Targeting the internal epitope of prostate-specific membrane antigen with 89Zr-7E11 immuno-PET

Abstract

The potential of the positron-emitting (89)Zr has been recently investigated for the design of radioimmunoconjugates for immuno-PET. In this study, we report the preparation and in vivo evaluation of (89)Zr-desferrioxamine B (DFO)-7E11, a novel (89)Zr-labeled monoclonal antibody (mAb) construct for targeted imaging of prostate-specific membrane antigen (PSMA), a prototypical cell surface marker highly overexpressed in prostate cancer. The ability of (89)Zr-DFO-7E11 to delineate tumor response to therapy was also investigated, because it binds to the intracellular epitope of PSMA, which becomes available only on membrane disruption in dead or dying cells.

Methods: 7E11 as a marker of dying cells was studied by flow cytometry and microscopy of cells after antiandrogen-, radio-, and chemotherapy in LNCaP and PC3 PSMA-positive cells. The in vivo behavior of (89)Zr-DFO-7E11 was characterized in mice bearing subcutaneous LNCaP (PSMA-positive) tumors by biodistribution studies and immuno-PET. The potential of assessing tumor response was evaluated in vivo after radiotherapy.

Results: In vitro studies correlated 7E11 binding with markers of apoptosis (7-amino-actinomycin-D and caspase-3). In vivo biodistribution experiments revealed high, target-specific uptake of (89)Zr-DFO-7E11 in LNCaP tumors after 24 h (20.35 ± 7.50 percentage injected dose per gram [%ID/g]), 48 h (22.82 ± 3.58 %ID/g), 96 h (36.94 ± 6.27 %ID/g), and 120 h (25.23 ± 4.82 %ID/g). Excellent image contrast was observed with immuno-PET. 7E11 uptake was statistically increased in irradiated versus control tumor as measured by immuno-PET and biodistribution studies. Binding specificity was assessed by effective blocking studies at 48 h.

Conclusion: These findings suggest that (89)Zr-DFO-7E11 displays high tumor-to-background tissue contrast in immuno-PET and can be used as a tool to monitor and quantify, with high specificity, tumor response in PSMA-positive prostate cancer.

FIGURE 1

PSMA-positive cell responses to different treatments in vitro. (A) mAb 7E11 binds to intracellular epitope of PSMA, labeling apoptotic or already dead cells, whose leaky cell membrane permits access of antibody to intracellular domain. mAb J591 recognizes extracellular domain of PSMA and thus binds to all PSMA-positive cells, regardless of their viability. Compared with control, higher percentage of 7AAD- and 7E11-stained cells is observed over time after treatment (P < 0.05 at all treatments and regimens, with exception of etoposide treatment at 24 h). Corresponding staining is observed for both 7E11 and 7AAD staining in all experiments (P > 0.05). Data are reported as percentage of labeled cells after treatment with etoposide (PC3-PSMA-positive) (B), flutamide (C), and radiation therapy (LNCap) (D).

FIGURE 2

Immunofluorescence assay of PC cells stained with DAPI, 7E11, and activated caspase-3 obtained at 96 h after treatment. (A) PC3-PSMA-positive cells treated with representative vehicle (control from etoposide study). Additional controls are in Supplemental Figure 2. (B) PC3-PSMA-positive cells after treatment with etoposide. (C) LNCaP cells receiving flutamide. (D) LNCaP cells treated with radiation therapy (×20). In all samples, colocalization of 7E11 and activated caspase-3 staining was observed.

FIGURE 3

In vivo imaging of therapy response with ⁸⁹Zr-7E11 immuno-PET in xenograft-bearing mice. (A) Representative transverse and coronal ⁸⁹Zr-7E11 immuno-PET images at different time points in LNCaP xenograft–bearing mouse treated with selective radiation to right side. Increased uptake of ⁸⁹Zr-7E11 was observed in selectively irradiated tumor (right), compared with control (left). Dashed line represents position of perpendicularly oriented image. (B) ⁸⁹Zr-7E11 uptake values (obtained from PET data as maximum %ID/g) were significantly higher in irradiated tumors than in control tumors at 24 h (P = 0.0376), 48 h (P = 0.0009), 72 h (P = 0.0086), 96 h (P = 0.01), and 120 h (P = 0.0075). Li = liver; T = tumor; Trans. = transverse.

FIGURE 4

Biodistribution study with blocking experiment. Bar chart showing selected tissue biodistribution data (%ID/g) for uptake of ⁸⁹Zr-7E11 in mice bearing LNCaP xenografts at 24, 48, 72, and 96 h. Blocking experiments with nonradiolabeled 7E11 mAb were also conducted at 48 h after injection. With exception of uptake at 24 h, all data points show statistically significant difference between irradiated and control tumors. Some uptake is observed in nonirradiated LNCaP tumors because they are PSMA-positive and experience baseline cell death.

FIGURE 5

Autoradiography of irradiated and control tumor corresponds with activated caspase-3. Uptake of ⁸⁹Zr-7E11 was observed in treated tumors, colocalizing with apoptotic or necrotic areas by H/E and caspase-3 staining as demonstrated by overlay of DAR and activated caspase-3 staining. Numbered rectangles in caspase 3 staining represent approximate localization of higher magnification images.