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Tumor-Targeting Antibody–Anticalin Fusion Proteins for in Vivo Pretargeting Applications

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Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Wolfgang-Pauli-Strasse 10, CH-8093 Zürich, Switzerland
*Tel: +41-44-6337401. E-mail: [email protected]
Cite this: Bioconjugate Chem. 2013, 24, 2, 234–241
Publication Date (Web):January 27, 2013
Copyright © 2013 American Chemical Society

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Pretargeting approaches rely on the injection of bispecific antibodies capable of recognizing both an accessible disease marker and a small ligand, which is typically administered at a later stage and which serves as delivery vehicle for a payload for imaging or therapy applications. In the oncology field, pretargeting strategies have exhibited extremely promising biodistribution results and in vivo selectivity, but have often relied on the cumbersome preparation of multispecific antibodies by chemical conjugation techniques. Here, we describe the design, production, and characterization of a novel class of bispecific multivalent antibody products, which contain both antibody fragments and an anticalin moiety for the simultaneous recognition of tumor-associated antigens and a small organic molecule. Anticalins are derivatives of the naturally occurring binding proteins lipocalins, which have been engineered to recognize a target molecule with high affinity. In particular, we produced and compared in vitro and in vivo different fusion proteins, which contained the anticalin FluA that selectively recognizes various different fluorescein derivatives and the F8 antibody specific to the alternatively spliced EDA domain of fibronectin (a marker of tumor angiogenesis). The selective accumulation of the most promising fusion-protein scFv(F8)-FluA-scFv(F8) on solid tumors and simultaneous binding of fluorescein derivatives could be visualized in vivo using a fluorescein-near-infrared fluorescent dye conjugate, confirming the potential of antibody-anticalin fusion proteins for pretargeting applications.

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SDS-PAGE, size exclusion chromatography, gel filtration, synthesis of compounds, and tumor imaging. This material is available free of charge via the Internet at

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Cited By

This article is cited by 16 publications.

  1. Jennifer R. DeRosa, Brandon S. Moyer, Ellie Lumen, Aaron J. Wolfe, Meegan B. Sleeper, Anthony H. Bianchi, Ashleigh Crawford, Connor McGuigan, Danique Wortel, Cheyanne Fisher, Kelsey J. Moody, Adam R. Blanden. RPtag as an Orally Bioavailable, Hyperstable Epitope Tag and Generalizable Protein Binding Scaffold. Biochemistry 2018, 57 (21) , 3036-3049.
  2. Madduri Srinivasarao and Philip S. Low . Ligand-Targeted Drug Delivery. Chemical Reviews 2017, 117 (19) , 12133-12164.
  3. Hye-Yeong Kim, Xiaolei Wang, Brendon Wahlberg, and W. Barry Edwards . Discovery of Hapten-Specific scFv from a Phage Display Library and Applications for HER2-Positive Tumor Imaging. Bioconjugate Chemistry 2014, 25 (7) , 1311-1322.
  4. Friedrich-Christian Deuschle, Elena Ilyukhina, Arne Skerra. Anticalin® proteins: from bench to bedside. Expert Opinion on Biological Therapy 2020, 10 , 1-10.
  5. Sophia Hober, Sarah Lindbo, Johan Nilvebrant. Bispecific applications of non-immunoglobulin scaffold binders. Methods 2019, 154 , 143-152.
  6. Mathias Kahl, Florian Settele, Paul Knick, Ulrich Haupts, Eva Bosse-Doenecke. Mabfilin and Fabfilin - New antibody-scaffold fusion formats for multispecific targeting concepts. Protein Expression and Purification 2018, 149 , 51-65.
  7. Asim Azhar, Ejaj Ahmad, Qamar Zia, Mohd. Ahmar Rauf, Mohammad Owais, Ghulam Md Ashraf. Recent advances in the development of novel protein scaffolds based therapeutics. International Journal of Biological Macromolecules 2017, 102 , 630-641.
  8. Xiaowen Yu, Yu-Ping Yang, Emre Dikici, Sapna K. Deo, Sylvia Daunert. Beyond Antibodies as Binding Partners: The Role of Antibody Mimetics in Bioanalysis. Annual Review of Analytical Chemistry 2017, 10 (1) , 293-320.
  9. Qi Yang, Christina L. Parker, Justin D. McCallen, Samuel K. Lai. Addressing challenges of heterogeneous tumor treatment through bispecific protein-mediated pretargeted drug delivery. Journal of Controlled Release 2015, 220 , 715-726.
  10. Rodrigo Vazquez-Lombardi, Tri Giang Phan, Carsten Zimmermann, David Lowe, Lutz Jermutus, Daniel Christ. Challenges and opportunities for non-antibody scaffold drugs. Drug Discovery Today 2015, 20 (10) , 1271-1283.
  11. Michaela Gebauer, Arne Skerra. Alternative Protein Scaffolds as Novel Biotherapeutics. 2015, 221-268.
  12. Kip P. Conner, Brooke M. Rock, Gayle K. Kwon, Joseph P. Balthasar, Lubna Abuqayyas, Larry C. Wienkers, Dan A. Rock. Evaluation of Near Infrared Fluorescent Labeling of Monoclonal Antibodies as a Tool for Tissue Distribution . Drug Metabolism and Disposition 2014, 42 (11) , 1906-1913.
  13. Shubhra Chaturvedi, Anil Kumar Mishra. Vectors for the delivery of radiopharmaceuticals in cancer therapeutics. Therapeutic Delivery 2014, 5 (8) , 893-912.
  14. Verena Strassberger, Katrin L. Gutbrodt, Nikolaus Krall, Christoph Roesli, Hitoshi Takizawa, Markus G. Manz, Tim Fugmann, Dario Neri. A comprehensive surface proteome analysis of myeloid leukemia cell lines for therapeutic antibody development. Journal of Proteomics 2014, 99 , 138-151.
  15. A. Richter, E. Eggenstein, A. Skerra. Anticalins: Exploiting a non-Ig scaffold with hypervariable loops for the engineering of binding proteins. FEBS Letters 2014, 588 (2) , 213-218.
  16. Benjamin J. Hackel. Alternative Protein Scaffolds for Molecular Imaging and Therapy. 2014, 343-364.

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