Cancer Research Cancer Medicine 8 Jordan
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gattenlöhner, S.
Right arrow Articles by Rossig, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gattenlöhner, S.
Right arrow Articles by Rossig, C.
[Cancer Research 66, 24-28, January 1, 2006]
© 2006 American Association for Cancer Research

Priority Reports

Rhabdomyosarcoma Lysis by T Cells Expressing a Human Autoantibody-Based Chimeric Receptor Targeting the Fetal Acetylcholine Receptor

Stefan Gattenlöhner1, Alexander Marx1, Birgit Markfort2, Sibylle Pscherer2, Silke Landmeier2, Heribert Juergens2, Hans-Konrad Müller-Hermelink1, Ian Matthews3, David Beeson3, Angela Vincent3 and Claudia Rossig2

1 Institute of Pathology, University of Wuerzburg, Wuerzburg, Germany; 2 Department of Pediatric Hematology and Oncology, University Children's Hospital Münster, Münster, Germany; and 3 Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom

Requests for reprints: Stefan Gattenlöhner, Institute of Pathology, University of Wuerzburg, Josef-Schneider-Str. 2, D-97080 Wuerzburg, Germany. Phone: 49-931-201-47793; Fax: 49-931-201-47440; E-mail: stefan.gattenloehner{at}mail.uni-wuerzburg.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Rhabdomyosarcomas are the most frequent malignant soft tissue tumors of childhood; however, because current multimodality treatments fail to improve the poor survival rate of children with metastatic rhabdomyosarcoma, new treatments are required. We previously identified the {gamma}-subunit of the fetal acetylcholine receptor (fAChR) as a specific cell surface target in rhabdomyosarcoma. Here, we engineered human T lymphocytes to express chimeric receptors composed of the antigen-binding domain of a human anti-fAChR antibody joined to the signaling domain of the human T-cell receptor {zeta}-chain. The interaction of fAChR{zeta}-transduced T cells with fAChR-positive rhabdomyosarcoma cell lines, but not with fAChR-negative control cells, induced T-cell activation characterized by strong secretion of IFN-{gamma} and delayed lysis of tumor cells. Importantly, we found that in six of six rhabdomyosarcoma patients, chemotherapy increased fAChR expression on residual tumor cells in vivo. Our observations suggest that these fully human chimeric fAChR{zeta}-transduced T cells, which should be well tolerated by the patient, have potential use in vivo both as a primary treatment for rhabdomyosarcoma and as a complementary approach to eradicate residual tumor cells after chemotherapy. (Cancer Res 2006; 66(1): 24-28)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Rhabdomyosarcomas are the most common soft tissue sarcomas of childhood. Despite intensified, multimodality treatments, the overall survival for high-risk populations has remained at 5% to 20% over the last decades (1, 2). Therapeutic targeting of cancers with adoptively transferred cytotoxic T cells recognizing tumor-specific antigens has had encouraging success in lymphomas (3) and melanoma (4). T cells may be a therapeutic option for rhabdomyosarcoma, and the PAX3-FKHR fusion protein in alveolar rhabdomyosarcoma (5) or MAGE antigens (6) are potential targets. However, rhabdomyosarcomas express only low levels of MHC molecules and resist the MHC-restricted recognition by T cells (6). To bypass immune resistance, T cells have been engineered to express tumor-specific, chimeric receptors (chRec) for antigen recognition (7). In these recombinant receptors, an antibody-derived, single-chain scFv fragment is linked to the cytoplasmic signaling domain of the TCR{zeta} chain. ChRec gene–modified T cells exhibit specific, MHC-independent in vitro lysis and cytokine secretion in response to antigen-expressing target cells and were immunoprotective in murine tumor xenograft models (7). A critical requirement is the presence of a tumor-specific target and availability of a specific antibody. A candidate target in rhabdomyosarcoma is the fetal type of the nicotinic acetylcholine receptor (fAChR; ref. 8). During development of the neuromuscular junction, a change from the fetal type ({alpha}2ß{gamma}{delta}) to the adult type ({alpha}2ß{varepsilon}{delta}) of the AChR occurs, with replacement of the {gamma}-subunit by the {varepsilon}-subunit (9). After birth, expression of fAChR is almost completely lost from mature muscle but is maintained in thymic myoid cells, some extraocular muscle fibers, and denervated muscle (8, 10). In addition, fAChRs are highly expressed in rhabdomyosarcoma, distinguishing them from normal muscle (8, 10). Moreover, cloned human Fab antibodies specific for fAChR are available (11). Here, we created chimeric T cells with specificity for fAChR and show that they can mediate specific T-cell lysis of rhabdomyosarcoma cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and antibodies. Cell lines were provided by Ewa Koscielniak (Olgahospital, Stuttgart, Germany; rhabdomyosarcoma: FL-OH, Ax-OH-1), Paul H. Sorensen (University of British Columbia, Vancouver, British Columbia, Canada; rhabdomyosarcoma: Birch), Gary P. Nolan (Massachusetts Institute of Technology, Cambridge, MA; Phoenix-eco), and American Type Culture Collection (Manassas, VA; rhabdomyosarcoma: TE-671, A-204; leiomyosarcoma: SK-LMS-1). The DNA encoding adult AChR ({alpha}12, ß, {delta}, {varepsilon}) and fetal AChR ({alpha}12, ß, {delta}, {gamma}) was generated as described (9). Murine monoclonal antibody B8 (MIB8) binds to the {gamma}-subunit of human AChR as shown previously (12). Santa Cruz Biotechnology (Heidelberg, Germany) provided the rabbit polyclonal antiserum (SC-1453) against the fAChR {gamma}-subunit.

Cloning of chRec genes. The human recombinant Fab fragment, Fab 35, specific for the extracellular domain of the fetal AChR {gamma}-chain, was isolated by phage display technology from a pregnant woman whose fetus was affected by in utero myasthenia gravis (11). The variable Fab domains VH and VL were assembled as scFv fragment by PCR. To generate the fAChR{zeta} chRec gene, the DNA encoding VH and VL were amplified by PCR and ligated into pRSV (provided by Zelig Eshhar, Rehovot, Israel) in frame with the DNA coding for the human IgG1 hinge domain and transmembrane and intracellular domain of the human TCR {zeta}-chain. The chimeric gene was subcloned into the retroviral vector SFG (13). Expression of the chRec in SFG-transduced cells was proved by reverse transcription-PCR (RT-PCR). The negative control CD19{zeta} chRec was cloned as reported previously (14).

Production of recombinant retrovirus. Fresh retroviral supernatants from transiently transfected Phoenix-eco cells were used to infect the packaging cell line PG-13 in the presence of polybrene (8 µg/mL) for 48 hours at 32°C. After overnight incubation at 37°C in fresh culture medium, infected cells were subjected to a second identical round of infection. Supernatants were generated on the resulting bulk producer cell lines by incubation for 24 hours at 32°C and used to transduce T cells.

Isolation and transduction of lymphocytes. Fresh peripheral blood mononuclear cells from five healthy donors were prestimulated with CD3- and CD28-specific antibodies and transduced as described (15). Bulk-transduced T cells containing >98% CD3+ T cells (coexpressing CD8 in 55-86%), but no detectable CD3–CD56+ natural killer cells, were used for all subsequent experiments without selection of chRec+ cells.

RT-PCR. Expression of chRec mRNA in transduced T cells was semiquantitatively assessed by RT-PCR using insert-specific primers. Amplification was done using a 5' primer annealing to the scFv domain of the chRec, combined with primer 3' Hinge, annealing to an 18 bp region within the spacer domain. F{gamma}-AChR and R{gamma}-AChR primers were used to determine the fAChR-specific {gamma}-subunit and the common {alpha}-subunit mRNA as surrogate markers for fAChR expression (8, 10).

Measurement of cytokine production. Triplicate samples of transduced effector T cells (5 x 104 per well) were cocultured with various tumor cells at a stimulator-to-effector ratio of 3:1 in the presence of rhIL-2 (100 IU/mL) in 96-well round-bottomed plates. After 72 hours, supernatants were analyzed for IFN-{gamma} (Hölzel Diagnostika, Cologne, Germany) by ELISA following the instructions of the manufacturer.

51Cr release assays. Various numbers of T cells were coincubated in triplicate with 2,500 target cells labeled with 100 µCi 51Cr / 0. 5 x 106 cells (PE Applied Biosystems, Foster City, CA) in a volume of 200 µL in a V-bottomed 96-well plate. After 4 hours of incubation at 37°C and 5% CO2, radioactivity of supernatants was counted in a {gamma}-counter. Maximum release was determined by Triton X lysis of target cells.

Long-term cytotoxicity assays. Target cells (rhabdomyosarcoma cell line cells and control SK-LMS-1 cells at 2 x 105 per well) were cocultured in 24-well plates with fAChR{zeta}-transduced or negative control (nontransduced or CD19-transduced) T cells at various ratios. Other negative control wells contained target cells alone. During coculture, the percentage of tumor cells was repeatedly determined by cytometry analysis after intracellular desmin staining. Nonvital cells were excluded by propidium iodide staining.

Granzyme B secretion assay. T cells (1 x 105 per well) and tumor target cells (5 x 105 per well) were added to triplicate wells coated with antihuman granzyme B capture antibody (ELISPOT Human Granzyme B kit, Becton Dickinson, Heidelberg, Germany). Negative controls consisted of T cells alone, target cells without T cells, and media alone. Following 4 hours of coincubation, granzyme B was detected following the recommendations of the manufacturer.

Biopsies. Fresh frozen prechemotherapeutic and postchemotherapeutic biopsies were available from six patients (two males, four females; age range 3-17 years, mean = 10.7 years) with either an alveolar (n = 2) or embryonal (n = 4) rhabdomyosarcoma. Chemotherapies followed CWS-96-based protocols (16).

In vitro chemotherapy. Subconfluent TE-671 cells grown on histologic slides or in cell culture flasks were subjected to single-agent chemotherapy for 3 days using either doxorubicin (250 nmol/L), etoposide (5 µmol/L), or cisplatinum (140 nmol/L). TE-671 cells on glass slides were fixed with acetone before immunofluorescence analysis. For RT-PCR and flow cytometry studies, TE-671 cells were released from cell culture flasks by trypsinization.

Immunohistochemistry and immunofluorescence studies. Immunohistochemistry and immunofluorescence studies on fresh frozen sections and rhabdomyosarcoma cells grown on histologic slides, respectively, followed standard protocols. Percentages of MIB8-immunoreactive cells were calculated from tumor cells counted in at least 10 high-power fields (at x400 magnification).

Statistical analysis. Student's t test was used to test for significance in each set of values, assuming equal variance. Mean values ± SD are given unless otherwise stated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
FAChR{zeta}-transduced T cells produce IFN-{gamma} in response to fAChR-expressing tumor cells. To show specific recognition of fAChR-expressing rhabdomyosarcoma cells by chR-transduced T cells, we measured IFN-{gamma} secretion after 72 hours of coculture. ChRec composed of the scFv domains of the anti-fAChR Fab 35 in VL-VH orientation fused to the signaling moiety of the {zeta}-chain (designated fAChR{zeta}) induced strong secretion of IFN-{gamma} (up to 19 ng/mL/106 cells) when cocultured with fAChR-positive rhabdomyosarcoma cell lines (TE-671, Birch, and FL-OH), but not with the fAChR-negative cell line A-204 (Fig. 1A). Nontransduced T cells (not shown) and T cells transduced with an unrelated anti-CD19-reactive scFv (Fig. 1B) secreted much less IFN-{gamma}. Thus, it seems that the interaction between fAChR{zeta} and fAChR triggers a functional and specific T-cell response associated with IFN-{gamma} secretion.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 1. IFN-{gamma} secretion by fAChR{zeta} transduced (A) compared with negative control CD19{zeta}-transduced (B) T cells cocultured with fAChR-positive rhabdomyosarcoma cell lines TE-671, Ax-OH-1, Birch, and FL-OH-1. A-204 is an fAChR-negative control rhabdomyosarcoma cell line. Surface fAChR expression is low on TE-671 cells (Fig. 2B) as well as Birch and FL-OH-1 cells (not shown), whereas it is higher on AX-OH-1 (Fig. 2B). IFN-{gamma} secretion was quantified in coculture supernatants by ELISA. One representative experiment of six done with T cells from four individual donors. Columns, means of three observations; bars, SD.

 


View larger version (21K):
[in this window]
[in a new window]
  		Figure 2. Short-term cytotoxicity of chRec+ (fAChR{zeta}) and nontransduced (NT; negative control) T cells toward different fAChR-positive and fAChR-negative rhabdomyosarcoma cell lines. A, cytolysis of fAChRhigh rhabdomyosarcoma cell line Ax-OH-1 (spontaneously fAChRhigh) and TE-671/{gamma} (fAChRhigh due to transfection) by fAChR{zeta}+ T cells was compared with cytolysis of nontransduced T cells and T cells transfected with the fAChR {varepsilon}-chain in a 4-hour 51Cr release assay. *, P < 0.01. One representative experiment of three. Points, percentage of the maximum 51Cr release following Triton X-100 treatment. E:T, ratio of effector (E) T cells to target (T) cell lines. B, expression of fAChRs on rhabdomyosarcoma cell lines TE-671, TE-671/{varepsilon}, TE-671/{gamma}, and Ax-OH-1 by flow cytometry analysis following staining with anti-fAChR antibody MIB8. C, granzyme B release assay with fAChR-positive rhabdomyosarcoma (TE-671, TE-671/{varepsilon}, TE-671/{gamma}, Ax-OH-1, FL-OH-1, and Birch) and fAChR-negative control (SK-LMS-1) cell lines. Granzyme B secretion is given as the number of GrB-positive spots per well using ELISPOT technology. One representative result of four experiments.

 
Increasing fAChR density on rhabdomyosarcoma cells confers higher susceptibility to fAChR{zeta}-mediated immediate cytotoxicity. Although fAChR{zeta}+ T cells exhibited fAChR-specific IFN-{gamma} production (Fig. 1A), they failed to mediate significant lysis of these rhabdomyosarcoma cell lines within the 4-hour incubation period typically used in 51Cr release assays (Fig. 2A). Therefore, we transiently transfected TE-671 cells with fetal AChR ({alpha}12, ß, {delta}, and {gamma}; henceforth called TE-671/{gamma}) to increase the surface fAChR expression level (Fig. 2B). Indeed, TE-671/{gamma} cells were consistently lysed better than native TE-671 cells (Fig. 2A) or TE-671 cells transfected with adult AChR ({alpha}12, ß, {delta}, {varepsilon}; TE-671/{varepsilon}). Furthermore, the native fAChRhigh rhabdomyosarcoma cell line Ax-OH-1 was even better lysed than TE-671/{gamma} cells (Fig. 2A). Moreover, secretion of granzyme B, an important mediator of immediate T-cell cytolysis, was lower in cocultures with fAChRlow (TE-671; TE-671/{varepsilon}; Birch; FL-OH-1) compared with fAChRhigh (TE-671/{gamma}; Ax-OH-1) rhabdomyosarcoma cell lines (Fig. 2C).

FAChR{zeta}-expressing T cells efficiently lyse fAChR-expressing tumor cells during long-term coincubation. The above findings showed resistance of low AChR–expressing rhabdomyosarcoma cells toward fAChR-specific cytolysis in short-term culture. Coincubation of rhabdomyosarcoma targets with fAChR{zeta}-transduced T cells for 7 days, however, resulted in gradual elimination of tumor cells (Fig. 3A-C). Tumor cell numbers declined after 24 hours of coculture, dropped steeply up to day 3, and were almost undetectable by day 7. FAChR-expressing tumor cells incubated with nontransduced T cells were not lysed during long-term cocultures (Fig. 3A and B) nor were fAChR-negative control SK-LMS-1 cells (not shown).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 3. fAChR{zeta}-transduced T cells lyse fAChR-positive rhabdomyosarcoma cells during 7 days of coculture. TE-671 cells (A, B, and C) and Ax-OH-1, Birch, and FL-OH-1 rhabdomyosarcoma cells (C) were cultured with nontransduced and fAChR{zeta}-transduced T cells at T cell-to-tumor cell ratios of 1:1 (A), 5:1 (B), and 1:2.5 (C). Tumor cells were quantified by flow cytometry at 1, 2, 3, and 7 days after intracellular desmin staining. Representative of five experiments.

 
FAChR expression in rhabdomyosarcoma after chemotherapy. It has been reported in a single case of rhabdomyosarcoma that AChR expression was increased after chemotherapy (17). To confirm this finding, we studied biopsies from six individual patients, both before and after chemotherapy, by immunohistochemistry and RT-PCR, using simultaneous amplification of the {alpha}- and {gamma}-subunit of the AChR as previously described (10). Whereas the primary biopsies exhibited variable percentages of fAChR+ tumor cells (35-60%, mean = 48.3%; Fig. 4A and B), postchemotherapy tumor cells showed strong fAChR expression in >95% of the cells (Fig. 4D and E), with increased fAChR {gamma}-subunit mRNA levels in all biopsies (Fig. 4C and F). In addition, in vitro, single-agent, short-term treatment with rhabdomyosarcoma-directed chemotherapeutics (doxorubicine, etoposide, and cisplatinum) resulted in an increased expression of AChR {gamma}-subunit protein and mRNA in fAChRlow TE-671 rhabdomyosarcoma cells (Fig. 4G-K; confirmatory flow cytometry findings not shown). Neither agent induced fAChR expression in fAChR-negative A-204 rhabdomyosarcoma cells (not shown).



View larger version (67K):
[in this window]
[in a new window]
  		Figure 4. Effect of in vivo (A-F) and in vitro (G-K) chemotherapy on fAChR expression in rhabdomyosarcoma. A biopsy of an embryonal rhabdomyosarcoma (lane 1) taken before chemotherapy shows heterogeneous expression of fAChR on ~35% of tumor cells (A, B) and characteristic expression of AChR {alpha}- and {gamma}-subunit mRNA (arrowheads {alpha} and {gamma}, respectively, in C, lane 1). In the same patient, the residual rhabdomyosarcoma infiltrates in a biopsy after chemotherapy exhibit increased cell size and a relative increase of {gamma}-subunit compared with {alpha}-subunit mRNA (F, lane 1). Analogous effects of chemotherapy were observed in a total of six patients with either embryonal (n = 4) or alveolar (n = 2) rhabdomyosarcoma (lanes 1-6 in C and F). Doxorubicin treatment (3 days, 250 nmol/L) has a profound influence on the in vitro morphology, including an increase in cell size (G and I) and fAChR {gamma}-subunit expression (H and J) of embryonal rhabdomyosarcoma cell line TE671. There was up-regulation of fAChR {alpha}- and {gamma}-subunit mRNA (arrowheads {alpha} and {gamma}, respectively) in TE-671 cells after 3 days of in vitro treatment with cisplatinum (Cis, 140 nmol/L), etoposide (Eto, 5 µmol/L), and doxorubicin (Doxo, 250 nmol/L) compared with untreated control cells (ctr), as revealed by semiquantitative RT-PCR (K). H&E staining in (A and D); immunoperoxidase in (B and E; x400). mRNA was detected by semiquantitative RT-PCR (ethidium bromide stained 1.5% agarose gels in C, F, and K; M, molecular size marker; ref. 10). The morphology and fAChR protein expression of TE-671 cells are shown by phase contrast (G and I; x200) and immunofluorescence (H and J) microscopy (x200; anti-AchR {gamma}-subunit antibody SC-1453), respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We show that a chimeric T-cell receptor consisting of a recombinant human-derived scFv, specific for human fAChR and fused to the T-cell receptor {zeta}-chain, specifically recognizes fAChR-expressing rhabdomyosarcoma cells as indicated by short-term IFN-{gamma} secretion and granzyme B release, and cytolysis of >90% of fAChR+ rhabdomyosarcoma cells after prolonged coculture. During short-term cultures, substantial cytolysis occurred only when rhabdomyosarcoma cells expressed higher fAChR levels, either spontaneously or by transfection. Moreover, we found that fAChR expression was increased after chemotherapy in biopsies of residual tumors of rhabdomyosarcoma patients compared with the respective tumors before chemotherapy. Based on these two findings, the fully human fAChR{zeta}-transduced T cell has some potential as a future treatment option for rhabdomyosarcoma and could, in addition, be helpful in eradicating residual cells after chemotherapy.

We chose to target the fAChR {gamma}-chain because it is the first antigen shown to be overexpressed on both embryonal and alveolar rhabdomyosarcoma (8, 10). Due to its restricted expression on only a few extraocular muscle fibers and thymic myoid cells but not on innervated skeletal muscle (10), immune targeting of fAChR in patients with rhabdomyosarcoma could be tumor selective and the risk of side effects by cross-reactivity with normal tissues should be limited. This is supported by the observation that maternal autoantibodies specific for the fAChR can be present in asymptomatic mothers while causing paralysis in their developing fetuses (18).

Besides tumor specificity, the successful immunotherapeutic use of chRec-redirected T cells requires two more antigen features. First, accessible extracellular domains and, second, stable expression on all or most tumor cells. The fAChR is a transmembrane protein with an extracellular domain that is a proved target for autoantibodies (9). In addition, although primary rhabdomyosarcoma tumor biopsies (8, 10) and rhabdomyosarcoma cell lines (Fig. 2B) show variable fAChR expression, most persisting rhabdomyosarcoma cells following chemotherapy exhibit strong fAChR expression (Fig. 4; ref. 17). Thus, the use of fAChR-specific T cells as an adjuvant to conventional chemotherapy should be considered.

One potential obstacle for the successful therapeutic use of chRec-expressing T cells is the limited survival of adoptively transferred xenogeneic T cells. The antigen-binding domains of most tumor-specific chRecs described to date are derived from murine antibodies, including the recently described immunotoxin that reacts with another target specifically expressed on rhabdomyosarcoma cells (19). Human anti-mouse antibody responses mediate rapid clearance of murine antibodies from the circulation, especially after repeated administration (20), and may thus limit the life span of T cells bearing murine antibody-derived recognition domains. Furthermore, humanization of mouse antibodies by complementarity determining region grafting does not completely abrogate immunogenicity. Our observation that the killing process in vitro requires prolonged contact times between sarcoma targets and effector T cells indicates that extended in vivo survival of chimeric T cells following adoptive transfer may be important for effective treatment of rhabdomyosarcoma. In addition, whereas immunotherapies against leukemias typically follow short-term treatment protocols (21), due to the easy accessibility of tumor cells, efficient T-cell homing to solid tumors may require prolonged survival or repeated application of the chimeric T cells (19, 20). Because the chimeric T-cell receptor described here is fully human, it should show enhanced persistence in vivo.

The molecular mechanisms underlying long-term cytotoxicity of fAChR{zeta}-transduced T cells toward fAChRlow rhabdomyosarcoma cell lines remain unclear. However, the Fas/FasL system seems not to be involved: Although we found virtually no expression of FAS on TE-671 or FL-OH-1 cells by flow cytometry, substantial FAS expression occurred on Birch or Ax-OH-1 cells (not shown). Nevertheless, fAChR{zeta}-transduced T cells killed the various rhabdomyosarcoma cell lines to a similar extent (Fig. 3).

In summary, we show for the first time that fAChR{zeta}-transduced T cells are specifically activated by tumor target cells expressing fAChR, and specifically eliminate rhabdomyosarcoma cells from long-term cocultures. Furthermore, we show that fAChR expression on rhabdomyosarcoma can be increased by chemotherapeutic agents in vitro and confirm the previous observation (17) that fAChR hyperexpression is characteristic of rhabdomyosarcoma cells surviving chemotherapy in rhabdomyosarcoma patients. Therefore, our findings open new perspectives for clinical trials to evaluate fAChR{zeta}-transduced T cells for potential immunotherapy of rhabdomyosarcoma and as a tool to eradicate chemoresistant residual disease in rhabdomyosarcoma patients.


   Acknowledgments
 
Grant support: Bundesministerium für Bildung und Forschung (C. Rossig), Dr. Mildred-Scheel-Stiftung der Deutschen Krebshilfe grant 10-1859 (C. Rossig), and Wilhelm-Sander-Stiftung grant 99.112.1 (S. Gattenlöhner and A. Marx), all in Germany.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Malcolm K. Brenner and Cliona A. Rooney for helpful discussions and Erwin Schmitt and Margrit Bonengel for expert technical assistance.


   Footnotes
 
Note: S. Gattenlöhner and A. Marx share first authorship.

Received 2/18/05. Revised 9/12/05. Accepted 11/11/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Breneman JC, Lyden E, Pappo AS, et al. Prognostic factors and clinical outcomes in children and adolescents with metastatic rhabdomyosarcoma—a report from the Intergroup Rhabdomyosarcoma Study IV. J Clin Oncol 2003;21:78–84.[Abstract/Free Full Text]
  2. Walterhouse DO, Lyden ER, Breitfeld PP, Qualman SJ, Wharam MD, Meyer WH. Efficacy of topotecan and cyclophosphamide given in a phase II window trial in children with newly diagnosed metastatic rhabdomyosarcoma: a Children's Oncology Group study. J Clin Oncol 2004;22:1398–403.[Abstract/Free Full Text]
  3. Rooney CM, Smith CA, Ng CY, et al. Use of gene-modified virus-specific T lymphocytes to control Epstein-Barr-virus-related lymphoproliferation. Lancet 1995;345:9–13.[CrossRef][Medline]
  4. Dudley ME, Wunderlich JR, Robbins PF, et al. Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science 2002;298:850–4.[Abstract/Free Full Text]
  5. Dagher R, Long LM, Read EJ, et al. Pilot trial of tumor-specific peptide vaccination and continuous infusion interleukin-2 in patients with recurrent Ewing sarcoma and alveolar rhabdomyosarcoma: an inter-institute NIH study. Med Pediatr Oncol 2002;38:158–64.[CrossRef][Medline]
  6. Tanzarella S, Lionello I, Valentinis B, Russo V, Lollini PL, Traversari C. Rhabdomyosarcomas are potential target of MAGE-specific immunotherapies. Cancer Immunol Immunother 2004;53:519–24.[CrossRef][Medline]
  7. Rossig C, Brenner MK. Genetic modification of T lymphocytes for adoptive immunotherapy. Mol Ther 2004;10:5–18.[CrossRef][Medline]
  8. Gattenloehner S, Vincent A, Leuschner I, et al. The fetal form of the acetylcholine receptor distinguishes rhabdomyosarcomas from other childhood tumors. Am J Pathol 1998;152:437–44.[Abstract]
  9. Beeson D, Vincent A, Morris A, et al. cDNA and genomic clones encoding the human muscle acetylcholine receptor. Ann N Y Acad Sci 1993;681:165–7.[Medline]
  10. Gattenlohner S, Schneider C, Thamer C, et al. Expression of foetal type acetylcholine receptor is restricted to type 1 muscle fibres in human neuromuscular disorders. Brain 2002;125:1309–19.[Abstract/Free Full Text]
  11. Matthews I, Sims G, Ledwidge S, et al. Antibodies to acetylcholine receptor in parous women with myasthenia: evidence for immunization by fetal antigen. Lab Invest 2002;82:1407–17.[Medline]
  12. Whiting PJ, Vincent A, Schluep M, Newsom-Davis J. Monoclonal antibodies that distinguish between normal and denervated human acetylcholine receptor. J Neuroimmunol 1986;11:223–35.[CrossRef][Medline]
  13. Riviere I, Brose K, Mulligan RC. Effects of retroviral vector design on expression of human adenosine deaminase in murine bone marrow transplant recipients engrafted with genetically modified cells. Proc Natl Acad Sci U S A 1995;92:6733–7.[Abstract/Free Full Text]
  14. Rischer M, Pscherer S, Duwe S, Vormoor J, Jurgens H, Rossig C. Human {gamma}{delta} T cells as mediators of chimeric-receptor redirected anti-tumour immunity. Br J Haematol 2004;126:583–92.[CrossRef][Medline]
  15. Rossig C, Bollard CM, Nuchtern JG, Merchant DA, Brenner MK. Targeting of G(D2)-positive tumor cells by human T lymphocytes engineered to express chimeric T-cell receptor genes. Int J Cancer 2001;94:228–36.[CrossRef][Medline]
  16. Schuck A, Mattke AC, Schmidt B, et al. Group II rhabdomyosarcoma and rhabdomyosarcoma-like tumors: is radiotherapy necessary? J Clin Oncol 2004;22:143–9.[Abstract/Free Full Text]
  17. Kirchner T, Geuder KI, Marx A, Muller-Hermelink HK. [Nicotinic acetylcholine receptors in tumors with rhabdomyomatous differentiation. Immunohistochemical and molecular genetic demonstration]. Verh Dtsch Ges Pathol 1990;74:409–14.[Medline]
  18. Vincent A, Newland C, Brueton L, et al. Arthrogryposis multiplex congenita with maternal autoantibodies specific for a fetal antigen. Lancet 1995;346:24–5.[CrossRef][Medline]
  19. Onda M, Wang QC, Guo HF, Cheung NK, Pastan I. In vitro and in vivo cytotoxic activities of recombinant immunotoxin 8H9(Fv)-PE38 against breast cancer, osteosarcoma, and neuroblastoma. Cancer Res 2004;64:1419–24.[Abstract/Free Full Text]
  20. Khazaeli MB, Conry RM, LoBuglio AF. Human immune response to monoclonal antibodies. J Immunother 1994;15:42–52.
  21. Kreitman RJ, Wilson WH, Bergeron K, et al. Efficacy of the anti-CD22 recombinant immunotoxin BL22 in chemotherapy- resistant hairy-cell leukemia. N Engl J Med 2001;345:241–7.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 INT J SURG PATHOLHome page
Bin Chang, Li Juan Pang, Yan Qi, Chun Xia Liu, Ying Cao, Hong An Li, Wen Hao Hu, Jin Fang Jiang, Wen Jie Zhang, and Feng Li
PAX--FKHR Fusion Genes and AChR-{gamma} in Chinese Patients With Rhabdomyosarcoma: Diagnosis Using Formalin-Fixed Archival Tissues
International Journal of Surgical Pathology, February 1, 2009; 17(1): 6 - 15.
[Abstract] [PDF]

Home page
 NEJMHome page
S. Gattenlohner, E.-B. Brocker, and H.-K. Muller-Hermelink
Malignant Melanoma with Metastatic Rhabdomyosarcomatoid Transdifferentiation
N. Engl. J. Med., February 7, 2008; 358(6): 649 - 650.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gattenlöhner, S.
Right arrow Articles by Rossig, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gattenlöhner, S.
Right arrow Articles by Rossig, C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online