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).
Highlights
The epidermal growth factor receptor (EGFR, ErbB1, HER1) is a member of the ErbB type I receptor tyrosine kinase (RTK) family
The binding moiety and valency toward Epidermal growth factor receptor (EGFR) was addressed by choosing a previously described scFv and a Designed Ankyrin Repeat Protein (DARPin), which were fused to an IgG1 Fc antibody domain
This study suggests that extremely high binding affinities of protein vehicles like DARPin E01 (KD = 0.5 nM) are disadvantageous for the ubiquitously expressed tumor marker EGFR in an appropriate xenograft model [9]
Summary
The epidermal growth factor receptor (EGFR, ErbB1, HER1) is a member of the ErbB type I receptor tyrosine kinase (RTK) family. Binding of a soluble ligand to the ectodomain of the EGFR induces homo- or heterodimerization, which triggers activation and internalization [1,2,3]. Lysosomal targeting or receptor recycling are the two main destinations upon EGFR internalization [4]. The. EGFR is the most studied RTK due to its general role in signal transduction related to cell proliferation and migration as well as its association with oncogenesis [4,6]. EGFR is an overexpressed and validated target in various cancers such as colorectal, gynecological and urological cancers, squamous cell carcinomas (SSC) of the head and neck, non-small cell lung cancer, renal and breast neoplasms and gliomas [7]
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