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

Abstract

Cellular quiescence is a reversible state of cell cycle arrest whereby cells are temporarily maintained in the nondividing phase. Inducing quiescence in cancer cells by targeting growth receptors is a treatment strategy to slow cell growth in certain aggressive tumors, which in turn increases the efficacy of treatments such as surgery or systemic chemotherapy. However, ligand interactions with cell receptors induce receptor-mediated endocytosis followed by proteolytic degradation, which limits the duration of cellular quiescence. Here, we report the effects of targeted covalent affibody photoconjugation to epidermal growth factor receptors (EGFR) on EGFRpositive MDA-MB-468 breast cancer cells. First, covalently conjugating affibodies to cells increased doubling time two-fold and reduced ERK activity by 30% as compared to cells treated with an FDA-approved anti-EGFR antibody Cetuximab, which binds to EGFR noncovalently. The distribution of cells in each phase of the cell cycle was determined, and cells conjugated with the affibody demonstrated an accumulation in the G1 phase, indicative of G1 cell cycle arrest. Finally, the proliferative capacity of the cells was determined by the incorporation of 5-ethynyl-2deoxyuridine and Ki67 Elisa assay, which showed that the percentage of proliferative cells with photoconjugated affibody was half of that found for the untreated control.

Effect of cetuximab plus FOLFOX4 regimen on clinical outcomes in advanced gastric carcinoma patients receiving evidence-based care

“…The RR and DCR were significantly higher in RG compared with CG, which indicates that CET + FOLFOX4 results in a superior treatment response and clinical efficacy for aGC patients who received EBC. CET exerts its anti-tumor effect by inducing G1 cell cycle arrest in tumor cells and inhibiting vascular endothelial growth factor production, tumor-related angiogenesis, and cancer cell invasion[ 20 , 21 ]. As reported by Lordick et al [ 22 ], the CET + FOLFOX4 regimen also has a high remission rate in the treatment of metastatic GC, suggesting the ability of CET to enhance the efficacy of the FOLFOX4 regimen, which is consistent with our findings.…”

Enzyme Prodrug Therapy with Photo-Cross-Linkable Anti-EGFR Affibodies Conjugated to Upconverting Nanoparticles

“…The affibody–enzyme ACD and the enzyme cytosine deaminase (CD) alone were expressed, synthesized, purified, and modified with BP by methods reported previously. , 250 nM of purified human extracellular domain (ECD) of EGFR (Thermo Fisher, 70 kDa) was mixed with 2.5 μM of ACD, 500 μg/mL of water-soluble UCNP for 30 min in microcentrifuge tubes, after which the reaction mixture was irradiated with an 808 nm NIR laser diode (Focuslight) at 0.8 W/cm 2 driver (Arroyo) power for 10 min. The other treatment groups were: (+)­ACD, (−)­EGFR, (+)­UCNP, (+)­NIR; (−)­ACD, (+)­EGFR, (+)­UCNP, (+)­NIR; (+)­ACD, (+)­EGFR, (−)­UCNP, (+)­NIR; and (+)­ACD, (+)­EGFR, (+)­UCNP, (−)­NIR.…”

“…Previously, we reported a non-genetic method of receptor−ligand chemical conjugation in which a benzophenone-modified affibody−enzyme (ACD) fusion protein could bind to cancer cells that overexpress EGFR. 26 We demonstrated in these earlier studies that photo-cross-linking the affibody−enzyme to EGFR on live cells prevented intracellular degradation of the targeting ligands and thus retained expression of enzymatic activity for up to 96 h. 27 The retained affibody−enzymes could then convert an inactive prodrug, 5-fluorocytosine (5-FC), to an active anticancer drug, 5-fluorouracil (5-FU), in vitro, which was cytotoxic to these EGFR-expressive cancer cells. 27 Translating this technology in vivo is difficult because of the poor penetration depths of UV light through animal skin and tissues.…”

“…More recent developments for receptor targeting include using small molecules like single-chain variable fragment (scFV) and affibodies for receptor augmentation. Previously, we reported a non-genetic method of receptor–ligand chemical conjugation in which a benzophenone-modified affibody–enzyme (ACD) fusion protein could bind to cancer cells that overexpress EGFR . We demonstrated in these earlier studies that photo-cross-linking the affibody–enzyme to EGFR on live cells prevented intracellular degradation of the targeting ligands and thus retained expression of enzymatic activity for up to 96 h .…”