Development of prostate specific membrane antigen targeted ultrasound microbubbles using bioorthogonal chemistry

Abstract

Prostate specific membrane antigen (PSMA) targeted microbubbles (MBs) were developed using bioorthogonal chemistry. Streptavidin-labeled MBs were treated with a biotinylated tetrazine (MBTz) and targeted to PSMA expressing cells using trans-cyclooctene (TCO)-functionalized anti-PSMA antibodies (TCO-anti-PSMA). The extent of MB binding to PSMA positive cells for two different targeting strategies was determined using an in vitro flow chamber. The initial approach involved pretargeting, where TCO-anti-PSMA was first incubated with PSMA expressing cells and followed by MBTz, which subsequently showed a 2.8 fold increase in the number of bound MBs compared to experiments performed in the absence of TCO-anti-PSMA. Using direct targeting, where TCO-anti-PSMA was linked to MBTz prior to initiation of the assay, a 5-fold increase in binding compared to controls was observed. The direct targeting approach was subsequently evaluated in vivo using a human xenograft tumor model and two different PSMA-targeting antibodies. The US signal enhancements observed were 1.6- and 5.9-fold greater than that for non-targeted MBs. The lead construct was also evaluated in a head-to-head study using mice bearing both PSMA positive or negative tumors in separate limbs. The human PSMA expressing tumors exhibited a 2-fold higher US signal compared to those tumors deficient in human PSMA. The results demonstrate both the feasibility of preparing PSMA-targeted MBs and the benefits of using bioorthogonal chemistry to create targeted US probes.

Six‐Transmembrane Epithelial Antigen of the Prostate‐1 (STEAP‐1)–Targeted Ultrasound Imaging Microbubble Improves Detection of Prostate Cancer In Vivo

“…In this study, the STEAP‐1–targeted SonoVue microbubble enhanced the ultrasound signal intensity remarkably compared with ordinary SonoVue microbubble. Our results are in agreement with the previous report of the PSMA target microbubble by Zlitni et al and Wang et al Therefore, STEAP‐1–targeted SonoVue microbubbles could bind with their targets expressed by tumors in the nude mouse xenograft models and enhanced the intensities of ultrasound imaging signals.…”

“…The 5‐year survival rate of PCa is around 99% in cases of early detection. When PCa has spread beyond the prostate, survival reduces significantly . Transrectal ultrasound‐guided biopsy is the gold standard for PCa diagnosis, which is generally performed in patients with elevated serum levels of prostate‐specific antigen.…”

Optimized Anti–Prostate‐Specific Membrane Antigen Single‐Chain Variable Fragment–Loaded Nanobubbles as a Novel Targeted Ultrasound Contrast Agent for the Diagnosis of Prostate Cancer

“…Prostate‐specific membrane antigen is highly expressed in prostatic intraepithelial neoplasia, hormone‐dependent PCa, non–hormone‐dependent PCa, and metastases . The PSMA antibody was developed to specifically bind to PCa cells in vivo, and some studies have shown that the application of PSMA‐targeting or other specific antigen‐targeting MBs can improve the diagnostic accuracy of US …”

“…13,14 The PSMA antibody was developed to specifically bind to PCa cells in vivo, 16,18 and some studies have shown that the application of PSMAtargeting or other specific antigen-targeting MBs can improve the diagnostic accuracy of US. [29][30][31] Although the vascular endothelial gap is expanded in some disease states, such as in tumors, only particles of less than 700 nm in diameter are allowed to pass through. 32 Targeted MBs have a wide range of clinical applications, but because they are larger than 1000 μm, it is difficult for MBs to enter tumor tissue through the vessel wall and specifically bind to tumor cells; in addition, the short in vivo halflife of MBs results in an insufficient time frame for imaging the target.…”

“…34 The goal of this work was to investigate novel PSMA-targeting NBs as a targeted UCA for the molecular imaging of PCa. Unlike previous studies of PSMAtargeting contrast agents, 12,30 we applied DPPC and biotinylated DSPE-PEG-2000 in the lipid shell of the NBs. Microbubbles made of DPPC combine with the ligand to form a dome shape with a larger binding area.…”