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

Visual 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 89 Zr or 211 At as a theranostic target in pancreatic ductal adenocarcinoma.Methods: GPC1 mAb clone 01a033 was labeled with 89 Zr or 211 At 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 5 6) were intravenously administered [ 89 Zr]GPC1 mAb (0.91 6 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 5 3) and a blocking study.DNA double-strand breaks were evaluated using the gH2AX antibody.The antitumor effect was evaluated by administering [ 211 At]GPC1 mAb (100 kBq) to PANC-1 xenograft mice (n 5 10).Results: GPC1 mAb clone 01a033 showed increased internalization ratios over time.One day after administration, a high accumulation of [ 89 Zr]GPC1 mAb was observed in the PANC-1 xenograft (SUV max , 3.856 0.10), which gradually decreased until day 7 (SUV max , 2.16 6 0.30).The uptake in the BxPC-3 xenograft was significantly higher than in the BxPC-3 GPC1-knockout xenograft (SUV max , 4.66 6 0.40 and 2.36 6 0.36, respectively; P 5 0.05).The uptake was significantly inhibited in the blocking group compared with the nonblocking group (percentage injected dose per gram, 7.3 6 1.3 and 12.4 6 3.0, respectively; P 5 0.05).DNA double-strand breaks were observed by adding 150 kBq of [ 211 At]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 [ 211 At]GPC1 mAb.Internalization inhibitors (prochlorperazine) significantly inhibited the therapeutic effect of [ 211 At]GPC1 mAb, suggesting an essential role in targeted a-therapy.Conclusion: [ 89 Zr]GPC1 mAb PET showed high tumoral uptake in the early phase after administration, and targeted a-therapy using [ 211 At]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.
Pancr eatic ductal adenocarcinoma (PDAC) is one of the most refractory cancers worldwide.Despite the development of many anticancer drugs, the 5-y survival rate remains lower than 10% (1).Detection of PDAC is sometimes difficult because abnormalities are not shown in CT images for small tumors (2).In addition, some PDACs are not [ 18 F]FDG-avid and cannot be detected using conventional [ 18 F]FDG PET (3).Therefore, new precise imaging techniques for the early detection of PDAC, as well as the development of new therapies in combination with theranostic approaches, are needed.
Glypicans are a family of heparan sulfate proteoglycans that play diverse roles in growth factor signaling, cell adhesion, and differentiation (4).Among the 6 known glypicans, glypican-1 (GPC1) has gained significant attention because of its aberrant expression in several types of cancer, including pancreatic, breast, and uterine cervical cancers, as well as esophageal squamous cell carcinoma and cholangiocarcinoma (5)(6)(7)(8)(9).GPC1 has also been implicated in cancer cell proliferation, invasion, and metastasis, but its expression is low in normal human tissues (10).High expression of GPC1 is reportedly a poor prognostic factor in esophageal cancer, pancreatic cancer, cholangiocarcinoma, and glioblastoma (7,8,11,12).In pancreatic cancer, GPC1 expression is observed not only in cancer cells but also in cancer-associated fibroblasts of the stroma (13).As a potential therapeutic target, an anti-GPC1 monoclonal antibody (GPC1 mAb) drug conjugate has been developed and has shown excellent preclinical in vivo antitumor effects in pancreatic cancer models (7,13).Therefore, GPC1 is a promising target for cancer diagnosis and treatment.
Immuno-PET has emerged as a promising noninvasive imaging modality that uses radiolabeled antibodies (14).It allows targeted detection and quantification of specific molecular targets expressed in cancer cells with high sensitivity and specificity.Among the various radioisotopes available for immuno-PET, 89 Zr has gained significant attention because of its favorable properties, such as its long physical half-life (78.4 h), labeling capability with deferoxamine, and derivative chelators (15). 89Zr-labeled antibodies have been investigated in preclinical and clinical studies for imaging various types of cancer (15).However, their availability for PDACs remains limited, possibly because of the abundant stroma in PDAC, which prevents the delivery of the antibody to the target; thus, there is a need for the development of new 89 Zr-labeled antibodies with higher sensitivity (16).
In recent years, emphasis has been placed not only on diagnostic imaging but also on the development of radionuclide therapy as a theranostic agent.Targeted a-therapy is gaining increasing attention, because it can deliver greater therapeutic efficacy than can conventional b-emitters (17).Among a-emitting radionuclides, 211 At is attracting attention because it can be produced by irradiating natural bismuth targets, which are abundant in nature, with a-beams using a 30-MeV cyclotron (18).In this study, the potential of a GPC1 mAb labeled with 89 Zr or 211 At as a theranostic target for PDACs was evaluated using a xenograft model.
[ 89 Zr]GPC1 mAb was obtained with a radiochemical yield of 17% on average and more than 90% radiochemical purity via radio-thin-layer chromatography analysis by purifying the reaction solution using a PD-10 column and 5 mg/mL gentisic acid in 0.25 M sodium acetate buffer (pH 5.5).

Evaluation of the Internalization Ability of GPC1 mAb
An internalization assay was performed using GPC1 mAbs with fluorescence conjugation in PANC-1 cells in the presence of dynasore.Dynasore is a drug that noncompetitively inhibits the guanosine triphosphatase activity of Dynamin1, a guanosine triphosphatase protein responsible for membrane fission during endocytosis.Detailed methods are described in the supplemental materials.
Male nude mice and female nonobese diabetic/severe combined immunodeficiency mice were purchased from Japan SLC Inc. and Charles River Japan Inc., respectively.Tumor xenograft models were established by subcutaneous injection of PANC-1 (1 3 10 7 cells in nude mice) or BxPC-3 (5 3 10 6 cells in nonobese diabetic/severe combined immunodeficiency mice) suspended in phosphate-buffered saline (0.1 mL) and Matrigel (1:1; BD Biosciences).The mice were assessed 3 wk after implantation when the tumor size reached approximately 1 cm in diameter.The protocol was approved by the Animal Care and Use Committee of the Osaka University Graduate School of Medicine (approval number 30-088-009).Euthanasia was performed under deep anesthesia by isoflurane inhalation when signs of intolerable suffering or a significant decrease in body weight were observed.
[ 89 Zr]GPC1 mAb PET Imaging and Analysis [ 89 Zr]GPC1 mAb (0.91 6 0.10 MBq, 27 6 3.0 mg of mouse GPC1 mAb, clone 01a033) was intravenously administered to PANC-1 xenograft mice (9 wk old; body weight, 23.4 6 0.9 g; n 5 6).PET/CT scanning was performed 1 h, 1 d, 2 d, 4 d, and 7 d after administration under isoflurane anesthesia using a small-animal PET scanner (Siemens Inveon PET/CT).After the PET scan, the mice were euthanized, and the radioactivity and weight of the major organs were determined using a well counter (BeWell; Molecular Imaging Laboratory).A comparison of tumoral uptake between GPC1-positive and GPC1-negative xenografts was performed to confirm the specificity of [ 89 Zr]GPC1 mAb accumulation for GPC1.All PET data were reconstructed using 3-dimensional ordered-subset expectation maximization (16 subsets and 2 iterations), followed by maximum a posteriori estimation with scatter and attenuation correction.The regional uptake of radioactivity was decay-corrected to the injection time and expressed as SUV.Ellipsoid sphere regions of interest were manually placed on the tumor, heart, lungs, liver, kidneys, spleen, and bone joints using PMOD (version 3.6; PMOD Technologies).
Targeted a-Therapy Using [ 211 At]GPC1 mAb DNA double-strand breaks were evaluated using the gH2AX antibody as previously described (20).PANC-1 cells were seeded in 35-mm glassbased dishes and treated with 100 mM dynasore for 30 min or left untreated.[ 211 At]GPC1 mAb (149.58 kBq) was added to these cells, which were then incubated for 120 min.After washing, Hoechst 33342 was added, and the cells were incubated for 10 min.The cells were washed and fixed with 4% paraformaldehyde phosphate buffer and then   ).Tumor size (mm 3 ) and body weight (g) were monitored to evaluate treatment effects and systemic side effects.The therapeutic effect of internalization inhibition was also evaluated by administering the endocytosis inhibitor prochlorperazine (21).Detailed methods for immunohistochemistry are described in the supplemental materials.

Statistical Analyses
Comparisons between the 2 groups were performed using the Mann-Whitney U test or t test in SPSS (version 25.0;IBM Corp.).Differences were considered statistically significant if the P value was less than 0.05.

RESULTS
The internalization ability of GPC1 mAb is shown in Figure 1 and Supplemental Figure 1.GPC1 mAb clone 01a033 showed  increased internalization ratios over time, whereas GPC1 mAb clone 1-12 showed no significant internalization (Fig. 1).A significant blocking effect of dynasore on internalization was observed for GPC1 mAb clone 01a033 (Supplemental Figs.1A-1C).Antibodies with high internalization abilities are suitable for targeted a-therapy because of their increased probability of interacting with DNA in the nucleus.Therefore, clone 01a033 was used for subsequent PET and a-therapy experiments.PET images of PANC-1 xenograft mice after the administration of [ 89 Zr]GPC1 mAb clone 01a033 are shown in Figure 2A.A high accumulation of [ 89 Zr]GPC1 mAb was observed in the tumor xenograft 1 d after administration (SUV max , 3.85 6 0.10), and this level then gradually decreased until day 7 (SUV max , 2.16 6 0.30; Figs.2A and  2B).Relatively high accumulation was observed in the liver, spleen, and bone (epiphysis) and was considered physiologic accumulation of GPC1 mAb or 89 Zr.The whole-body distribution of [ 89 Zr]GPC1 mAb clone 01a033 is shown in Figure 2C.High uptake was observed in the liver (16.7 6 2.9 percentage injected dose [%ID]/g), spleen (17.7 6 4.3 %ID/g), and PANC-1 tumor (8.2 6 1.0 %ID/g), suggesting that the liver and spleen could be at-risk organs considering the residence time.Accumulation in the spleen was considered physiologic accumulation of IgG rather than GPC1-mediated accumulation.
DNA double-strand breaks were observed in PANC-1 cells by adding 150 kBq of [ 211 At]GPC1 mAb (Figs.5A  and 5B).Double-strand break induction was 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 xenograft mice after the administration of [ 211 At]GPC1 mAb clone 01a033 (antibodies with internalization ability) compared with nonradiolabeled GPC1 mAb clone 01a033 (Fig. 5C).However, [ 211 At]GPC1 mAb clone 1-12 (antibodies without internalization ability) showed no treatment effect compared with the nonradiolabeled GPC1 mAb clone 1-12.Internalization inhibitors (prochlorperazine) significantly inhibited the therapeutic effect of [ 211 At]GPC1 mAb clone 01a033 (Fig. 5D).There was no significant change in body weight after the administration of each mAb (Fig. 5D).

DISCUSSION
In this study, the whole-body distribution of [ 89 Zr]GPC1 mAb was evaluated using a xenograft model, and the therapeutic effect of [ 211 At]GPC1 mAb with internalization ability was assessed.
[ 89 Zr]GPC1 mAb demonstrated high tumoral uptake via PET as early as day 1, which gradually decreased until day 7. Through a comparative study using GPC1-negative tumors and a blocking study, the in vivo specificity of [ 89 Zr]GPC1 mAb against GPC1 was confirmed, which was compatible with the high expression of GPC1 in the PANC-1 xenografts.
Immuno-PET using full antibodies labeled with 89 Zr typically requires a long time to reach maximum uptake.Previous studies using [ 89 Zr]trastuzumab demonstrated that it took more than 3-5 d to achieve maximum uptake on PET in both xenograft models and patients with cancer (22)(23).Considering the physical half-life of 211 At (7.2 h), it is more suitable than 225 Ac for the labeling of GPC1 mAb, which has a longer half-life of 10 d.In addition, the low expression of GPC1 in normal organs is a favorable characteristic for targeted a-therapy to reduce the side effects caused by off-target accumulation.
The GPC1 mAb used in this study possesses an internalization ability, which may contribute to its high uptake in the early phase.Moreover, this study demonstrated that the internalization ability is essential for inducing therapeutic effects in the targeted a-therapy of [ 211 At]GPC1 mAb (Fig. 5).Given that the range of a-radiation is as short as 100 mM, it is more efficient to irradiate from the inside of the cell to increase the likelihood of targeting the DNA within the nucleus.
[ 18 F]FDG PET is commonly used for staging and detecting recurrence in patients with PDAC.However, in some PDACs, [ 18 F]FDG PET may show only faint or no abnormal uptake at the primary site or in metastatic lesions (3).[ 18 F]FAPI-74 PET is more useful than [ 18 F]FDG PET in detecting the metastatic lesion in PDAC, but caution is required, because it also shows high accumulation in pancreatitis (3).Although further translational research is needed to prove its clinical potential, [ 89 Zr]GPC1 mAb PET may be used for the precise staging or early detection of PDAC.Furthermore, GPC1-targeted mAb drug conjugates have been developed and have shown great potential for the effective treatment of refractory PDAC and esophageal cancer (5,11,13).
[ 89 Zr]GPC1 mAb may serve as a companion diagnostic modality for selecting suitable patients before administering a therapeutic GPC1 mAb drug conjugate.Although we previously detected the expression of GPC1 in the liver metastasis of PDAC ( 5), the expression levels of the target molecule may exhibit heterogeneity among individual metastatic lesions within the same patient.Therefore, whole-body evaluation using [ 89 Zr]GPC1 mAb PET is an ideal method compared with biopsy from a single lesion or blood biomarker, because the former can assess only a portion of 1 lesion and the latter can provide averaged information irrespective of the overall tumor burden.
In a comparison between a BxPC-3 and a BxPC-3 GPC1knockout tumor, mild accumulation was observed 24 h after the administration of [ 89 Zr]GPC1 mAb.This can be attributed to the enhanced permeability and retention effect, wherein antibodies tend to accumulate nonspecifically in tumor regions with increased vascular permeability.In addition, it is possible that the accumulation of [ 89 Zr]GPC1 mAb was observed in mouse-derived, cancerassociated fibroblasts present in the stroma of BxPC-3 GPC1knockout tumors because of the expression of GPC1 (Supplemental Fig. 3).The uptake of [ 89 Zr]GPC1 mAb PET was more than 2-fold higher in the BxPC-3 xenograft than in the BxPC-3 GPC1-knockout model, suggesting that specific uptake is mediated by GPC1.

211
At is a promising a-emitter because of its production capability using natural bismuth targets with a cyclotron.A clinical trial has been initiated using [ 211 At]NaAt for refractory thyroid cancer at Osaka University Hospital (NCT05275946).This trial is expected to be expanded to 211 At-labeled compounds and antibodies in the near future.[ 211 At]GPC1 mAb may be a promising therapeutic as a targeted a-therapy for PDAC and other GPC1-expressing tumors.For clinical application, it is necessary to confirm the safety of GPC1 mAb in humans and ensure its stable supply.We are aiming for clinical companion diagnostic use of [ 89 Zr]GPC1 mAb PET ahead of [ 211 At]GPC1 mAb in the future clinical trial of the GPC1 mAb drug conjugate.
This study had certain limitations.First, the number of animals in each experiment was limited, and further confirmation of reproducibility is necessary.Second, the biodistribution and potential side effects of [ 211 At]GPC1 mAb were not fully evaluated.Future studies should conduct a detailed evaluation, including histologic analysis of the organs at risk, such as the liver and spleen.Third, the chelator-to-antibody ratio of the deferoxamine is still under investigation and should be clarified for future research.Lastly, the tumor xenograft model used in this study exhibited limited stromal formation, which differs from cancer lesions observed in patients.Considering that GPC1 expression is also observed in the stroma of PDAC, it is important to assess the accumulation of GPC1 mAbs labeled with 89 Zr or 211 At in patient-derived tumor xenograft models before proceeding to clinical translation.

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THE JOURNAL OF NUCLEAR MEDICINE Vol.64 No. 12 December 2023 CONCLUSION [ 89 Zr]GPC1 mAb PET demonstrated high tumoral uptake in the early phase after administration, indicating its feasibility as a PET probe for detecting GPC1 expression.[ 211 At]GPC1 mAb with internalization ability exhibited tumor growth suppression, highlighting the potential of a theranostic approach targeting GPC1.GPC1 mAb holds promise for future applications, from the precise diagnosis of PDAC to GPC1-targeted a-therapy.

DISCLOSURE
This study was funded by the QiSS program of JST OPERA (grant number JPMJOP1721) and JSPS KAKENHI (grant numbers 16H06278 and 20H05675).Satoshi Serada and Tetsuji Naka declare that a patent for US10077316B2 has been issued and a patent for WO2015098112A1 is pending, which is related to the GPC1 mAb.No other potential conflict of interest relevant to this article was reported.

FIGURE 1 .
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 mm in upper row and 10 mm in lower row.Graph shows average intracellular internalization rates, with error bars indicating SD. **P , 0.01.***P , 0.001.ns 5 not significant.