACS analysis. Cytotoxicity of modified T-cells towards RMS cells was recorded by MTT-based viability tests; expression of co-stimulatory molecules and anti-apoptotic genes was studied by FACS and qRT-PCR analysis. ResultsCo-stimulatory molecules were expressed in low levels on RMS cells giving the rationale to generate a CD28-CD3ζ signalling CAR (chimeric antigen receptor) for redirecting T-cells. T-cells were successfully engineered with the “second generation” AChR-specific chimeric antigen receptor. Despite of high CAR expression engineered T-cells showed low killing efficiency towards RMS compared to redirected killing of CD20+ lymphoma or CEA-expressing adenocarcinoma cell lines when redirected by CD20- and/or CEA-specific CAR. ConclusionsData suggest that RMS cells exhibit resistance to a T-cell attack redirected by a fAChR-specific CAR. Inhibition of anti-apoptotic pathways in those cells may improve sensitivity to conventional as well as T-cell-based therapeutics. SchlüsselwörterRhabdomyosarkom-Fetaler Acetylcholinrezeptor-AChR-γ-T-Zellen-Chimärer Antigenrezeptor KeywordsRhabdomyosarcoma-Acetylcholine receptor, fetal-AChR-γ-T-cells-Antigen receptor, chimeric
  Der Pathologe 04/2012; 31:215-220. · 0.67 Impact Factor
• Article: Variable Resistance of RMS to Interferon γ Signaling.

  Katja Simon-Keller, Katharina Mößinger, Anna-Lena Bohlender, Philipp Ströbel, Alexander Marx
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  ABSTRACT: Aims. Chimeric T cells directed to the γ-subunit of the fetal acetylcholine receptor (fAChR) produce large amounts of interferon-γ (IFNγ) on coculture with fAChR-expressing rhabdomyosarcoma (RMS) cells prior to RMS cell death. The aim of this study was to elucidate whether IFNγ blocks proliferation and survival of RMS cells and modulates expression of genes with relevance for cytotoxicity of chimeric T cells. Methods. Expression levels of IFNγ receptor (IFNGR), AChR, MHCI, MHCII, and CIITA (class II transactivator) by RMS were checked by flow cytometry, qRT-PCR, and western blot. Proliferation and cell survival were investigated by annexin V and propidium iodide staining and MTT (thiazolyl-blue-tetrazolium-bromide) assay. Key phosphorylation and binding sites of IFNGRs were checked by DNA sequencing. Results. IFNγ treatment blocked proliferation in 3 of 6 RMS cell lines, but reduced survival in only one. IFNGR was expressed at levels comparable to controls and binding sites for JAK and STAT1 were intact. Induction of several target genes (e.g., AChR, MHCI, and MHCII) by IFNγ was detected on the RNA level but not protein level. Conclusions. IFNγ does not significantly contribute to the killing of RMS cells by fAChR directed chimeric T cells. Signalling downstream of the IFNR receptor, including the posttranscriptional level, is impaired in most RMS cell lines.
  ISRN oncology. 01/2012; 2012:789152.
• Article: [Adoptive T-cell therapy of rhabdomyosarcoma].

  K Simon-Keller, A Paschen, S Eichmüller, S Gattenlöhner, S Barth, E Koscielniak, I Leuschner, P Stöbel, A Hombach, H Abken, A Marx
  [show abstract] [hide abstract]
  ABSTRACT: To improve survival of patients with advanced rhabdomyosarcomas (RMS), we aimed to adoptively transfer T-cells with redirected specificity for the fetal acetylcholine receptor (AChR), an RMS-specific cell surface antigen. A "second generation" chimeric antigen receptor (CAR) with a combined CD28-CD3ζ signaling domain was derived from our previously described chimeric antigen receptor composed of an extracellular human anti-fAChR antibody fragment, an Fc hinge region, and the intracellular T-cell receptor zeta chain. Lymphocytes from the peripheral blood were modified by retroviral transduction and monitored by FACS analysis. Cytotoxicity of modified T-cells towards RMS cells was recorded by MTT-based viability tests; expression of co-stimulatory molecules and anti-apoptotic genes was studied by FACS and qRT-PCR analysis. Co-stimulatory molecules were expressed in low levels on RMS cells giving the rationale to generate a CD28-CD3ζ signalling CAR (chimeric antigen receptor) for redirecting T-cells. T-cells were successfully engineered with the "second generation" AChR-specific chimeric antigen receptor. Despite of high CAR expression engineered T-cells showed low killing efficiency towards RMS compared to redirected killing of CD20+ lymphoma or CEA-expressing adenocarcinoma cell lines when redirected by CD20- and/or CEA-specific CAR. Data suggest that RMS cells exhibit resistance to a T-cell attack redirected by a fAChR-specific CAR. Inhibition of anti-apoptotic pathways in those cells may improve sensitivity to conventional as well as T-cell-based therapeutics.
  Der Pathologe 10/2010; 31 Suppl 2:215-20. · 0.67 Impact Factor