Research Papers:

Generation and evaluation of antibody agents for molecular imaging of CD44v6-expressing cancers

Anna-Karin Haylock, Johan Nilvebrant _, Anja Mortensen, Irina Velikyan, Marika Nestor and Ronny Falk

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Oncotarget. 2017; 8:65152-65170. https://doi.org/10.18632/oncotarget.17996

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Anna-Karin Haylock1,2, Johan Nilvebrant3, Anja Mortensen2, Irina Velikyan4, Marika Nestor2,* and Ronny Falk5,*

1Department of Surgical Sciences, Uppsala University, Uppsala, Sweden

2Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden

3Division of Protein Technology, School of Biotechnology, Royal Institute of Technology, Stockholm, Sweden

4Department of Medicinal Chemistry, Uppsala University, Uppsala, Sweden

5Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden

*These authors shared senior authorship

Correspondence to:

Johan Nilvebrant, email: [email protected]

Marika Nestor, email: [email protected]

Keywords: scFv, recombinant antibody formats, CD44v6, squamous cell carcinoma, molecular imaging

Received: December 21, 2016    Accepted: April 26, 2017    Published: May 18, 2017


Aim: The aim of this study was to generate and characterize scFv antibodies directed to human CD44v6, as well as to radiolabel and evaluate top candidates in vitro and in vivo for their potential use in CD44v6-targeted molecular imaging in cancer patients.

Materials and methods: Phage display selections were used to isolate CD44v6-specific scFvs. A chain shuffling strategy was employed for affinity maturation based on a set of CD44v6-specific first-generation clones. Two second-generation scFv clones were then chosen for labeling with 111In or 125I and assessed for CD44v6-specific binding on cultured tumor cells. In vivo uptake and distribution was evaluated in tumor-bearing mice using a dual tumor model. Finally, a proof-of-concept small animal PET-CT study was performed on one of the candidates labeled with 124I.

Results: Two affinity-matured clones, CD44v6-scFv-A11 and CD44v6-scFv-H12, displayed promising binding kinetics. Seven out of eight radiolabeled conjugates demonstrated CD44v6-specific binding. In vivo studies on selected candidates demonstrated very advantageous tumor-to-organ ratios, in particular for iodinated conjugates, where 125I-labeled scFvs exhibited favorable kinetics and tumor-to-blood ratios above five already at 24 hours p.i.. The small animal PET-CT study using 124I-labeled CD44v6-scFv-H12 was in line with the biodistribution data, clearly visualizing the high CD44v6-expressing tumor.

Conclusion: The single chain fragments, CD44v6-scFv-A11 and CD44v6-scFv-H12 specifically bind to CD44v6, and the radiolabeled counterparts provide high tumor-to-blood ratios and fast clearance from organs and blood. We conclude that radioiodinated CD44v6-scFv-A11 and CD44v6-scFv-H12 possess features highly suitable for stringent molecular imaging.

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