Bivalent EGFR-Targeting DARPin-MMAE Conjugates

  • Int J Mol Sci. 2022 Feb 23;23(5):2468. doi: 10.3390/ijms23052468.
Lennard Karsten  1 ,  Nils Janson  2 ,  Vadim Le Joncour  3 ,  Sarfaraz Alam  4 ,  Benjamin Müller  5 ,  Jayendrakishore Tanjore Ramanathan  3 ,  Pirjo Laakkonen  3  6 ,  Norbert Sewald  2 ,  Kristian M Müller  1
Affiliations
  • 1. Cellular and Molecular Biotechnology, Faculty of Technology, Bielefeld University, 33615 Bielefeld, Germany.
  • 2. Organic and Bioorganic Chemistry, Faculty of Chemistry, Bielefeld University, 33615 Bielefeld, Germany.
  • 3. Translational Cancer Medicine Research Program, Faculty of Medicine, University of Helsinki, 00290 Helsinki, Finland.
  • 4. Biochemistry III, Faculty of Chemistry, Bielefeld University, 33615 Bielefeld, Germany.
  • 5. Biofidus AG, 33615 Bielefeld, Germany.
  • 6. Laboratory Animal Center, Helsinki Institute of Life Science (HiLIFE), University of Helsinki, 00290 Helsinki, Finland.
PMID: 35269611 DOI: 10.3390/ijms23052468
Abstract

Epidermal growth factor receptor (EGFR) is a validated tumor marker overexpressed in various cancers such as squamous cell carcinoma (SSC) of the head and neck and gliomas. We constructed protein-drug conjugates based on the anti-EGFR Designed Ankyrin Repeat Protein (DARPin) E01, and compared the bivalent DARPin dimer (DD1) and a DARPin-Fc (DFc) to the monomeric DARPin (DM) and the antibody derived scFv425-Fc (scFvFc) in Cell Culture and a mouse model. The modular conjugation system, which was successfully applied for the preparation of protein-drug and -dye conjugates, uses bio-orthogonal protein-aldehyde generation by the formylglycine-generating enzyme (FGE). The generated carbonyl moiety is addressed by a bifunctional linker with a pyrazolone for a tandem Knoevenagel reaction and an azide for strain-promoted azide-alkyne cycloaddition (SPAAC). The latter reaction with a PEGylated linker containing a dibenzocyclooctyne (DBCO) for SPAAC and monomethyl Auristatin E (MMAE) as the toxin provided the stable conjugates DD1-MMAE (drug-antibody ratio, DAR = 2.0) and DFc-MMAE (DAR = 4.0) with sub-nanomolar cytotoxicity against the human squamous carcinoma derived A431 cells. In vivo imaging of Alexa Fluor 647-dye conjugates in A431-xenografted mice bearing subcutaneous tumors as the SCC model revealed unspecific binding of bivalent DARPins to the ubiquitously expressed EGFR. Tumor-targeting was verified 6 h post-injection solely for DD1 and scFvFc. The total of four administrations of 6.5 mg/kg DD1-MMAE or DFc-MMAE twice weekly did not cause any sequela in mice. MMAE conjugates showed no significant anti-tumor efficacy in vivo, but a trend towards increased necrotic areas (p = 0.2213) was observed for the DD1-MMAE (n = 5).

Keywords
DARPin; EGFR; Knoevenagel ligation; MMAE conjugates; SCC model; antibody-drug conjugates; cytotoxicity; formylglycine-generating enzyme; in vivo imaging; xenograft.
Products