Immuno-PET and Targeted α-Therapy Using Anti-Glypican-1 Antibody Labeled with 89Zr or 211At: A Theranostic Approach for Pancreatic Ductal Adenocarcinoma

Abstract

Glypican-1 (GPC1) is overexpressed in several solid cancers and is associated with tumor progression, whereas its expression is low in normal tissues. This study aimed to evaluate the potential of an anti-GPC1 monoclonal antibody (GPC1 mAb) labeled with ⁸⁹Zr or ²¹¹At as a theranostic target in pancreatic ductal adenocarcinoma. Methods: GPC1 mAb clone 01a033 was labeled with ⁸⁹Zr or ²¹¹At with a deferoxamine or decaborane linker, respectively. The internalization ability of GPC1 mAb was evaluated by fluorescence conjugation using a confocal microscope. PANC-1 xenograft mice (n = 6) were intravenously administered [⁸⁹Zr]GPC1 mAb (0.91 ± 0.10 MBq), and PET/CT scanning was performed for 7 d. Uptake specificity was confirmed through a comparative study using GPC1-positive (BxPC-3) and GPC1-negative (BxPC-3 GPC1-knockout) xenografts (each n = 3) and a blocking study. DNA double-strand breaks were evaluated using the γH2AX antibody. The antitumor effect was evaluated by administering [²¹¹At]GPC1 mAb (∼100 kBq) to PANC-1 xenograft mice (n = 10). Results: GPC1 mAb clone 01a033 showed increased internalization ratios over time. One day after administration, a high accumulation of [⁸⁹Zr]GPC1 mAb was observed in the PANC-1 xenograft (SUV_max, 3.85 ± 0.10), which gradually decreased until day 7 (SUV_max, 2.16 ± 0.30). The uptake in the BxPC-3 xenograft was significantly higher than in the BxPC-3 GPC1-knockout xenograft (SUV_max, 4.66 ± 0.40 and 2.36 ± 0.36, respectively; P = 0.05). The uptake was significantly inhibited in the blocking group compared with the nonblocking group (percentage injected dose per gram, 7.3 ± 1.3 and 12.4 ± 3.0, respectively; P = 0.05). DNA double-strand breaks were observed by adding 150 kBq of [²¹¹At]GPC1 and were significantly suppressed by the internalization inhibitor (dynasore), suggesting a substantial contribution of the internalization ability to the antitumor effect. Tumor growth suppression was observed in PANC-1 mice after the administration of [²¹¹At]GPC1 mAb. Internalization inhibitors (prochlorperazine) significantly inhibited the therapeutic effect of [²¹¹At]GPC1 mAb, suggesting an essential role in targeted α-therapy. Conclusion: [⁸⁹Zr]GPC1 mAb PET showed high tumoral uptake in the early phase after administration, and targeted α-therapy using [²¹¹At]GPC1 mAb showed tumor growth suppression. GPC1 is a promising target for future applications for the precise diagnosis of pancreatic ductal adenocarcinoma and GPC1-targeted theranostics.

Keywords: astatine; glypican-1; immuno-PET; pancreatic ductal adenocarcinoma; targeted α-therapy; theranostics; zirconium.

Graphical abstract

FIGURE 1.

Evaluation of internalization ability in PANC-1 cells using imaging analysis. Changes in internalization ratios of mouse GPC1 mAb are shown for clone 01a033 (A) and clone 1-12 (B). White scale bars indicate 50 μm in upper row and 10 μm in lower row. Graph shows average intracellular internalization rates, with error bars indicating SD. **P < 0.01. ***P < 0.001. ns = not significant.

FIGURE 2.

(A) [⁸⁹Zr]GPC1 mAb PET images (clone 01a033) in PANC-1 xenograft mice (arrows indicate PANC-1 xenografts). (B) Quantitative analyses of [⁸⁹Zr]GPC1 mAb PET (clone 01a033) in PANC-1 xenograft mice. Uptake in spleen at 1 h could not be evaluated because of spillover from high uptake in liver. (C) Biodistribution of [⁸⁹Zr]GPC1 PET (clone 01a033) in PANC-1 xenograft mice (day 7).

FIGURE 3.

(A) [⁸⁹Zr]GPC1 mAb PET images (clone T2) in BxPC-3 and BxPC-3 GPC1-knockout xenograft mice (arrows indicate tumor xenografts). (B) Quantitative analyses of tumoral uptake on [⁸⁹Zr]GPC1 mAb PET (clone T2). (C) Biodistribution of [⁸⁹Zr]GPC1 mAb clone T2 24 h after administration. *P = 0.05 using Mann–Whitney U test. KO = knockout.

FIGURE 4.

(A) Comparison of [⁸⁹Zr]GPC1 mAb PET/CT images (clone T2) with and without injection of nonradiolabeled GPC1 mAb (300 μg) before injection of [⁸⁹Zr]GPC1 mAb clone T2 in PANC-1 xenograft mice (arrows indicate PANC-1 xenografts). (B) Comparison of tumoral uptake on [⁸⁹Zr]GPC1 mAb PET (clone T2) and biodistribution. (C) Whole-body distribution of [⁸⁹Zr]GPC1 mAb clone T2 24 h after administration. *P = 0.05 compared with blocking group.

FIGURE 5.

(A and B) Evaluation of DNA double-strand breaks with and without inhibitors (dynasore). White scale bars indicate 100 μm, n = 3. **P < 0.01. ***P < 0.001. (C) Tumor growth curves after administration of [²¹¹At]GPC1 mAb or nonradiolabeled mAb (left, clone 01a033; right, clone 1-12). (D) Tumor growth curves and changes in body weight after administration of [²¹¹At]GPC1 mAb or nonradiolabeled mAb clone 01a033 with or without administering endocytosis inhibitor. PCZ = prochlorperazine.

FIGURE 6.

(A) GPC1 immunostaining in PANC-1 xenograft (left, low magnification, ×200; right, high magnification, ×400). (B) Negative controls of corresponding sections without adding primary GPC1 mAb.