The effect of a dimeric Affibody molecule (ZEGFR:1907)2 targeting EGFR in combination with radiation in colon cancer cell lines

Abstract

Abstract. The epidermal growth factor receptor (EGFR) is frequently overexpressed in colorectal cancer and is therefore an attractive target for treatment. (Z EGFR:1907 ) 2 is a newly developed dimeric affibody molecule with high affinity to the extracellular part of EGFR. In this study, we evaluated the cytotoxic effects of (Z EGFR:1907 ) 2 in combination with external radiation and the possible inhibitory effects in the EGFR signalling pathways in the colon cancer cell lines HT-29 and HCT116. The effects were compared with an EGFR antibody (cetuximab) and the tyrosine kinase inhibitors (erlotinib and sunitinib). These cell lines are genotypically different with respect to e.g. KRAS and BRAF mutational status, recently shown to be of clinical significance for therapeutic effects. Both cell lines express approximately 100,000-150,000 EGFRs per cell but differ in the radiation response (HCT116, SF2=0.28 and HT-29, SF2=0.70). Exposure to (Z EGFR:1907 ) 2 produced a small, but significant, reduction in survival in HCT116 but did not affect HT-29 cells. Similar results were obtained after exposure to EGF and the EGFR antibody cetuximab. The EGFR tyrosine kinase targeting inhibitor erlotinib and the multi-tyrosine kinase inhibitor sunitinib reduced survival in both cell lines. However, none of the drugs had any significant radiosensitizing effects in combination with radiation. Akt and Erk are central proteins in the EGFR downstream signalling and in the cellular response to ionizing radiation. The activation of Akt (Ser 473) and Erk (Thr202/ Tyr204) by radiation was both dose-and time-dependent. However the activation of EGFR was not clearly affected by radiation. Neither (Z EGFR:1907 ) 2 nor any of the other drugs were able to completely inactivate Akt or Erk. On the contrary, erlotinib stimulated Akt phosphorylation in both cell lines and in HCT116 cells Erk was activated. Overall the results illustrate the complexity in response to radiation and drugs in cells with differential phenotypic status.

Comparison of radiobiological parameters for 90Y radionuclide therapy (RNT) and external beam radiotherapy (EBRT) in vitro

“…The HT29 and HCT116 cell lines were the most radioresistant and radiosensitive cell lines, respectively. This is also consistent with previous studies [38, 39]. Similar sensitivity for both cell lines was observed for 90 Y irradiation results.…”

“…For both EBRT and 90 Y, the HT29 and HCT116 cell lines were the most radioresistance and radiosensitive cell lines, respectively. HT-29 cells have the TP53 mutation which may increase the cell resistance to radiation [38]. The α / β derived from the HDR– 90 Y cell survival curve was larger than that derived from EBRT by a factor of ≈ 8 for all three cell lines.…”

The influence of AKT isoforms on radiation sensitivity and DNA repair in colon cancer cell lines

“…There are also indications that Ser473 is autophosphorylated. It has previously been shown by us and others that AKT activation is radiation dose and time dependent [27], and it is believed to be involved in the DNA repair after exposure to ionizing radiation [16, 28]. …”

Affibody-Binding Ligands

“…A conspicuous body of literature is now available on the application of affibodies in the bioanalytical and medicinal fields. For example, affibodies were used as (i) alternatives to antibodies to develop cancer therapeutics (e.g., breast [20] and colorectal [21]) or ELISA kits for quantifying human plasma proteins [22], and to functionalize ProteOn TM GLM sensor chips for detecting human epidermal growth factor receptor 2 (HER2)-binding affibody (ZHER2) and ZHER3 breast cancer markers [23], (ii) radiological tracers for in vivo medical imaging, owing to their lower circulation time, higher tissue permeation, and better imaging contrast as compared to antibodies [24], (iii) drug vectors, either in polyethylene glycol (PEG)-ylated or poly(phenylene sulfone) (PAS)-ylated forms to prevent fast kidney clearance, for radiometal-based therapy, or to decorate vesicles loaded with anti-cancer small interfering RNA (siRNA) [25], and finally, (iv) as ligands for the purification of blood factors [26] and antibodies [27] by affinity chromatography.…”

“…Sci. 2020,21, 3769 of Representative fluorescence microscope images of library beads binding red-and greenlabeled affibodies at different binding ratios.…”