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

Prostate specific membrane antigen (PSMA) targeted microbubbles (MBs) were developed using bioorthogonal chemistry. Streptavidin-labeled MBs were treated with a biotinylated tetrazine (MB_Tz) 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 MB_Tz, 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 MB_Tz 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.