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Journal of Cancer Research and Clinical Oncology

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01-06-2015 | Original Article – Cancer Research

In vitro effects and ex vivo binding of an EGFR-specific immunotoxin on rhabdomyosarcoma cells

Authors: Judith Niesen, Hannes Brehm, Christoph Stein, Nina Berges, Alessa Pardo, Rainer Fischer, Andre ten Haaf, Stefan Gattenlöhner, Mehmet K. Tur, Stefan Barth

Published in: Journal of Cancer Research and Clinical Oncology | Issue 6/2015

Abstract

Purpose

Rhabdomyosarcoma (RMS) is a rare and aggressive soft tissue sarcoma with limited treatment options and a high failure rate during standard therapy. New therapeutic strategies based on targeted immunotherapy are therefore much in demand. The epidermal growth factor receptor (EGFR) has all the characteristics of an ideal target. It is overexpressed in up to 80 % of embryonal RMS and up to 50 % of alveolar RMS tumors. We therefore tested the activity of the EGFR-specific recombinant immunotoxin (IT) 425(scFv)-ETA′ against EGFR⁺ RMS cells in vitro and ex vivo.

Methods

We tested the specific binding and internalization behavior of 425(scFv)-ETA′ in RMS cell lines in vitro by flow cytometry, compared to the corresponding imaging probe 425(scFv)-SNAP monitored by live cell imaging. The cytotoxic activity of 425(scFv)-ETA′ was tested using cell viability and apoptosis assays. Specific binding of the IT was confirmed on formalin-fixed paraffin-embedded tissue samples from two RMS patients.

Results

We confirmed the specific binding of 425(scFv)-ETA′ to RMS cells in vitro and ex vivo. Both the IT and the corresponding imaging probe were rapidly internalized. The IT killed EGFR⁺ RMS cells in a dose-dependent manner, while showing no effect against control cells. It showed specific apoptotic activity against one selected RMS cell line.

Conclusions

This is the first study showing the promising therapeutic potential of a recombinant, EGFR-targeting, ETA′-based IT on RMS cells. We confirmed the selective killing with IC₅₀ values of up to 50 pM, and immunohistochemical staining confirmed the specific ex vivo binding to primary RMS material.

Abraham J et al (2011) Preclinical testing of erlotinib in a transgenic alveolar rhabdomyosarcoma mouse model. Sarcoma 2011:130484. doi:10.1155/2011/130484 CrossRefPubMedCentralPubMed

Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NB, Hamid M (2012) scFv antibody: principles and clinical application. Clin Dev Immunol 2012:980250. doi:10.1155/2012/980250 CrossRefPubMedCentralPubMed

Allen TM (2002) Ligand-targeted therapeutics in anticancer therapy. Nat Rev Cancer 2:750–763. doi:10.1038/nrc903 CrossRefPubMed

Amoury M et al (2013) SNAP-tag based agents for preclinical in vitro imaging in malignant diseases. Curr Pharm Des 19:5429–5436CrossRefPubMed

Antignani A, Fitzgerald D (2013) Immunotoxins: the role of the toxin. Toxins 5:1486–1502. doi:10.3390/toxins5081486 CrossRefPubMedCentralPubMed

Armistead PM et al (2007) Expression of receptor tyrosine kinases and apoptotic molecules in rhabdomyosarcoma: correlation with overall survival in 105 patients. Cancer 110:2293–2303. doi:10.1002/cncr.23038 CrossRefPubMed

Azemar M et al (2000) Recombinant antibody toxins specific for ErbB2 and EGF receptor inhibit the in vitro growth of human head and neck cancer cells and cause rapid tumor regression in vivo International journal of cancer. J Int Cancer 86:269–275CrossRef

Bachran D et al (2010) Epidermal growth factor receptor expression affects the efficacy of the combined application of saponin and a targeted toxin on human cervical carcinoma cells. Int J Cancer 127:1453–1461. doi:10.1002/ijc.25123 CrossRefPubMed

Barth S (2002) Technology evaluation: BL22. NCI current opinion in molecular therapeutics 4:72–75

Barth S, Huhn M, Matthey B, Klimka A, Galinski EA, Engert A (2000) Compatible-solute-supported periplasmic expression of functional recombinant proteins under stress conditions. Appl Environ Microbiol 66:1572–1579CrossRefPubMedCentralPubMed

Becker N, Benhar I (2012) Antibody-based immunotoxins for the treatment of cancer. Antibodies 1:39–69. doi:10.3390/antib1010039 CrossRef

Biberacher V et al (2012) The cytotoxicity of anti-CD22 immunotoxin is enhanced by bryostatin 1 in B-cell lymphomas through CD22 upregulation and PKC-betaII depletion. Haematologica 97:771–779. doi:10.3324/haematol.2011.049155 CrossRefPubMedCentralPubMed

Bisogno G et al (2012) Rhabdomyosarcoma in adolescents: a report from the AIEOP soft tissue sarcoma committee. Cancer 118:821–827. doi:10.1002/cncr.26355 CrossRefPubMed

Brehm H et al (2014) A CSPG4-specific immunotoxin kills rhabdomyosarcoma cells and binds to primary tumor tissues Cancer Lett. doi:10.1016/j.canlet.2014.07.006

Bruell D et al (2003) The recombinant anti-EGF receptor immunotoxin 425(scFv)-ETA′ suppresses growth of a highly metastatic pancreatic carcinoma cell line. Int J Oncol 23:1179–1186PubMed

Bruell D et al (2005) Recombinant anti-EGFR immunotoxin 425(scFv)-ETA′ demonstrates anti-tumor activity against disseminated human pancreatic cancer in nude mice. Int J Mol Med 15:305–313PubMed

Camp ER, Summy J, Bauer TW, Liu W, Gallick GE, Ellis LM (2005) Molecular mechanisms of resistance to therapies targeting the epidermal growth factor receptor. Clin Cancer Res 11:397–405PubMed

Cen L et al (2007) Phosphorylation profiles of protein kinases in alveolar and embryonal rhabdomyosarcoma. Mod Pathol 20:936–946. doi:10.1038/modpathol.3800834 CrossRefPubMed

Chandramohan V, Bigner D (2013) A novel recombinant immunotoxin-based therapy targeting wild-type and mutant EGFR improves survival in murine models of glioblastoma. OncoImmunology 2(12):e26852CrossRefPubMedCentralPubMed

Chong CR, Janne PA (2013) The quest to overcome resistance to EGFR-targeted therapies in cancer. Nat Med 19:1389–1400. doi:10.1038/nm.3388 CrossRefPubMedCentralPubMed

Cizeau J, Grenkow DM, Brown JG, Entwistle J, MacDonald GC (2009) Engineering and biological characterization of VB6-845, an anti-EpCAM immunotoxin containing a T-cell epitope-depleted variant of the plant toxin bouganin. J Immunother 32:574–584. doi:10.1097/CJI.0b013e3181a6981c CrossRefPubMed

Colcher D, Pavlinkova G, Beresford G, Booth BJ, Choudhury A, Batra SK (1998) Pharmacokinetics and biodistribution of genetically-engineered antibodies. Q J Nucl Med 42:225–241PubMed

Davicioni E et al (2009) Molecular classification of rhabdomyosarcoma–genotypic and phenotypic determinants of diagnosis: a report from the Children’s Oncology Group. Am J Pathol 174:550–564. doi:10.2353/ajpath.2009.080631 CrossRefPubMedCentralPubMed

De Giovanni C et al (1996) Antisense epidermal growth factor receptor transfection impairs the proliferative ability of human rhabdomyosarcoma cells. Cancer Res 56:3898–3901PubMed

Dittmann K, Mayer C, Fehrenbacher B, Schaller M, Kehlbach R, Rodemann HP (2010) Nuclear EGFR shuttling induced by ionizing radiation is regulated by phosphorylation at residue Thr654. FEBS Lett 584:3878–3884. doi:10.1016/j.febslet.2010.08.005 CrossRefPubMed

Ganti R et al (2006) Expression and genomic status of EGFR and ErbB-2 in alveolar and embryonal rhabdomyosarcoma. Mod Pathol 19:1213–1220. doi:10.1038/modpathol.3800636 CrossRefPubMed

Gattenlohner S et al (2010) A human recombinant autoantibody-based immunotoxin specific for the fetal acetylcholine receptor inhibits rhabdomyosarcoma growth in vitro and in a murine transplantation model. J Biomed Biotechnol 2010:187621. doi:10.1155/2010/187621 CrossRefPubMedCentralPubMed

Ghetie MA, Tucker K, Richardson J, Uhr JW, Vitetta ES (1994) Eradication of minimal disease in severe combined immunodeficient mice with disseminated Daudi lymphoma using chemotherapy and an immunotoxin cocktail. Blood 84:702–707PubMed

Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York

Hawkins DS, Gupta AA, Rudzinski ER (2014) What is new in the biology and treatment of pediatric rhabdomyosarcoma? Curr Opin Pediatr 26:50–56. doi:10.1097/MOP.0000000000000041 CrossRefPubMedCentralPubMed

Hristodorov D et al (2013) Microtubule-associated protein tau facilitates the targeted killing of proliferating cancer cells in vitro and in a xenograft mouse tumour model in vivo. Br J Cancer 109:1570–1578. doi:10.1038/bjc.2013.457 CrossRefPubMedCentralPubMed

Hussain AF, Amoury M, Barth S (2013) SNAP-tag technology: a powerful tool for site specific conjugation of therapeutic and imaging agents. Curr Pharm Des 19:5437–5442CrossRefPubMed

Jain S, Xu R, Prieto VG, Lee P (2010) Molecular classification of soft tissue sarcomas and its clinical applications Int J. Clin Exp Pathol 3:416–428

Kamat V et al (2008) Enhanced EGFR inhibition and distinct epitope recognition by EGFR antagonistic mAbs C225 and 425. Cancer Biol Ther 7:726–733CrossRefPubMed

Kampmeier F, Ribbert M, Nachreiner T, Dembski S, Beaufils F, Brecht A, Barth S (2009) Site-specific, covalent labeling of recombinant antibody fragments via fusion to an engineered version of 6-O-alkylguanine DNA alkyltransferase. Bioconjug Chem 20:1010–1015. doi:10.1021/bc9000257 CrossRefPubMed

Kampmeier F et al (2010) Rapid optical imaging of EGF receptor expression with a single-chain antibody SNAP-tag fusion protein. Eur J Nucl Med Mol Imaging 37:1926–1934. doi:10.1007/s00259-010-1482-5 CrossRefPubMed

Koefoed K et al (2011) Rational identification of an optimal antibody mixture for targeting the epidermal growth factor receptor. mAbs 3:584–595. doi:10.4161/mabs.3.6.17955 CrossRefPubMedCentralPubMed

Kreitman RJ (2006) Immunotoxins for targeted cancer therapy. AAPS j 8:E532–E551. doi:10.1208/aapsj080363 CrossRefPubMedCentralPubMed

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMed

Mazzoleni S et al (2010) Epidermal growth factor receptor expression identifies functionally and molecularly distinct tumor-initiating cells in human glioblastoma multiforme and is required for gliomagenesis. Cancer Res 70:7500–7513. doi:10.1158/0008-5472.can-10-2353 CrossRefPubMed

Mendelsohn J (2002) Targeting the epidermal growth factor receptor for cancer therapy. J Clin Oncol 20:1s–13sPubMed

Missiaglia E et al (2012) PAX3/FOXO1 fusion gene status is the key prognostic molecular marker in rhabdomyosarcoma and significantly improves current risk stratification. J Clin Oncol 30:1670–1677. doi:10.1200/jco.2011.38.5591 CrossRefPubMed

Muller KM, Arndt KM, Strittmatter W, Pluckthun A (1998) The first constant domain (C(H)1 and C(L)) of an antibody used as heterodimerization domain for bispecific miniantibodies. FEBS Lett 422:259–264CrossRefPubMed

Murthy U, Basu A, Rodeck U, Herlyn M, Ross AH, Das M (1987) Binding of an antagonistic monoclonal antibody to an intact and fragmented EGF-receptor polypeptide. Arch Biochem Biophys 252:549–560CrossRefPubMed

Pardo A, Stocker M, Kampmeier F, Melmer G, Fischer R, Thepen T, Barth S (2012) In vivo imaging of immunotoxin treatment using Katushka-transfected A-431 cells in a murine xenograft tumour model. Cancer Immunol Immunother 61:1617–1626. doi:10.1007/s00262-012-1219-3 CrossRefPubMed

Pastan I, FitzGerald D (1991) Recombinant toxins for cancer treatment. Science 254:1173–1177CrossRefPubMed

Pastan I, Chaudhary V, FitzGerald DJ (1992) Recombinant toxins as novel therapeutic agents. Annu Rev Biochem 61:331–354. doi:10.1146/annurev.bi.61.070192.001555 CrossRefPubMed

Pastan I, Hassan R, FitzGerald DJ, Kreitman RJ (2007) Immunotoxin treatment of cancer. Annu Rev Med 58:221–237. doi:10.1146/annurev.med.58.070605.115320 CrossRefPubMed

Pedersen MW, Jacobsen HJ, Koefoed K, Hey A, Pyke C, Haurum JS, Kragh M (2010) Sym004: a novel synergistic anti-epidermal growth factor receptor antibody mixture with superior anticancer efficacy. Cancer Res 70:588–597. doi:10.1158/0008-5472.CAN-09-1417 CrossRefPubMed

Ratti M, Tomasello G (2014) Emerging combination therapies to overcome resistance in EGFR-driven tumors. Anticancer Drugs 25:127–139. doi:10.1097/cad.0000000000000035 CrossRefPubMed

Ray A, Huh WW (2012) Current state-of-the-art systemic therapy for pediatric soft tissue sarcomas. Curr Oncol Rep 14:311–319. doi:10.1007/s11912-012-0243-y CrossRefPubMed

Ricci C et al (2002) HER/erbB receptors as therapeutic targets of immunotoxins in human rhabdomyosarcoma cells. J Immunother 25:314–323CrossRefPubMed

Schiffer S et al (2013) Species-dependent functionality of the human cytolytic fusion proteins granzyme B-H22(scFv) and H22(scFv)-angiogenin in macrophages. Antibodies 2:9–18. doi:10.3390/antib2010009 CrossRef

Sequist LV, Lynch TJ (2008) EGFR tyrosine kinase inhibitors in lung cancer: an evolving story. Annu Rev Med 59:429–442. doi:10.1146/annurev.med.59.090506.202405 CrossRefPubMed

Simon-Keller K, Barth S, Vincent A, Marx A (2013) Targeting the fetal acetylcholine receptor in rhabdomyosarcoma. Expert Opin Ther Targets 17:127–138. doi:10.1517/14728222.2013.734500 CrossRefPubMed

Singh R, Samant U, Hyland S, Chaudhari PR, Wels WS, Bandyopadhyay D (2007) Target-specific cytotoxic activity of recombinant immunotoxin scFv(MUC1)-ETA on breast carcinoma cells and primary breast tumors. Mol Cancer Ther 6:562–569. doi:10.1158/1535-7163.MCT-06-0604 CrossRefPubMed

Singh R, Zhang Y, Pastan I, Kreitman RJ (2012) Synergistic antitumor activity of anti-CD25 recombinant immunotoxin LMB-2 with chemotherapy. Clin Cancer Res 18:152–160. doi:10.1158/1078-0432.ccr-11-1839 CrossRefPubMedCentralPubMed

Stahnke B et al (2008) Granzyme B-H22(scFv), a human immunotoxin targeting CD64 in acute myeloid leukemia of monocytic subtypes. Mol Cancer Ther 7:2924–2932. doi:10.1158/1535-7163.MCT-08-0554 CrossRefPubMed

Stocker M, Tur MK, Sasse S, Krussmann A, Barth S, Engert A (2003) Secretion of functional anti-CD30-angiogenin immunotoxins into the supernatant of transfected 293T-cells. Protein Expr Purif 28:211–219CrossRefPubMed

Sumegi J, Streblow R, Frayer RW, Dal Cin P, Rosenberg A, Meloni-Ehrig A, Bridge JA (2010) Recurrent t(2;2) and t(2;8) translocations in rhabdomyosarcoma without the canonical PAX-FOXO1 fuse PAX3 to members of the nuclear receptor transcriptional coactivator family. Genes Chromosom Cancer 49:224–236. doi:10.1002/gcc.20731 PubMedCentralPubMed

Tebbutt N, Pedersen MW, Johns TG (2013) Targeting the ERBB family in cancer: couples therapy. Nat Rev Cancer 13:663–673. doi:10.1038/nrc3559 CrossRefPubMed

Thorpe SJ, Turner C, Heath A, Feavers I, Vatn I, Natvig JB, Thompson KM (2003) Clonal analysis of a human antimouse antibody (HAMA) response. Scand J Immunol 57:85–92CrossRefPubMed

Tur MK et al (2003) Recombinant CD64-specific single chain immunotoxin exhibits specific cytotoxicity against acute myeloid leukemia cells. Cancer Res 63:8414–8419PubMed

Wachtel M et al (2006) Subtype and prognostic classification of rhabdomyosarcoma by immunohistochemistry. J Clin Oncol 24:816–822. doi:10.1200/jco.2005.03.4934 CrossRefPubMed

Walter D et al (2011) CD133 positive embryonal rhabdomyosarcoma stem-like cell population is enriched in rhabdospheres. PLoS ONE 6:e19506. doi:10.1371/journal.pone.0019506 CrossRefPubMedCentralPubMed

Wayne AS, Fitzgerald DJ, Kreitman RJ, Pastan I (2014) Immunotoxins for leukemia. Blood 123:2470–2477. doi:10.1182/blood-2014-01-492256 CrossRefPubMedCentralPubMed

Wei BR, Ghetie MA, Vitetta ES (2000) The combined use of an immunotoxin and a radioimmunoconjugate to treat disseminated human B-cell lymphoma in immunodeficient mice. Clin Cancer Res 6:631–642PubMed

Weidle UH, Tiefenthaler G, Schiller C, Weiss EH, Georges G, Brinkmann U (2014) Prospects of bacterial and plant protein-based immunotoxins for treatment of cancer. Cancer Genomics Proteomics 11:25–38PubMed

Weiss A, Gill J, Goldberg J, Lagmay J, Spraker-Perlman H, Venkatramani R, Reed D (2014) Advances in therapy for pediatric sarcomas. Curr Oncol Rep 16:395. doi:10.1007/s11912-014-0395-z CrossRefPubMed

Weldon JE, Pastan I (2011) A guide to taming a toxin–recombinant immunotoxins constructed from Pseudomonas exotoxin A for the treatment of cancer. FEBS J 278:4683–4700. doi:10.1111/j.1742-4658.2011.08182.x CrossRefPubMedCentralPubMed

Wels W et al (2004) Recombinant immunotoxins and retargeted killer cells: employing engineered antibody fragments for tumor-specific targeting of cytotoxic effectors. Cancer Immunol Immunother 53:217–226. doi:10.1007/s00262-003-0482-8 CrossRefPubMed

Williamson D et al (2010) Fusion gene-negative alveolar rhabdomyosarcoma is clinically and molecularly indistinguishable from embryonal rhabdomyosarcoma. J Clin Oncol 28:2151–2158. doi:10.1200/jco.2009.26.3814 CrossRefPubMed

Yamamoto Y et al (2013) Cetuximab promotes anticancer drug toxicity in rhabdomyosarcomas with EGFR amplification in vitro. Oncol Rep 30:1081–1086. doi:10.3892/or.2013.2588 PubMed

Yewale C, Baradia D, Vhora I, Patil S, Misra A (2013) Epidermal growth factor receptor targeting in cancer: a review of trends and strategies. Biomaterials 34:8690–8707. doi:10.1016/j.biomaterials.2013.07.100 CrossRefPubMed

Yip WL, Weyergang A, Berg K, Tonnesen HH, Selbo PK (2007) Targeted delivery and enhanced cytotoxicity of cetuximab-saporin by photochemical internalization in EGFR-positive cancer cells. Mol Pharm 4:241–251. doi:10.1021/mp060105u CrossRefPubMed

Yokota T, Milenic DE, Whitlow M, Schlom J (1992) Rapid tumor penetration of a single-chain Fv and comparison with other immunoglobulin forms. Cancer Res 52:3402–3408PubMed

Yun CH et al (2008) The T790 M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proc Natl Acad Sci USA 105:2070–2075. doi:10.1073/pnas.0709662105 CrossRefPubMedCentralPubMed

Zanola A, Rossi S, Faggi F, Monti E, Fanzani A (2012) Rhabdomyosarcomas: an overview on the experimental animal models. J Cell Mol Med 16:1377–1391. doi:10.1111/j.1582-4934.2011.01518.x CrossRefPubMedCentralPubMed

Title: In vitro effects and ex vivo binding of an EGFR-specific immunotoxin on rhabdomyosarcoma cells
Authors: Judith Niesen
Hannes Brehm
Christoph Stein
Nina Berges
Alessa Pardo
Rainer Fischer
Andre ten Haaf
Stefan Gattenlöhner
Mehmet K. Tur
Stefan Barth
Publication date: 01-06-2015
Publisher: Springer Berlin Heidelberg
Published in: Journal of Cancer Research and Clinical Oncology / Issue 6/2015
Print ISSN: 0171-5216
Electronic ISSN: 1432-1335
DOI: https://doi.org/10.1007/s00432-014-1884-z

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