An affinity matured minibody for PET imaging of prostate stem cell antigen (PSCA)-expressing tumors.

PURPOSE: Prostate stem cell antigen (PSCA), a cell surface glycoprotein expressed in normal human prostate and bladder, is over-expressed in the majority of localized prostate cancer and most bone metastases. We have previously shown that the hu1G8 minibody, a humanized anti-PSCA antibody fragment (single-chain Fv-C(H)3 dimer, 80 kDa), can localize specifically and image PSCA-expressing xenografts at 21 h post-injection. However, the humanization and antibody fragment reformatting decreased its apparent affinity. Here, we sought to evaluate PET imaging contrast with affinity matured minibodies. METHODS: Yeast scFv display, involving four rounds of selection, was used to generate the three affinity matured antibody fragments (A2, A11, and C5) that were reformatted into minibodies. These three affinity matured anti-PSCA minibodies were characterized in vitro, and following radiolabeling with (124)I were evaluated in vivo for microPET imaging of PSCA-expressing tumors. RESULTS: The A2, A11, and C5 minibody variants all demonstrated improved affinity compared to the parental (P) minibody and were ranked as follows: A2 > A11 > C5 > P. The (124)I-labeled A11 minibody demonstrated higher immunoreactivity than the parental minibody and also achieved the best microPET imaging contrast in two xenograft models, LAPC-9 (prostate cancer) and Capan-1 (pancreatic cancer), when evaluated in vivo. CONCLUSION: Of the affinity variant minibodies tested, the A11 minibody that ranked second in affinity was selected as the best immunoPET tracer to image PSCA-expressing xenografts. This candidate is currently under development for evaluation in a pilot clinical imaging study.

Human prostate sphere-forming cells represent a subset of basal epithelial cells capable of glandular regeneration in vivo.

BACKGROUND: Prostate stem/progenitor cells function in glandular development and maintenance. They may be targets for tumor initiation, so characterization of these cells may have therapeutic implications. Cells from dissociated tissues that form spheres in vitro often represent stem/progenitor cells. A subset of human prostate cells that form prostaspheres were evaluated for self-renewal and tissue regeneration capability in the present study. METHODS: Prostaspheres were generated from 59 prostatectomy specimens. Lineage marker expression and TMPRSS-ERG status was determined via immunohistochemistry and fluorescence in situ hybridization (FISH). Subpopulations of prostate epithelial cells were isolated by cell sorting and interrogated for sphere-forming activity. Tissue regeneration potential was assessed by combining sphere-forming cells with rat urogenital sinus mesenchyme (rUGSM) subcutaneously in immunocompromised mice. RESULTS: Prostate tissue specimens were heterogeneous, containing both benign and malignant (Gleason 3-5) glands. TMPRSS-ERG fusion was found in approximately 70% of cancers examined. Prostaspheres developed from single cells at a variable rate (0.5-4%) and could be serially passaged. A basal phenotype (CD44+CD49f+CK5+p63+CK8-AR-PSA-) was observed among sphere-forming cells. Subpopulations of prostate cells expressing tumor-associated calcium signal transducer 2 (Trop2), CD44, and CD49f preferentially formed spheres. In vivo implantation of sphere-forming cells and rUGSM regenerated tubular structures containing discreet basal and luminal layers. The TMPRSS-ERG fusion was absent in prostaspheres derived from fusion-positive tumor tissue, suggesting a survival/growth advantage of benign prostate epithelial cells. CONCLUSION: Human prostate sphere-forming cells self-renew, have tissue regeneration capability, and represent a subpopulation of basal cells.

Humanized radioiodinated minibody for imaging of prostate stem cell antigen-expressing tumors.

PURPOSE: Prostate stem cell antigen (PSCA) is a cell surface glycoprotein that is overexpressed in prostate cancer, including hormone refractory disease. Previous preclinical studies showed the intact anti-PSCA antibodies, 1G8 and hu1G8, localized specifically to PSCA-expressing xenografts. Optimal micro positron emission tomography (microPET) imaging using hu1G8, however, required a delay of 168 hours postinjection. In this study, the 2B3 minibody (an 80-kDa engineered antibody fragment) has been produced for rapid targeting and imaging. EXPERIMENTAL DESIGN: A gene encoding a PSCA-specific minibody, V(L)-linker-V(H)-hinge-huIgG1 C(H)3, was assembled. The minibody was expressed by secretion from mammalian cells and purified by cation exchange chromatography. Relative affinity and specificity were determined by competition ELISA and flow cytometry. Serial microPET imaging using a 124I-labeled minibody was conducted at 4 and 21 hours in mice bearing LAPC-9 AD, LAPC-9 AI, PC-3, and LNCaP-PSCA human prostate cancer xenografts. Tumor and tissue biodistribution was determined, and region of interest analysis of the images was conducted. RESULTS: Yields of 20 mg/L purified 2B3 minibody were obtained that showed specific binding to LNCaP-PSCA cells. Purified 2B3 minibody showed specific binding to LNCaP-PSCA cells with an apparent affinity of 46 nmol/L. Radioiodinated 2B3 minibody showed rapid nontarget tissue and blood clearance kinetics (t1/2beta = 11.2 hours). MicroPET scanning using the 124I-2B3 minibody showed both androgen-dependent and -independent tumors as early as 4 hours and excellent high contrast images at 21 hours postinjection. CONCLUSIONS: Imaging PSCA-positive prostate cancer is feasible using an intermediate size antibody fragment at 21 hours.