Efficient targeting of CD13 on cancer cells by the immunotoxin scFv13–ETA′ and the bispecific scFv [13xds16]
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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.
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.
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.
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.
KeywordsCD13 Single-chain variable fragment Antibody derivative Immunotoxin Bispecific scFv
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.
Compliance with ethical standards
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.
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.
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