Abstract 111: In search of a lead fluorescent tracer for PSCA-expressing prostate cancer: A comprehensive analysis of imaging agents and dyes
Authors: Jiang Ziyue Karen , Wu Anna , Reiter Robert 
Published: May 2015

Abstract
Prostate stem cell antigen (PSCA) is over-expressed on the surface of many prostate cancer cells especially in castration-resistant and metastatic lesions. Thus, it is of great interest to develop PSCA-targeted imaging probes for cancer detection. To this end our group has generated anti-PSCA antibody fragments including minibodies and diabodies that can be fluorescently labeled via either site-specific or non-specific conjugations. We here report a detailed optimization of such labeling protocols and propose a lead imaging probe for PSCA+ prostate tumors. Male nude mice were bilaterally implanted with PSCA over-expressing and parental PSCAlow 22rv1 cells. All probes were injected intravenously and imaging was performed at 6 (diabody) or 48 (minibody) hours post injection.
Anti-PSCA A2 diabody contains a cysteine tag in the linker region that is suitable for site-specific conjugation via maleimide chemistry. Two fluorescent dyes with maleimide moiety were tested: a far-red dye Cy5 (ex: 650 nm; em: 670 nm) and a near infrared IRDye800CW (ex: 774 nm; em: 789 nm). Our results revealed that although having a better tissue penetration rate, A2-800CW was mainly trapped in the kidney potentially due to the renal clearance of diabodies and thus failed to achieve PSCA-specific cancer imaging; whereas A2-Cy5 was able to delineate the PSCA-over-expressing tumor after skin removal (PSCA + to - tumor ratio: 1.58 ± 0.10). In vivo imaging in live animals was not feasible due to the interference of autofluorescence with Cy5 even though the mice were kept on an alfalfa-free diet. Such discrepancy in the behavior of the two probes might be attributed to the differential physical properties of the dyes, which could lead to altered 3-D structure and PSCA-binding affinity of the diabody after conjugation.
Minibodies are larger molecules than diabodies and thus their binding affinity to PSCA is likely more resilient to non-specific labeling. We here used Cy5, IRDye800CW and an FDA-approved near infrared dye 4XPEG-ICG (ex: 774 nm; em: 805 nm) to label an anti-PSCA minibody A11. All three dyes were targeted to amines that are present mainly in lysine residues via NHS ester chemistry. Corroborating the diabody results, only the two near infrared dyes but not Cy5 permitted in vivo imaging in live animals. Further, A11-ICG enabled distinct tumor detection by achieving a PSCA + to - tumor ratio of 2.09 ± 0.37. However, heightened background in the liver and draining lymph nodes was observed. On the other hand, A11-800CW generated very impressive tumor contrasts with the average ratio of PSCA + to - tumors being 2.73 ±0.71.
Overall, PSCA-targeted diabodies and minibodies are versatile imaging tools that can be linked to various fluorescent dyes. However, A11 minibody conjugated to IRDye800CW appeared to have reached the highest contrast in imaging and thus is a better candidate for translational imaging trials.
Citation Format: Ziyue Karen Jiang, Anna Wu, Robert Reiter. In search of a lead fluorescent tracer for PSCA-expressing prostate cancer: A comprehensive analysis of imaging agents and dyes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 111. doi:10.1158/1538-7445.AM2014-111