Evaluation of Site-Specific Radioiodination of Anti-EGFR-Targeted DARPin E01 Using (4-Hydroxyphenyl)ethyl Maleimide

Non-invasive radionuclide molecular imaging of epidermal growth factor receptor (EGFR) expression can guide patient stratification for EGFR-targeted therapies. The designed ankyrin repeat protein (DARPin) E01, which binds EGFR ectodomain III with sub-nanomolar affinity, is a promising scaffold for single-photon emission computed tomography (SPECT) imaging probes. In the present study, we compared site-specific radioiodination of DARPin E01 using the bifunctional prosthetic group (4-hydroxyphenyl)ethyl maleimide (HPEM) with site-unspecific radioiodination via [¹²³I]I-para-iodobenzoate (PIB). [¹²³I]I-HPEM was conjugated to the C-terminus of DARPin E01 via Glu-Glu-Glu-Cys ([¹²³I]I-E01-E₃C-HPEM) or Gly-Gly-Gly-Cys ([¹²³I]I-E01-G₃C-HPEM) linkers. Radiolabelling yields were 6 ± 2% and 13 ± 5%, respectively. Size-exclusion purification provided radiochemical purity > 98%. Both HPEM conjugates retained nanomolar EGFR-binding affinity (K_D: 3.2 ± 0.6 and 4.8 ± 0.9 nM) and demonstrated EGFR-specific tumour accumulation in A-431 xenografts. Cellular processing was characterised by rapid binding, slow internalisation, and non-residualising behaviour of all variants. Kidney uptake was lower for the site-specifically labelled variants. However, site-specific labelling evidently elevated hepatobiliary excretion and uptake in Na/I-symporter-expressing organs compared to [¹²³I]I-(HE)₃-E01-PIB, while linker composition (E₃C vs. G₃C) did not significantly alter biodistribution. Site-unspecific radioiodination with [¹²³I]I-PIB remains the preferred approach for clinical SPECT imaging of EGFR expression with DARPin E01.