Novel PSCA targeting scFv-fusion proteins for diagnosis and immunotherapy of prostate cancer
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Despite great progress in the diagnosis and treatment of localized prostate cancer (PCa), there remains a need for new diagnostic markers that can accurately distinguish indolent and aggressive variants. One promising approach is the antibody-based targeting of prostate stem cell antigen (PSCA), which is frequently overexpressed in PCa. Here, we show the construction of a molecular imaging probe comprising a humanized scFv fragment recognizing PSCA genetically fused to an engineered version of the human DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT), the SNAP-tag, enabling specific covalent coupling to various fluorophores for diagnosis of PCa. Furthermore, the recombinant immunotoxin (IT) PSCA(scFv)-ETA′ comprising the PSCA(scFv) and a truncated version of Pseudomonas exotoxin A (PE, ETA′) was generated.
We analyzed the specific binding and internalization behavior of the molecular imaging probe PSCA(scFv)-SNAP in vitro by flow cytometry and live cell imaging, compared to the corresponding IT PSCA(scFv)-ETA′. The cytotoxic activity of PSCA(scFv)-ETA′ was tested using cell viability assays. Specific binding was confirmed on formalin-fixed paraffin-embedded tissue specimen of early and advanced PCa.
Alexa Fluor® 647 labeling of PSCA(scFv)-SNAP confirmed selective binding to PSCA, leading to rapid internalization into the target cells. The recombinant IT PSCA(scFv)-ETA′ showed selective binding leading to internalization and efficient elimination of target cells.
Our data demonstrate, for the first time, the specific binding, internalization, and cytotoxicity of a scFv-based fusion protein targeting PSCA. Immunohistochemical staining confirmed the specific ex vivo binding to primary PCa material.
KeywordsProstate cancer Prostate stem cell antigen (PSCA) Immunotoxin (IT) Single-chain fragment variable (scFv) Pseudomonas exotoxin A (ETA′) SNAP-tag
This work was funded by the ForSaTum project, sponsored within the NRW-EU Ziel 2-Programm “Regionale Wettbewerbsfähigkeit und Beschäftigung 2007–2013” (ERFE). We would like to thank Radoslav Mladenov and Nina Berges (Department of Experimental Medicine and Immunotherapy, Institute of Applied Medical Engineering, RWTH Aachen University Clinic, Aachen, Germany) for their help with immunohistochemistry and confocal microscopy. We also thank Dr. Richard M. Twyman for support in preparation of the manuscript draft.
Compliance with ethical standards
Conflict of interest
The authors declare no conflicts of interest.
In accordance with the Helsinki Declaration of 1975, primary tissue samples were obtained during routine clinical practice at the University Hospital Giessen approved by the appropriate ethics committee.
- 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 CrossRefPubMedGoogle Scholar
- Elsasser-Beile U, Wolf P, Gierschner D, Buhler P, Schultze-Seemann W, Wetterauer U (2006) A new generation of monoclonal and recombinant antibodies against cell-adherent prostate specific membrane antigen for diagnostic and therapeutic targeting of prostate cancer. Prostate 66:1359–1370. doi: 10.1002/pros.20367 CrossRefPubMedGoogle Scholar
- Hassan R et al (2014) Phase 1 study of the antimesothelin immunotoxin SS1P in combination with pemetrexed and cisplatin for front-line therapy of pleural mesothelioma and correlation of tumor response with serum mesothelin, megakaryocyte potentiating factor, and cancer antigen 125. Cancer 120:3311–3319. doi: 10.1002/cncr.28875 CrossRefPubMedGoogle Scholar
- Hristodorov D, Nordlohne J, Mladenov R, Huhn M, Fischer R, Thepen T, Barth S (2014) Human microtubule-associated protein tau mediates targeted killing of CD30(+) lymphoma cells in vitro and inhibits tumour growth in vivo. Br J Haematol 164:251–257. doi: 10.1111/bjh.12626 CrossRefPubMedGoogle Scholar
- Hussain AF, Kampmeier F, von Felbert V, Merk HF, Tur MK, Barth S (2011) SNAP-tag technology mediates site specific conjugation of antibody fragments with a photosensitizer and improves target specific phototoxicity in tumor cells. Bioconjug Chem 22:2487–2495. doi: 10.1021/bc200304k CrossRefPubMedGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar
- Singh CK, Ojha A, Bhatanagar RK, Kachru DN (2008) Detection and characterization of recombinant DNA expressing vip3A-type insecticidal gene in GMOs-standard single, multiplex and construct-specific PCR assays. Anal Bioanal Chem 390:377–387. doi: 10.1007/s00216-007-1714-0 CrossRefPubMedGoogle Scholar
- Taylor RM, Severns V, Brown DC, Bisoffi M, Sillerud LO (2012) Prostate cancer targeting motifs: expression of alphanu beta3, neurotensin receptor 1, prostate specific membrane antigen, and prostate stem cell antigen in human prostate cancer cell lines and xenografts. Prostate 72:523–532. doi: 10.1002/pros.21454 CrossRefPubMedGoogle Scholar
- Weidle UH, Georges G, Brinkmann U (2012) Fully human targeted cytotoxic fusion proteins: new anticancer agents on the horizon. Cancer Genom Proteom 9:119–133Google 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 Genom Proteom 11:25–38Google Scholar
- Wu AM, Reiter RE, Lepin EJ, Marks JD, Zhou Y (2010) High affinity anti-prostate stem cell antigen (PSCA) antibodies for cancer targeting and detection. Google PatentsGoogle Scholar