Targeted elastin-like polypeptide fusion protein for near-infrared imaging of human and canine urothelial carcinoma

Abstract

Cystoscopic visualization of bladder cancer is an essential method for initial bladder cancer detection and diagnosis, transurethral resection, and monitoring for recurrence. We sought to develop a new intravesical imaging agent that is more specific and sensitive using a polypeptide based NIR (near-infrared) probe designed to detect cells bearing epidermal growth factor receptors (EGFR) that are overexpressed in 80% of urothelial carcinoma (UC) cases. The NIR imaging agent consisted of an elastin like polypeptide (ELP) fused with epidermal growth factor (EGF) and conjugated to Cy5.5 to give Cy5.5-N24-EGF as a NIR contrast agent. In addition to evaluation in human cells and tissues, the agent was tested in canine cell lines and tissue samples with naturally occurring invasive UC. Flow cytometry and confocal microscopy were used to test cell-associated fluorescence of the probe in T24 human UC cells, and in K9TCC-SH (high EGFR expression) and K9TCC-Original (low EGF expression) canine cell lines. The probe specifically engages these cells through EGFR within 15 min of incubation and reached saturation within a clinically relevant 1 h timeframe. Furthermore, ex vivo studies with resected canine and human bladder tissues showed minimal signal from normal adjacent tissue and significant NIR fluorescence labeling of tumor tissue, in good agreement with our in vitro findings. Differential expression of EGFR ex vivo was revealed by our probe and confirmed by anti-EGFR immunohistochemical staining. Taken together, our data suggests Cy5.5-ELP-EGF is a NIR probe with improved sensitivity and selectivity towards BC that shows excellent potential for clinical translation.

Unravelling the Mechanism of Elastin-like Polypeptide-Enzyme Fusion Stabilization in Organic Solvents

“…This method was shown to isolate pure ELP in a molecular weight- and charge-independent manner. The approach was also effective in purifying ELP fusion proteins with either CryS96 or epidermal growth factor domains that retained their chaperone function in vitro and epidermal growth factor receptor binding affinity in EGFR+ cells and tissues in vivo (mouse) and ex vivo (human and canine), respectively. − Purification of functional proteins, especially enzymes, via organic solvent extraction may seem highly counterintuitive since the presence of organic solvents can impact the solubility and functional properties of enzymes in a manner that leads to aggregation or loss of activity. , We sought to develop a better understanding of how ELP fusion proteins can retain their function during the organic solvent extraction–precipitation process.…”