Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors

Abstract

A significant challenge for solid tumor treatment is ensuring that a sufficient concentration of therapeutic agent is delivered to the tumor site at doses that can be tolerated by the patient. Biomolecular targeting can bias accumulation in tumors by taking advantage of specific interactions with receptors overexpressed on cancerous cells. However, while antibody-based immunoconjugates show high binding to specific cells, their low dissociation constants ( K) and large Stokes radii hinder their ability to penetrate deep into tumor tissue, leading to incomplete cell killing and tumor recurrence. To address this, we demonstrate the design and production of a photo-cross-linkable affibody that can form a covalent bond to epidermal growth factor receptor (EGFR) under near UV irradiation. Twelve cysteine mutations were created of an EGFR affibody and conjugated with maleimide-benzophenone. Of these only one exhibited photoconjugation to EGFR, as demonstrated by SDS-PAGE and Western blot. Next this modified affibody was shown to not only bind EGFR expressing cells but also show enhanced retention in a 3D tumor spheroid model, with minimal loss up to 24 h as compared to either unmodified EGFR-binding affibodies or nonbinding, photo-cross-linkable affibodies. Finally, in order to show utility of photo-cross-linking at clinically relevant wavelengths, upconverting nanoparticles (UCNPs) were synthesized that could convert 980 nm light to UV and blue light. In the presence of UCNPs, both direct photoconjugation to EGFR and enhanced retention in tumor spheroids could be obtained using near-infrared illumination. Thus, the photoactive affibodies developed here may be utilized as a platform technology for engineering new therapy conjugates that can penetrate deep into tumor tissue and be retained long enough for effective tumor therapy.

Effect of covalent photoconjugation of affibodies to epidermal growth factor receptor (EGFR) on cellular quiescence

“…After 6 h, ERK activity was reduced by 30% in cells crosslinked to N23BP (Figure 4b) when compared with the untreated control, which correlated well with the cell growth inhibition data shown in Figure 1. For samples treated with uncrosslinkable proteins, an initial reduction in ERK was observed that reversed within 5 h. We hypothesize that the initial reduction in ERK in these samples was caused by the strong binding of these biomolecules to EGFR (WT: 7 nM, N23BP: 22 nM and Cetuximab: 0.4 nM) (Brasino et al, 2018;García-foncillas et al, 2019). Notably, at earlier timepoints the stronger-binding Cetuximab appeared to block ERK activity more effectively than the more weakly-binding WT and N23BP.…”

“…The photo-cross-linkable affibody N23BP was synthesized as explained in Brasino et al (2018). Briefly, an EGFR binding WT affibody Z EGFR:1907 in a pET21b+ vector with a 6XHis Tag at the C-terminus, was procured from Integrated DNA Technologies (IDT) which was then modified to contain a cysteine using a Q5 site-directed mutagenesis kit (New England Biolabs [NEB]), leading to the construction of the N23C plasmid.…”

“…Recently, we introduced a method to photo-cross-link EGFR binding affibodies to cell-expressed EGFR by using a benzophenonemodified, engineered affibody (N23BP) (Brasino et al, 2018). Upon UV irradiation, the affibodies were covalently conjugated to cellexpressed EGFR and remained expressed at the surface of EGFRpositive cancer cells for at least 24 h both in two-dimensional cells and three-dimensional spheroid models (Brasino et al, 2018).…”

“…Recently, we introduced a method to photo-cross-link EGFR binding affibodies to cell-expressed EGFR by using a benzophenonemodified, engineered affibody (N23BP) (Brasino et al, 2018). Upon UV irradiation, the affibodies were covalently conjugated to cellexpressed EGFR and remained expressed at the surface of EGFRpositive cancer cells for at least 24 h both in two-dimensional cells and three-dimensional spheroid models (Brasino et al, 2018). In comparison, wild-type affibodies were only retained within cells for only 2-4 h. We found that covalently photo-cross-linking affibodies increased their retention time within cells because the crosslinked affibodies could bypass the endolytic pathway, as confirmed by confocal and flow cytometry studies (Roy et al, 2020).…”

Light control of RTK activity: from technology development to translational research

“… 14 In the second one, RTK is activated optochemically using semi-genetically encoded RTK chimeras in which dimerization or conformational changes are put under the control of photocaged small molecules. 15 , 16 Other optochemical techniques include photocaging of amino acid residues in the kinase domain 17 and photocaging of RTK activators like DNA aptamers, 7 RTK inhibition with light-activatable anti-RTK antibodies (photobodies) 18 , 19 and RTK degradation with an opto-PROTAC (proteolysis targeting chimera) technique. 20 …”