Skip to main content

Advertisement

SpringerLink
Log in

Efficient targeting of CD13 on cancer cells by the immunotoxin scFv13–ETA′ and the bispecific scFv [13xds16]

  • Original Article – Cancer Research
  • Published:
Journal of Cancer Research and Clinical Oncology Aims and scope Submit manuscript

Abstract

Purpose

Treatment of cancer using standard chemotherapy still offers a poor prognosis combined with severe side effects. Novel antibody-based therapies have been shown to overcome low efficiency and lack of selectivity by targeting cancer-associated antigens, such as aminopeptidase CD13.

Methods

We isolated a high-affinity CD13-specific single-chain fragment variable (scFv13) from a phage display library of V-genes from mice immunized with soluble antigen. An immunotoxin comprising the scFv13 and a truncated version of the exotoxin A of Pseudomonas aeruginosa (ETA′, scFv13–ETA′) and a bispecific scFv targeting CD13 and CD16 simultaneously (bsscFv[13xds16]) was generated and investigated for their therapeutic potential.

Results

Both fusion proteins bound specifically to target cells with high affinity. Furthermore, scFv13–ETA′ inhibited the proliferation of human cancer cell lines efficiently at low concentrations (IC50 values of 408 pM–7 nM) and induced apoptosis (40–85% of target cells). The bsscFv triggered dose-dependent antibody-dependent cell-mediated cytotoxicity, resulting in the lysis of up to 23.9% A2058 cells, 18.0% MDA-MB-468 cells and 19.1% HL-60 cells.

Conclusion

The provided data demonstrate potent therapeutic activity of the scFv13–ETA′ and the bsscFv[13xds16]. The CD13-specific scFv is therefore suitable for the direct and specific delivery of both cytotoxic agents and effector cells to cancer-derived cells, making it ideal for further therapeutic evaluation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Alewine C, Hassan R, Pastan I (2015) Advances in anticancer immunotoxin therapy. Oncologist 20:176–185. doi:10.1634/theoncologist.2014-0358

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Asano R, Hagiwara Y, Koyama N, Masakari Y, Orimo R, Arai K, Ogata H, Furumoto S, Umetsu M, Kumagai I (2013) Multimerization of anti-(epidermal growth factor receptor) IgG fragments induces an antitumor effect: the case for humanized 528 scFv multimers. FEBS J 280:4816–4826. doi:10.1111/febs.12451

    Article  CAS  PubMed  Google Scholar 

  • Bargou R, Leo E, Zugmaier G, Klinger M, Goebeler M, Knop S, Noppeney R, Viardot A, Hess G, Schuler M, Einsele H, Brandl C, Wolf A, Kirchinger P, Klappers P, Schmidt M, Riethmuller G, Reinhardt C, Baeuerle PA, Kufer P (2008) Tumor regression in cancer patients by very low doses of a T cell-engaging antibody. Science (New York, NY) 321:974–977. doi:10.1126/science.1158545

    Article  CAS  Google Scholar 

  • 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 66:1572–1579

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bauvois B, Dauzonne D (2006) Aminopeptidase-N/CD13 (EC 3.4.11.2) inhibitors: chemistry, biological evaluations, and therapeutic prospects. Med Res Rev 26:88–130. doi:10.1002/med.20044

    Article  CAS  PubMed  Google Scholar 

  • Benedict CA, MacKrell AJ, Anderson WF (1997) Determination of the binding affinity of an anti-CD34 single-chain antibody using a novel, flow cytometry based assay. J Immunol Methods 201:223–231

    Article  CAS  PubMed  Google Scholar 

  • Bruenke J, Fischer B, Barbin K, Schreiter K, Wachter Y, Mahr K, Titgemeyer F, Niederweis M, Peipp M, Zunino SJ, Repp R, Valerius T, Fey GH (2004) A recombinant bispecific single-chain Fv antibody against HLA class II and FcgammaRIII (CD16) triggers effective lysis of lymphoma cells. Br J Haematol 125:167–179. doi:10.1111/j.1365-2141.2004.04893.x

    Article  CAS  PubMed  Google Scholar 

  • Bruenke J, Barbin K, Kunert S, Lang P, Pfeiffer M, Stieglmaier K, Niethammer D, Stockmeyer B, Peipp M, Repp R, Valerius T, Fey GH (2005) Effective lysis of lymphoma cells with a stabilised bispecific single-chain Fv antibody against CD19 and FcgammaRIII (CD16). Br J Haematol 130:218–228. doi:10.1111/j.1365-2141.2005.05414.x

    Article  CAS  PubMed  Google Scholar 

  • Costello RT, Fauriat C, Sivori S, Marcenaro E, Olive D (2004) NK cells: innate immunity against hematological malignancies? Trends Immunol 25:328–333. doi:10.1016/j.it.2004.04.005

    Article  CAS  PubMed  Google Scholar 

  • Cremer C, Braun H, Mladenov R, Schenke L, Cong X, Jost E, Brummendorf TH, Fischer R, Carloni P, Barth S, Nachreiner T (2015) Novel angiogenin mutants with increased cytotoxicity enhance the depletion of pro-inflammatory macrophages and leukemia cells ex vivo. Cancer Immunol Immunother 64:1575–1586. doi:10.1007/s00262-015-1763-8

    Article  CAS  PubMed  Google Scholar 

  • Daeron M (1997) Fc receptor biology. Annu Rev Immunol 15:203–234. doi:10.1146/annurev.immunol.15.1.203

    Article  CAS  PubMed  Google Scholar 

  • Dondossola E, Rangel R, Guzman-Rojas L, Barbu EM, Hosoya H, St John LS, Molldrem JJ, Corti A, Sidman RL, Arap W, Pasqualini R (2013) CD13-positive bone marrow-derived myeloid cells promote angiogenesis, tumor growth, and metastasis. Proc Natl Acad Sci USA 110:20717–20722. doi:10.1073/pnas.1321139110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Durocher Y, Perret S, Kamen A (2002) High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res 30:E9

    Article  PubMed  PubMed Central  Google Scholar 

  • Haraguchi N, Ishii H, Mimori K, Tanaka F, Ohkuma M, Kim HM, Akita H, Takiuchi D, Hatano H, Nagano H, Barnard GF, Doki Y, Mori M (2010) CD13 is a therapeutic target in human liver cancer stem cells. J Clin Invest 120:3326–3339. doi:10.1172/jci42550

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hollevoet K, Mason-Osann E, Muller F, Pastan I (2015) Methylation-associated partial down-regulation of mesothelin causes resistance to anti-mesothelin immunotoxins in a pancreatic cancer cell line. PLoS ONE 10:e0122462. doi:10.1371/journal.pone.0122462

    Article  PubMed  PubMed Central  Google Scholar 

  • Hristodorov D, Mladenov R, Pardo A, Pham AT, Huhn M, Fischer R, Thepen T, Barth S (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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kellner C, Bruenke J, Stieglmaier J, Schwemmlein M, Schwenkert M, Singer H, Mentz K, Peipp M, Lang P, Oduncu F, Stockmeyer B, Fey GH (2008) A novel CD19-directed recombinant bispecific antibody derivative with enhanced immune effector functions for human leukemic cells. J Immunother (Hagerstown, Md: 1997) 31:871–884. doi:10.1097/CJI.0b013e318186c8b4

    Article  CAS  Google Scholar 

  • Krebber A, Bornhauser S, Burmester J, Honegger A, Willuda J, Bosshard HR, Pluckthun A (1997) Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J Immunol Methods 201:35–55

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mina-Osorio P (2008) The moonlighting enzyme CD13: old and new functions to target. Trends Mol Med 14:361–371. doi:10.1016/j.molmed.2008.06.003

    Article  CAS  PubMed  Google Scholar 

  • Mladenov R, Hristodorov D, Cremer C, Hein L, Kreutzer F, Stroisch T, Niesen J, Brehm H, Blume T, Brummendorf TH, Jost E, Thepen T, Fischer R, Stockmeyer B, Barth S, Stein C (2015) The Fc-alpha receptor is a new target antigen for immunotherapy of myeloid leukemia. Int J Cancer 137:2729–2738. doi:10.1002/ijc.29628

    Article  CAS  PubMed  Google Scholar 

  • Monnier PP, Vigoroux RJ, Tassew NG (2013) In vivo applications of single chain Fv (variable domain) (scFv) fragments. Antibodies 2:193–208. doi:10.3390/antib2020193

    Article  CAS  Google Scholar 

  • Niesen J, Stein C, Brehm H, Hehmann-Titt G, Fendel R, Melmer G, Fischer R, Barth S (2015) Novel EGFR-specific immunotoxins based on panitumumab and cetuximab show in vitro and ex vivo activity against different tumor entities. J Cancer Res Clin Oncol 141:2079–2095. doi:10.1007/s00432-015-1975-5

    Article  CAS  PubMed  Google Scholar 

  • Niesen J, Hehmann-Titt G, Woitok M, Fendel R, Barth S, Fischer R, Stein C (2016) A novel fully-human cytolytic fusion protein based on granzyme B shows in vitro cytotoxicity and ex vivo binding to solid tumors overexpressing the epidermal growth factor receptor. Cancer Lett 374:229–240. doi:10.1016/j.canlet.2016.02.020

    Article  CAS  PubMed  Google Scholar 

  • Nohara S, Kato K, Fujiwara D, Sakuragi N, Yanagihara K, Iwanuma Y, Kajiyama Y (2016) Aminopeptidase N (APN/CD13) as a target molecule for scirrhous gastric cancer. Clin Res Hepatol Gastroenterol 40:494–503. doi:10.1016/j.clinre.2015.11.003

    Article  CAS  PubMed  Google Scholar 

  • Pasqualini R, Koivunen E, Kain R, Lahdenranta J, Sakamoto M, Stryhn A, Ashmun RA, Shapiro LH, Arap W, Ruoslahti E (2000) Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis. Can Res 60:722–727

    CAS  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Piedfer M, Dauzonne D, Tang R, N’Guyen J, Billard C, Bauvois B (2011) Aminopeptidase-N/CD13 is a potential proapoptotic target in human myeloid tumor cells. FASEB J 25:2831–2842. doi:10.1096/fj.11-181396

    Article  CAS  PubMed  Google Scholar 

  • Qiu Y, Lv W, Xu M, Xu Y (2016) Single chain antibody fragments with pH dependent binding to FcRn enabled prolonged circulation of therapeutic peptide in vivo. J Control Release 229:37–47. doi:10.1016/j.jconrel.2016.03.017

    Article  CAS  PubMed  Google Scholar 

  • Raghavan M, Chen MY, Gastinel LN, Bjorkman PJ (1994) Investigation of the interaction between the class I MHC-related Fc receptor and its immunoglobulin G ligand. Immunity 1:303–315

    Article  CAS  PubMed  Google Scholar 

  • Schiffer S, Hansen HP, Hehmann-Titt G, Huhn M, Fischer R, Barth S, Thepen T (2013) Efficacy of an adapted granzyme B-based anti-CD30 cytolytic fusion protein against PI-9-positive classical Hodgkin lymphoma cells in a murine model. Blood Cancer J 3:e106. doi:10.1038/bcj.2013.4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schubert I, Kellner C, Stein C, Kugler M, Schwenkert M, Saul D, Stockmeyer B, Berens C, Oduncu FS, Mackensen A, Fey GH (2012) A recombinant triplebody with specificity for CD19 and HLA-DR mediates preferential binding to antigen double-positive cells by dual-targeting. MAbs 4:45–56. doi:10.4161/mabs.4.1.18498

    Article  PubMed  PubMed Central  Google Scholar 

  • Schubert I, Saul D, Nowecki S, Mackensen A, Fey GH, Oduncu FS (2014) A dual-targeting triplebody mediates preferential redirected lysis of antigen double-positive over single-positive leukemic cells. MAbs 6:286–296. doi:10.4161/mabs.26768

    Article  PubMed  Google Scholar 

  • Schwenkert M, Birkholz K, Schwemmlein M, Kellner C, Kugler M, Peipp M, Nettelbeck DM, Schuler-Thurner B, Schaft N, Dorrie J, Ferrone S, Kampgen E, Fey GH (2008) A single chain immunotoxin, targeting the melanoma-associated chondroitin sulfate proteoglycan, is a potent inducer of apoptosis in cultured human melanoma cells. Melanoma Res 18:73–84. doi:10.1097/CMR.0b013e3282f7c8f9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Scott AM, Wolchok JD, Old LJ (2012) Antibody therapy of cancer. Nat Rev Cancer 12:278–287. doi:10.1038/nrc3236

    Article  CAS  PubMed  Google Scholar 

  • Sheng W, Shang Y, Li L, Zhen Y (2014) An EGFR/CD13 bispecific fusion protein and its enediyne-energized analog show potent antitumor activity. Anticancer Drugs 25:82–91. doi:10.1097/cad.0000000000000029

    Article  CAS  PubMed  Google Scholar 

  • Singer H, Kellner C, Lanig H, Aigner M, Stockmeyer B, Oduncu F, Schwemmlein M, Stein C, Mentz K, Mackensen A, Fey GH (2010) Effective elimination of acute myeloid leukemic cells by recombinant bispecific antibody derivatives directed against CD33 and CD16. J Immunother (Hagerstown, Md: 1997) 33:599–608. doi:10.1097/CJI.0b013e3181dda225

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Stein C, Kellner C, Kugler M, Reiff N, Mentz K, Schwenkert M, Stockmeyer B, Mackensen A, Fey GH (2010) Novel conjugates of single-chain Fv antibody fragments specific for stem cell antigen CD123 mediate potent death of acute myeloid leukaemia cells. Br J Haematol 148:879–889. doi:10.1111/j.1365-2141.2009.08033.x

    Article  CAS  PubMed  Google Scholar 

  • Stewart BW, Wild CP (2014) World Cancer Report 2014. International Agency for Research on Cancer, Lyon

    Google Scholar 

  • 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 57:85–92

    Article  CAS  PubMed  Google Scholar 

  • Tur MK, Huhn M, Thepen T, Stocker M, Krohn R, Vogel S, Jost E, Osieka R, van de Winkel JG, Fischer R, Finnern R, Barth S (2003) Recombinant CD64-specific single chain immunotoxin exhibits specific cytotoxicity against acute myeloid leukemia cells. Can Res 63:8414–8419

    CAS  Google Scholar 

  • Tur MK, Huhn M, Jost E, Thepen T, Brummendorf TH, Barth S (2011) In vivo efficacy of the recombinant anti-CD64 immunotoxin H22(scFv)–ETA′ in a human acute myeloid leukemia xenograft tumor model. Int J Cancer 129:1277–1282. doi:10.1002/ijc.25766

    Article  CAS  PubMed  Google Scholar 

  • Wang WW, Das D, Tang XL, Budzynski W, Suresh MR (2005) Antigen targeting to dendritic cells with bispecific antibodies. J Immunol Methods 306:80–92. doi:10.1016/j.jim.2005.07.023

    Article  CAS  PubMed  Google Scholar 

  • 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 11:25–38

    PubMed  Google Scholar 

  • Wels W, Moritz D, Schmidt M, Jeschke M, Hynes NE, Groner B (1995) Biotechnological and gene therapeutic strategies in cancer treatment. Gene 159:73–80

    Article  CAS  PubMed  Google Scholar 

  • Wickstrom M, Larsson R, Nygren P, Gullbo J (2011) Aminopeptidase N (CD13) as a target for cancer chemotherapy. Cancer Sci 102:501–508. doi:10.1111/j.1349-7006.2010.01826.x

    Article  PubMed  Google Scholar 

  • Yang F, Wen W, Qin W (2016) Bispecific antibodies as a development platform for new concepts and treatment strategies. Int J Mol Sci. doi:10.3390/ijms18010048

    Google Scholar 

  • Zhang Q, Wang J, Zhang H, Zhao D, Zhang Z, Zhang S (2015) Expression and clinical significance of aminopeptidase N/CD13 in non-small cell lung cancer. J Cancer Res Ther 11:223–228. doi:10.4103/0973-1482.138007

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Reinhard Rosinke, Severin Schmies, Kai Fuhrmann, Anh-Tuan Pham and Nicole Kündgen-Redding (Fraunhofer IME, Aachen) for technical support and Dr. Richard M Twyman for critically reading the manuscript. The plasmids pAK100 and pAK400 were kindly provided through a material transfer agreement (Krebber et al. 1997). Mira Woitok was supported by the RWTH Aachen University scholarship of Young Researchers at RWTH Aachen University (RFwN). BHK-21 and CD16+ BHK-21 cells were kindly provided by Dr. Christian Kellner (Christian-Albrechts-University Kiel). This work was funded by the Fraunhofer MAVO-project MultiNaBeL.

Author information

Authors and Affiliations

  1. Department of Immunotherapy, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstr. 6, 52074, Aachen, Germany

    Elena Grieger, Gerrit Gresch, Judith Niesen, Mira Woitok, Stefan Barth, Rainer Fischer, Rolf Fendel & Christoph Stein

  2. Institute for Applied Medical Engineering, University Hospital RWTH Aachen, Pauwelsstr. 20, 52074, Aachen, Germany

    Elena Grieger, Gerrit Gresch, Stefan Barth & Christoph Stein

  3. Department of Integrative Biomedical Sciences, Faculty of Health Sciences, University of Cape Town, Anzio Road Observatory, Cape Town, 7925, South Africa

    Stefan Barth

Authors
  1. Elena Grieger
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gerrit Gresch
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Judith Niesen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mira Woitok
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Stefan Barth
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Rainer Fischer
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Rolf Fendel
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Christoph Stein
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Elena Grieger.

Ethics declarations

Funding

Mira Woitok was supported by the RWTH Aachen University scholarship of Young Researchers at RWTH Aachen University (RFwN).

Conflict of interest

All authors declare that they have no conflicts of interest.

Ethical approval

In accordance with the Helsinki Declaration of 1964 and its later amendments, primary blood samples were obtained during routine clinical practice at the University Hospital Aachen after receiving informed consent and with the approval of the Clinical Research Ethics Board of the University of Aachen. The experimental use of mice was approved by the responsible local authorities and all European guidelines for the protection of laboratory animals were followed.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Grieger, E., Gresch, G., Niesen, J. et al. Efficient targeting of CD13 on cancer cells by the immunotoxin scFv13–ETA′ and the bispecific scFv [13xds16]. J Cancer Res Clin Oncol 143, 2159–2170 (2017). https://doi.org/10.1007/s00432-017-2468-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00432-017-2468-5

Keywords

Search

Navigation