Theranostic application of 64Cu/177Lu-labeled anti-Trop2 monoclonal antibody in pancreatic cancer tumor models

doi:10.21203/rs.3.rs-1754367/v1

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Research Article

Theranostic application of 64Cu/177Lu-labeled anti-Trop2 monoclonal antibody in pancreatic cancer tumor models

https://doi.org/10.21203/rs.3.rs-1754367/v1

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Purpose

Pancreatic cancer is a malignant tumor with high degree of malignancy, strong heterogeneity and high lethality. Trop2 is a transmembrane glycoprotein associated with the occurrence, development and poor prognosis of pancreatic cancer. This study aims to develop ⁶⁴Cu/¹⁷⁷Lu-labeled anti-Trop2 monoclonal antibody (IMB1636) for positron emission tomography (PET) imaging and radioimmunotherapy (RIT) application in pancreatic cancer tumor models.

Methods

The binding kinetics of IMB1636 to Trop2 was measured by Biolayer interferometry (BLI). Western blotting and flow cytometry were used to screen the Trop2 expression of pancreatic cancer cell lines. IMB1636 were conjugated with p-SCN-Bn-NOTA (NOTA) and DOTA-NHS-ester (DOTA) for ⁶⁴Cu and ¹⁷⁷Lu radiolabeling respectively. ImmunoPET imaging and RIT studies were performed using ⁶⁴Cu-NOTA-IMB1636 and ¹⁷⁷Lu-DOTA-IMB1636 in subcutaneous pancreatic cancer tumor models.

Results

IMB1636 had strong binding affinity to Trop2 according to the results of BLI. T3M-4 cell line showed strongest expression of Trop2 and specific binding ability of IMB1636. The radiochemical purity of ⁶⁴Cu-NOTA-IMB1636 and ¹⁷⁷Lu-DOTA-IMB1636 exceeded 95%. PET imaging showed gradually accumulation of ⁶⁴Cu-NOTA-IMB1636 in T3M-4 tumor models. The peak tumor uptake was 8.95 ± 1.07%ID/g (n = 4) at 48 h post injection (p.i.), which had significant differences with T3M-4-blocked and PaTu8988 negative groups (P < 0.001). High dose ¹⁷⁷Lu-DOTA-IMB1636 demonstrated strongest tumor suppression with standardized tumor volume about 94.24 ± 14.62% (n = 5) at 14 days p.i., significantly smaller than other groups (P = 0.000 ~ 0.047). Ex vivo Biodistribution and histological staining verified the in vivo PET imaging and RIT results.

Conclusions

This study demonstrated that ⁶⁴Cu/¹⁷⁷Lu labeled IMB1636 could noninvasively evaluate the expression level of Trop2 and inhibit the Trop2-overexpressed tumor growth in pancreatic cancer tumor models. Further clinical evaluation and translation of Trop2-targeted drug may be of great help in the stratification and management of pancreatic cancer patients.

pancreatic cancer

Trop2

immunoPET

radioimmunotherapy (RIT)

Cu-64

Lu-177

Pancreatic cancer is a malignant tumor with high degree of malignancy, strong heterogeneity, and high lethality [1]. The latest global cancer statistics report shows that pancreatic cancer ranks 14th among all new cases of cancer and 7th among all cancer-related death [2]. The 5-year overall survival rate of pancreatic cancer is less than 5%. More than half of patients were diagnosed with distant metastases, whose 5-year survival rate is only 2.6% [3]. The stage of cancer at the time of diagnosis is an important factor affecting the survival rate of patients. Surgical resection is the most effective treatment, but 80% ~ 90% of patients are diagnosed at an advanced stage and cannot undergo surgery [4]. Existing radiotherapy and chemotherapy for pancreatic cancer have poor efficacy. Early diagnosis and treatment are still the key to improving survival rate.

Conventional imaging modalities such as ultrasonography, magnetic resonance imaging (MRI) and computed tomography (CT) are widely used in the diagnosis of cancer, but their sensitivity and specificity are limited [5]. Positron emission tomography (PET) is a highly sensitive functional imaging modality, and ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) is the most widely used radiotracer in current clinical practice [6]. However, ¹⁸F-FDG PET is no more effective than conventional methods in the early diagnosis or small metastases and can’t differentiate pancreatic cancer from pancreatitis. The demand for new highly specific and sensitive imaging methods is increasing due to the development of precision medicine. ImmunoPET imaging combined the high specificity and affinity of monoclonal antibodies (mAbs) with the high sensitivity of PET, could noninvasively visualize the expression and distribution of targeted antigens in vivo, thus changing the status that only a few patients can benefit from immunotherapy [7]. Radioimmunotherapy (RIT) based on molecular nuclear medicine has strong killing effect on the tumor site and little side effects on normal tissues, which can solve the problems of heterogeneity of tumor cell surface antigen expression, poor permeability of macromolecular mAb and repeated administration of conventional antitumor drugs [8].

The human trophoblast cell surface antigen 2 (Trop2) is a transmembrane protein with a molecular weight of 36 kDa and overexpressed in a variety of solid epithelioid cancers including pancreatic cancer [9]. The overexpression of Trop2 is related to the occurrence, progression and malignant degree of pancreatic cancer. Trop2 is a predictor of poor prognosis and may become a new biomarker of pancreatic cancer [10], but there are few researches in pancreatic cancer targeting Trop2. In this study, we used positron radionuclide ⁶⁴Cu (t_1/2 = 12.7 h) labeled humanized anti-Trop2 mAb IMB1636, which is an ideal carrier for Trop2-targeted diagnosis and therapy developed independently by our groups, for immunoPET imaging to evaluate Trop2 expression level of pancreatic cancer tumor models. RIT and single photon emission computed tomography (SPECT) imaging were performed with therapeutic radionuclide ¹⁷⁷Lu (t_1/2 =6.7 d) labeled IMB1636 to achieve the integration of diagnosis and treatment. The purpose of this study is to screen out the Trop2-overexpressed pancreatic cancer by immunoPET, and provide a novel theranostic method through RIT.

Cell culture and animal models

Pancreatic cancer cell lines T3M-4, BXPC-3, PaTu8988, MiaPaCa-2, PANC-1, AsPC-1 and immortalized human pancreatic duct-derived cell line HPNE were provided by our laboratory. AsPC-1 was cultured in RPMI 1640 medium, and other cells were cultured in DMEM medium (Gibco). Culture mediums were supplemented with 10% fetal bovine serum (Biological Industries) and 1% penicillin/streptomycin (KeyGEN BioTECH). Cell lines were incubated at 37°C in a humidified atmosphere of 5% CO₂. When the cell density was around 75%~90%, cells were used for ex vivo and in vivo experiments.

All animal studies were approved by the Institutional Animal Care and Use Committee of Beijing Friendship Hospital Capital Medical University (No. 21-2049). BALB/c nude female mice (5 weeks old), purchased from Beijing HFK Bioscience CO., LTD, were used to establish subcutaneous pancreatic cancer tumor models. Approximately 1 × 10⁷ cells were resuspended in 100 μL phosphate-buffered saline (PBS, pH 7.4, Corning) and then injected subcutaneously at the right flank and armpit of each mouse for immunoPET imaging and RIT experiments, respectively. The mice's health status and tumor volume were monitored every other day. When the tumor diameter was about 1 cm, in vivo studies were performed.

Western blotting

Proteins were collected, electrophoresed and transferred to the nitrocellulose (NC) membrane following a standard protocol [11].After being blocked with 5% skimmed milk dissolved in TBST buffer, the NC membrane was incubated with rabbit anti-human monoclonal Trop2 (Abcam) and rabbit anti-human β-actin (ZSGB-BIO) primary antibodies (1:1000) overnight at 4 °C. Then the NC membrane was incubated with goat anti-rabbit IgG H&L (HRP, ZSGB-BIO) secondary antibodies (1:1500) for 40 min at room temperature. Exposures were performed with ChemiDoc^TM MP Imaging System (Bio-RAD) using the Affinity ECL kit (ThermoFisher Scientific).

Biolayer interferometry (BLI)

The affinity between IMB1636 and Trop2 antigen (His-tag, Sino Biological) were measured by BLI using GatorTM LabelFree Bioanalysis System (GatorBio). All experiments were performed at 25 °C with shaking at 1000 rpm. Anti-human IgG Fc biosensors were preequilibrated in Q buffer, containing PBS, 0.02 % Tween-20, and 0.2 % BSA, for at least 300 s before using in experiments. IMB1636 was loaded onto anti-human IgG Fc biosensors at 10 μg/mL and flowed the Trop2 antigen at different concentrations of 1.56 nM, 3.12 nM, 6.25 nM, 12.50 nM, 25 nM for 300 s, respectively. The data analyzed using Gator™ System Analysis.

Flow cytometry

Cells were collected and resuspended in cold PBS containing 1% BSA at the concentration of 1 × 10⁶ cells/tube (200 μL). IMB1636 (10 μg/mL) was incubated with cells at 4°C for 1 h while IgG was used as a nonspecific control. Cells were washed twice with cold PBS and then incubated with FITC-labeled anti-human IgG secondary antibody (ZSGB-BIO). After washing three times, cells were analyzed using a LSRFortessa cell analyzer (BD Biosciences). Fluorescence intensities were processed by FlowJo analysis software.

Radiolabeling

Anti-Trop2 mAb IMB1636 first conjugated with p-SCN-Bn-NOTA (NOTA, Macrocyclic) and DOTA-NHS-ester (DOTA, Macrocyclic) at molar ratio about 1:20 following a standard protocol [12, 13].For ⁶⁴Cu and ¹⁷⁷Lu labeling, the purified NOTA-IMB1636 and DOTA-IMB1636 were incubated with ⁶⁴CuCl₂ and ¹⁷⁷LuCl₃ in a sodium acetate buffer (pH 4.5 ~ 5.5) at 37 °C for 1 h under constant shaking, respectively. The ratio of IMB1636 and radionuclide was not below 300 MBq/mg. ⁶⁴CuCl₂ and ¹⁷⁷LuCl₃were purchased from HTA Co., Ltd. PD-10 columns (GE Healthcare) were used to purify ⁶⁴Cu-NOTA-IMB1636 and ¹⁷⁷Lu-DOTA-IMB1636 with PBS as a mobile phase. Radiochemical purity was evaluated by radio-thin-layer chromatography (radio-TLC, BIOSCAN), using ethylene diamine tetraacetic acid (EDTA, 50 mM, pH 4.5) as the mobile phase.

ImmunoPET imaging of ⁶⁴Cu-NOTA-IMB1636

Pancreatic cancer tumor models were intravenously injected with ⁶⁴Cu-NOTA-IMB1636 (~7.4 MBq per mouse) for PET imaging. The mice of T3M-4-blocked group were injected with 20 mg/kg IMB1636 at the same time with ⁶⁴Cu-NOTA-IMB1636. The mice were anesthetized with 2% isoflurane, and small animal PET (MOLECUBES, Belgium) scans were performed for 10 min at 4 h, 24 h, and 48 h p.i., respectively. All images were reconstructed and attenuation corrected by the workstation. Quantitative region of interest (ROI) analysis of tumor, heart (blood), liver, and kidney were performed on PET images by VivoQuant software. Radioactive uptakes of tumors and organs were presented as the percentage of injected dose per gram (%ID/g).

RIT monitoring and SPECT imaging

The Trop2-positve pancreatic cancer tumor models were randomly divided into 5 groups (n = 5). The high-¹⁷⁷Lu-IMB1636 (~11.1 MBq) and low-¹⁷⁷Lu-IMB1636 (~3.7 MBq) were treatment groups, and the ¹⁷⁷Lu-only (~11.1 MBq), IMB1636-only (~200 μg), and PBS (~200 μL) were control groups according to our previous study [12].Photographing, measuring tumor size, body weighing, and in vivo SPECT imaging (MOLECUBES, Belgium) were performed at 4 h, 2 days, 4 days, 6 days, 8 days, 10 days, 12 days, and 14 days after a single-time tail vein injection. Tumor volume was calculated by 1/2 × length × wildth². The standard tumor volume and body weight were calculated by dividing the volume and weight at each time point by the initial volume and weight and then multiplying 100, represented as %. SPECT images were reconstructed and attenuation corrected by the workstation and VivoQuant software.

Ex vivo biodistribution studies of immunoPET imaging and RIT experiments

Mice were euthanized after the terminal time point of PET and SPECT imaging. Tumors and major organs including heart, blood, liver, spleen, lung, kidney, stomach, intestine, pancreas, bladder, muscle, bone, brain, and skin were all collected and weighed for biodistribution study. An automatic gamma counter (PerkinElmer) was used to measure the radioactivity counts. Biodistribution data was presented as %ID/g.

¹⁸F-FDG PET Imaging

¹⁸F-FDG PET imaging was performed at 14 days to evaluate the RIT therapeutic effect in high-¹⁷⁷Lu-IMB1636 and ¹⁷⁷Lu-only groups. The mice were kept fasting for at least 6 h before PET imaging. Each mouse was intravenously injected with ~7.4 MBq of ¹⁸F-FDG and was anesthetized with isoflurane for about 40 ~ 60 minutes before PET imaging. Quantitative analysis was performed by drawing ROI of tumor.

Biodistribution, hematological analysis and body weight monitoring of ¹⁷⁷Lu-DOTA-IMB1636 in normal mice

Normal BALB/c nude female mice (5 weeks old, Beijing HFK Bioscience CO., LTD) were euthanized for biodistribution and hematological analysis. Biodistribution was performed at 4 hours, 4 days, 7 days, 10 days, and 14 days after the injection of ¹⁷⁷Lu-DOTA-IMB1636 (~11.1 MBq, n = 3). Biodistribution procedure was the same as before. Hematological analysis was performed before and after the injection of ¹⁷⁷Lu-DOTA-IMB1636 at 4 days, 7 days, 10 days, and 14 days (~11.1 MBq, n = 3). At least 100 μL of venous blood was collected in an anticoagulant tube for whole blood cell analysis including white blood cell (WBC), red blood cell (RBC), platelet (PLT) and hemoglobin (HGB). Body weight was measured before and after the injection of ¹⁷⁷Lu-DOTA-IMB1636 at 2 days, 4 days, 6 days, 8 days,10 days, 12 days, and 14 days (~11.1 MBq, n = 3).

Histological staining

Tissue histological analysis included Hematoxylin-eosin (H&E) and immunohistochemical (IHC) staining following standard procedure [14].H&E staining was performed on tumor, heart, liver, spleen, lung, and kidney of immunoPET imaging and RIT experiments mice. IHC staining of Trop2 was performed only in tumor. IHC staining of tumor tissue was incubated with IMB1636 (10 μg/mL) as primary antibodies. The brown color represents staining level. All images were acquired using an A1R confocal microscope (Nikon).

Statistical analysis

All quantitative data were presented as mean ± standard deviation (SD). Repeated-measures ANOVA and student's t-test were used for comparisons among groups. P values less than 0.05 were considered statistically significant.

Cellular screening of Trop2 expression

Western blotting results of pancreatic cancer cell lines showed that T3M-4 and BXPC-3 had high Trop2 expression level, while PaTu8988, MiaPaCa-2, PANC-1, AsPC-1 and HPNE had low level (Fig. 1a). The semi-quantitative analysis of Trop2 divided by β-actin showed that the expression level of Trop2 in T3M-4 was higher than BXPC-3. Therefore, T3M-4 with higher Trop2 expression level and PaTu8988 with low level were selected for the evaluation of cell-antibody binding ability.

Antibodies binding kinetics

The binding kinetics of IMB1636 to Trop2 was measured by BLI. IMB1636 was loaded onto anti-human IgG Fc biosensors at 10 μg/mL and the Trop2 concentration was 1.56 nM, 3.12 nM, 6.25 nM, 12.50 nM, and 25 nM respectively. IMB1636 had strong binding affinity to Trop2 antigen according to the results of BLI. The association rate constant Kon is 6.08×10⁵ Ms^-1, the dissociation rate constant Koff is 1.59×10^-3 s^-1, and the corresponding equilibrium dissociation constant KD is 2.61×10^-9 M (Fig. 1b).

Cellular binding ability

Shown by flow cytometry, T3M-4 cells had significant signal shift after incubating with IMB1636 at a concentration of 10 μg/mL, compared to non-specific IgG, FITC-labeled secondary antibody-only, cells-only groups, and PaTu8988 cell line (Fig. 1c). The result indicated that IMB1636 had a specific binding capacity with Trop2 on the cell surface of T3M-4. We finally chose T3M-4 and PaTu8988 cell lines to construct subcutaneous positive and negative pancreatic cancer tumor models.

Radiolabeling of ⁶⁴Cu/¹⁷⁷Lu-labeled IMB1636

IMB1636 was first conjugated with NOTA and DOTA, and then labeled with ⁶⁴Cu and ¹⁷⁷Lu used for immunoPET imaging and RIT experiments respectively (Fig. 1d). The radiochemical purity of ⁶⁴Cu-NOTA-IMB1636 and ¹⁷⁷Lu-DOTA-IMB1636 exceeded 95% and the range of specific activity was about 10 ~ 20 MBq/mg.

ImmunoPET imaging and biodistribution of ⁶⁴Cu-NOTA-IMB1636

The maximum intensity projection (MIP) images showed that ⁶⁴Cu-NOTA-IMB1636 in T3M-4 positive group (n = 4) could clearly display the tumor morphology at 4 h post injection (p.i.), and the tumor radioactive uptake gradually accumulated with time. The tumor uptake of ⁶⁴Cu-NOTA-IMB1636 in T3M-4-blocked group (n=3) slightly increased, while PaTu8988 negative group (n = 4) was similar with background (Fig. 2a).

We further delineated the ROI and quantitatively analyzed the radioactive uptake of tumor, heart (blood), liver and kidney (Fig. 2b). The peak tumor uptake of ⁶⁴Cu-NOTA-IMB1636 in T3M-4 was about 8.95 ± 1.07 %ID/g at 48 h p.i., which had significant differences withT3M-4-blocked and PaTu8988 groups (3.56 ± 0.18 %ID/g, 2.08 ± 0.19 %ID/g; P < 0.001). And there was significant difference between tumor uptake of ⁶⁴Cu-NOTA-IMB1636 in T3M-4 and T3M-4-blocked groups (P = 0.003). The radioactive uptake of the heart (blood), liver and kidney gradually decreased with time. The liver uptake of ⁶⁴Cu-NOTA-IMB1636 in T3M-4 was lower than T3M-4-blocked group (P = 0.004), and there was no statical difference between T3M-4-blocked and PaTu8988 group. There were no significant differences among the three groups in heart (blood) and kidney (P >0.05).

After the terminal time point imaging, the mice were euthanized for ex vivo biodistribution analysis (Fig. 2c). The tumor uptake of ⁶⁴Cu-NOTA-IMB1636 in T3M-4, T3M-4-blocked, and PaTu8988 was 9.95 ± 0.47 %ID/g, 4.00 ± 0.52 %ID/g, and 1.45 ± 0.25 %ID/g respectively, which had significant differences among three groups (P < 0.001) and consistent with the in vivo PET imaging results. The radioactive uptake of blood, heart, spleen, and kidney in T3M-4-blocked group was higher than T3M-4 (P < 0.05), which may be caused by higher tumor uptake of ⁶⁴Cu-NOTA-IMB1636 in T3M-4 group. There were no statistical differences in lung, stomach, intestine, pancreas, bladder, muscle, bone, brain, and skin among three groups (P > 0.05).

RIT of ¹⁷⁷Lu-labeled IMB1636

The Trop2-overexpresing T3M-4 mice were photographed at 4 h, 2 days, 4 days, 6 days, 8 days, 10 days, 12 days, and 14 days after a single-time tail vein injection, which visually displayed the changes of tumor size in 5 groups (Fig. 3a). Meanwhile, tumor volume and body weight of mice were measured. High-¹⁷⁷Lu-IMB1636 group demonstrated strongest tumor suppression with standardized tumor volume about 94.24 ± 14.62 % at 14 days p.i., significantly smaller than other groups (low-¹⁷⁷Lu-IMB1636 = 172.89 ± 30.03 %, ¹⁷⁷Lu-only = 391.57 ± 70.11 %, IMB1636-only = 282.84 ± 49.98 %, and PBS = 402.41 ± 66.06 %; P = 0.000 ~ 0.047; Fig. 3b). The tumor volume of high-¹⁷⁷Lu-IMB1636 groups reached the maximum at 4 days p.i. (109.57 ± 6.19 %) and then gradually decreased with time, which may be related to the large molecular weight and the long blood circulation cycle of intact antibody. There was no statistically significant difference in tumor volume changes between the ¹⁷⁷Lu-only and PBS groups (P = 0.866), but both were significantly different from the IMB1636-only group (P = 0.009 and 0.006).

The body weight of tumor-bearing mice in all groups showed a downward trend (Fig. 3c). The IMB1636-only group had the smallest change about 95.19 ± 6.46 % at 14 days, and had significant differences with high-¹⁷⁷Lu-IMB1636, ¹⁷⁷Lu-only, and PBS groups (89.98 ± 4.72 %, 90.34 ± 3.82 %, and 91.31 ± 10.37 %; P = 0.023, 0.016 and 0.027, respectively). There were no statistical differences among high-, low-¹⁷⁷Lu-IMB1636, ¹⁷⁷Lu-only, and PBS groups (P > 0.05).

SPECT imaging and distribution of ¹⁷⁷Lu-labeled IMB1636

We performed in vivo SPECT imaging in high-¹⁷⁷Lu-IMB1636 and ¹⁷⁷Lu-only groups while tumor size monitoring (Fig. 4a). The high-¹⁷⁷Lu-IMB1636 group could show tumor morphology at 2 days p.i., while the ¹⁷⁷Lu-only group couldn’t display the tumor throughout. The radioactive uptakes of liver were high and gradually decreased over time.

Biodistribution analysis of high-, low-¹⁷⁷Lu-IMB1636 and ¹⁷⁷Lu-only groups were performed after the terminal imaging timepoint (Fig. 4b). The tumor radioactive uptake of high- and low-¹⁷⁷Lu-IMB1636 groups was 5.72 ± 0.68 %ID/g and 4.04 ± 0.76 %ID/g respectively (P = 0.005). The tumor uptake of ¹⁷⁷Lu-only was only 0.65 ± 0.52 %ID/g, which had significant difference with high- and low-¹⁷⁷Lu-IMB1636 groups (P < 0.001). The liver and spleen radioactive uptake of ¹⁷⁷Lu-only group was 31.52 ± 4.23 %ID/g and 31.02 ± 1.92 %ID/g respectively, which was significant different from high- and low-¹⁷⁷Lu-IMB1636 groups (P < 0.001). There were no statistical differences in heart, lung, intestine, pancreas, bladder, brain, and skin among three groups (P > 0.05). SPECT imaging enabled visualization and dynamic monitoring of the RIT therapeutic efficacy of ¹⁷⁷Lu-labeled IMB1636.

¹⁸F-FDG PET imaging

The high-¹⁷⁷Lu-IMB1636 and ¹⁷⁷Lu-only groups of RIT experiments underwent ¹⁸F-FDG PET imaging at 14 days p.i. (n = 3). The MIP images showed that the tumor of ¹⁷⁷Lu-only group had higher ¹⁸F-FDGuptake than high-¹⁷⁷Lu-IMB1636 (Fig. 4c). Quantitative ROI analysis was performed of tumors, the ¹⁸F-FDGuptake value of the high-¹⁷⁷Lu-IMB1636 group was 2.74 ± 0.50 %ID/g and ¹⁷⁷Lu-only was 4.19 ± 0.67 %ID/g, representing the RIT reduced the glucose metabolism of tumors, but there was no significant statistical difference between the two groups (P > 0.05, Fig. 4d).

Biodistribution and safety evaluation of ¹⁷⁷Lu-DOTA-IMB1636 in normal mice

Normal BALB/c nude mice were euthanized for biodistribution at 4 hours, 4 days, 7 days, 10 days, and 14 days after the injection of ¹⁷⁷Lu-DOTA-IMB1636 (~11.1 MBq, n = 3). Biodistribution results (Fig. 5a) showed that the radioactive uptake of blood, heart, lung, kidney, stomach, intestine, pancreas, bladder, muscle, brain, and skin gradually decreased with time, and the liver and spleen reached the peak at 7 days and 10 days p.i. (23.40 ± 0.26 %ID/g and 19.74 ± 1.29 %ID/g, respectively).

The hematological indexes of WBC, RBC, PLT and HGB gradually decreased within 4 days but recovered to normal level within 14 days, indicating that high-dose ¹⁷⁷Lu-DOTA-IMB1636 had a certain myelosuppressive effect in the short term (Fig. 5b). The body weight changes ranging from 96.83 ± 4.49 % to 101.67 ± 2.36 %, and reached a minimum about 96.83 ± 4.49 % at 8 days p.i. (n = 3, Fig. 5c). These results suggesting no significant long-term blood toxicity of ¹⁷⁷Lu-DOTA-IMB1636.

Histological staining

H&E and IHC staining were performed in major organs of T3M-4 and PaTu8988 tumor models (Fig. 6). The H&E staining results showed that there were no differences in heart, liver, kidney, lung, and stomach. There were different degrees of hemosiderin deposition in the spleen of ¹⁷⁷Lu-only, high- and low-¹⁷⁷Lu-IMB1636 goups, which may be the reason for the decrease in WBC and PLT p.i.. ¹⁷⁷Lu-only group developed intestinal mucosal sloughing and submucosal telangiectasis, indicating that ¹⁷⁷Lu may be more metabolized through intestine and resulting in radiation enteritis compared to other groups. The tumor H&E staining of high- and low-¹⁷⁷Lu-IMB1636 groups had hyperchromatic nucleolus and hypochromatic cytoplasm, indicating that ¹⁷⁷Lu-DOTA-IMB1636 may induce apoptosis through RIT. The Trop2 IHC staining of high-, low-¹⁷⁷Lu-IMB1636 and IMB1636-only groups were lighter than that in other groups, illustrating the therapeutic effect of IMB1636 targeting Trop2. The tumor IHC staining of PaTu8988 showed almost no expression of Trop2, which was also consistent with the western blotting, flow cytometry and in vivo immunoPET imaging results.

Pancreatic cancer is a fatal disease with no feasible early screening methods, and currently followed treatment regimens couldn’t significantly prolong patient survival. The main challenges in the treatment of pancreatic cancer include highly aggressive of tumor, advanced stage at diagnosis, and complex tumor microenvironment, resulting in the lack of effective therapeutic targets [15]. Searching for novel molecular markers of pancreatic cancer is a hot research topic with the development of immunotherapy [16]. The overexpression of Trop2 is related to the occurrence, development and malignancy of pancreatic cancer, which is a predictor of poor prognosis, and may become a novel biomarker of pancreatic cancer [17]. In our study, ⁶⁴Cu-NOTA-IMB1636 could noninvasively evaluate the expression of Trop2 in vivo, and high dose ¹⁷⁷Lu-labeled IMB1636 inhibited tumor through RIT in Trop2-overexpressing pancreatic cancer tumor models.

Trop2 has stem cell-like properties to promote organ formation and embryonic development, and it plays a similar role in tumor cells participating in tumor growth, proliferation, and cell migration. The possible molecular mechanisms of Trop2 in tumor include regulating calcium signaling pathways and cyclin expression, participating in epithelial-mesenchymal transition (EMT) to reduce cell adhesion, and enabling tumor cells to acquire invasion and migration characteristics [18, 19]. Trop2 is not or rarely expressed in normal tissues, making it an attractive therapeutic target due to limited toxicity [18]. Recently, antibody-drug conjugate (ADC) sacituzumab govitecan (SG, IMMU-132), composed of anti-Trop2 mAb hRS7 and irinotecan metabolite SN-38, had been approved by the US Food and Drug Administration (FDA) for treatment of disseminated triple negative breast cancer (TNBC) refractory to previous chemotherapies due to good clinical trial results [20]. The result of clinical trial of SG confirmed that Trop2 is a broad target in solid epithelioid carcinomas. Although the objective response rate of SG increased to 35% compared with 5% of conventional chemotherapy [20], how to further screen the beneficiaries and improve the clinical benefit is still a problem that we need to solve. ImmunoPET imaging could noninvasively visualize the expression and distribution of target antigens in vivo, screen individuals overexpressing Trop2 for immunotherapy or RIT, and improve the therapeutic efficacy [8].

Recently, ⁸⁹Zr-labeled anti-Trop2 mAb (⁸⁹Zr-DFO-AF650) had been used to achieve noninvasively evaluation of Trop2 in both subcutaneous and orthotopic pancreatic cancer tumor models. ⁸⁹Zr-DFO-AF650 could efficiently distinguish primary tumors in the orthotopic cancer model, showing high correlation between PET imaging and biodistribution [21]. In our study, ⁶⁴Cu-NOTA-IMB1636 in T3M-4 positive group could clearly display the tumor morphology at 4 h p.i., and the tumor uptake reached highest at 48 h about 8.95 ± 1.07%ID/g, which had significant differences with T3M-4-blocked and PaTu8988 groups (P < 0.001). The biodistribution and IHC results were consistent with immunoPET imaging, indicating that Trop2-targeted immunoPET is feasible for the diagnosis and monitoring of Trop2-positive pancreatic cancer. ⁶⁴Cu-labeled tracer can obtain better image quality similar to that of ¹⁸F and lower individual radiation dose compared with ⁸⁹Zr, which is beneficial to further clinical translation [22]. ⁶⁴Cu-NOTA-IMB1636 in T3M-4-blocked groups exhibited nonspecific tumor uptake but still lower than T3M-4 at all time points (P = 0.003). The reason for tumor nonspecific uptake may be attributed to the enhanced permeability and retention effect depends on vascular hyperpermeability, resulting in passive tumor targeting, which means fast growing tumors may have higher nonspecific uptake [23]. The tumor uptake of ⁶⁴Cu-NOTA-IMB1636 in the T3M-4 positive group was significantly higher than that in the other groups. The results suggesting that immunoPET imaging can evaluate the Trop2 expression in vivo.

In addition to Trop2, similar overexpressed proteins have also been investigated as potential targets for immunoPET imaging in pancreatic cancer. Studies had shown that the overexpression of GRP78 was associated with tumor growth, metastasis, and drug resistance. ⁶⁴Cu labeled GRP78-targeting mAb Mab159 accumulated in GRP78-positive subcutaneous pancreatic cancer tumor models [24]. Transferrin receptor (TfR) is involved in iron uptake and is associated with poor prognosis of pancreatic cancer. Sugyo et al. [25] sed ⁸⁹Zr-labeled TfR-targeting mAb TSP-A01 for imaging of murine pancreatic cancer tumor models, and the result showed that ⁸⁹Zr-labeled TSP-A01 could clearly display MiaPaCa-2 tumor, which had high TfR expression. Overexpression of mesothelin (MSLN) was a predictor of poor prognosis of pancreatic cancer. ⁸⁹Zr-labeled MSLN-targeting antibody was used to perform immunoPET imaging in patients with pancreatic cancer and ovarian cancer before relevant ADC treatments, and the tumor lesions as well as antibody biodistribution could be visualized [26]. These studies demonstrate that antibody-based specific immunoPET imaging can facilitate the early detection, efficacy evaluation and monitoring of treatment response of pancreatic cancer.

Positive progress had been achieved by immunotherapy targeting Trop2. SG achieved significant tumor growth inhibition in pancreatic cancer tumor-bearing mice and patients overexpressing Trop2 [27]. A study used the Fab fragments of anti-Trop2 mAb to conjugate doxorubicin to construct an ADC, and found that it inhibited the proliferation and migration of pancreatic cancer cells in a dose-dependent manner, and inhibited the growth of pancreatic cancer xenograft [28]. Photosensitizer IR700 conjugated with humanized anti-Trop2 mAb (Trop2-IR700) could kill the cells overexpressing Trop2, and inhibit the tumor growth of pancreatic cancer and cholangiocarcinoma tumor models after tail vein injection [29]. 2L-Rap(Q)-hRS7, immunotoxin constructed by conjugating mutant Ranpirnase (Rap) with hRS7, could inhibited the tumor growth of murine non-small cell lung cancer xenograft, and the cell growth of variety tumor cell lines [30]. ¹³¹I-IMP-R4-labeled hRS7 was used for RIT in subcutaneous breast cancer tumor models, and 5/11 mice had complete response [31]. However, there are no RIT studies of pancreatic cancer targeting Trop2.

In this study, we used ¹⁷⁷Lu-DOTA-IMB1636 for RIT of subcutaneous pancreatic cancer tumor models. The tumor of high-¹⁷⁷Lu-IMB1636 group had shrinkage and reached a stable disease state (94.24 ± 14.62% at 14 days). Although the tumor volume in the low-¹⁷⁷Lu-IMB1636 group increased (172.89 ± 30.03% at 14 days), it was significantly different from other control groups (P < 0.05). Combined the hematological analysis and body weight results, ¹⁷⁷Lu-labeled IMB1636 could inhibits tumor growth and had no significant long-term blood toxicity. The tumor volume of high-¹⁷⁷Lu-IMB1636 groups reached the maximum at 4 days p.i. and then gradually decreased with time, while the tumor volume of the other three control groups gradually increased, which may be related to the large molecular weight (~ 150 kDa) and the long blood circulation cycle of intact antibody [11]. The decay half-life of ¹⁷⁷Lu is 6.7 d, which is enough for the tumor to produce sufficient RIT effect. ¹⁷⁷Lu emits β-particles with 3 energies of 497 keV (78.6%), 384 keV (9.1%) and 176 keV (12.2%). Due to the relatively low energy, it can radiate to the lesions and has mild bone marrow suppression. At the same time, ¹⁷⁷Lu also emits γ-rays for SPECT imaging to achieve the purpose of theranostic [32]. In the future, we can construct F(ab')₂ or Fab fragments of IMB1636 (~ 110 kDa and ~ 50 kDa, respectively), which can reduce the molecular weight of the intact mAb while retaining the high affinity and specificity, shortening its circulation time and obtaining a higher tumor-target ratio to achieve immunoPET imaging within one day and better RIT efficacy [33].

We demonstrated that ⁶⁴Cu-NOTA-IMB1636 could noninvasively evaluate the expression level of Trop2, and ¹⁷⁷Lu-DOTA-IMB1636 could inhibit the tumor growth through RIT in pancreatic cancer tumor models. Trop2 is expected to become a potential theranostic target of pancreatic cancer. ImmunoPET imaging may be used to screen the beneficiary and provide a new treatment method for pancreatic cancer overexpressing Trop2 through RIT.

Funding This work was supported by the Natural Science Foundation of Beijing Municipality (Grant numbers 7224365 and 7202133), the National Natural Science Foundation of China (Grant numbers 82171970, 81871385, 81971642, and 82104052), the Beijing Science Foundation for Distinguished Young Scholars (Grant number JQ21025), the Peking University Medicine Fund of Fostering Young Scholars’ Scientific & Technological Innovation (Grant number BMU2022PY006), the Clinical Medicine Plus X- Youth Scholars Project of Peking University (Grant number PKU2020LCXQ023), CAMS Innovation Fund for Medical Sciences (CIFMS, Grant number 2021-I2M-1-026), and Natural Science Foundation of Shandong Province (Grant number ZR2020QH201).

Conflict of Interest: The authors declare no conflict of interest.

Ethical Approval: All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer Statistics. 2021. CA: A Cancer Journal for Clinicians. 2021;71:7–33. doi:10.3322/caac.21654.
Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71:209–49. doi:10.3322/caac.21660.
Ilic M, Ilic I. Epidemiology of pancreatic cancer. World J Gastroenterol. 2016;22:9694–705. doi:10.3748/wjg.v22.i44.9694.
Oberstein PE, Olive KP. Pancreatic cancer: why is it so hard to treat? Th Adv Gastroenterol. 2013;6:321–37. doi:10.1177/1756283X13478680.
Wu M, Shu J. Multimodal Molecular Imaging: Current Status and Future Directions. Contrast Media Mol Imaging. 2018;2018:1382183. doi:10.1155/2018/1382183.
Avril S, Muzic RF Jr, Plecha D, Traughber BJ, Vinayak S, Avril N. (1)(8)F-FDG PET/CT for Monitoring of Treatment Response in Breast Cancer. J Nucl Med. 2016;57(Suppl 1):34S-9S. doi:10.2967/jnumed.115.157875.
Wei W, Rosenkrans ZT, Liu J, Huang G, Luo QY, Cai W. ImmunoPET: Concept, Design, and Applications. Chem Rev. 2020;120:3787–851. doi:10.1021/acs.chemrev.9b00738.
Filippi L, Nervi C, Proietti I, Pirisino R, Potenza C, Martelli O, et al. Molecular imaging in immuno-oncology: current status and translational perspectives. Expert Rev Mol Diagn. 2020;20:1199–211. doi:10.1080/14737159.2020.1854090.
Goldenberg DM, Stein R, Sharkey RM. The emergence of trophoblast cell-surface antigen 2 (TROP-2) as a novel cancer target. Oncotarget. 2018;9:28989–9006. doi:10.18632/oncotarget.25615.
Zeng P, Chen MB, Zhou LN, Tang M, Liu CY, Lu PH. Impact of TROP2 expression on prognosis in solid tumors: A Systematic Review and Meta-analysis. Sci Rep. 2016;6:33658. doi:10.1038/srep33658.
Li C, Yang Q, Chen Z, Qiu Y, Du Y, Wang R, et al. Noninvasive Evaluation of Multidrug Resistance via Imaging of ABCG2/BCRP Multidrug Transporter in Lung Cancer Xenograft Models. Mol Pharm. 2022. doi:10.1021/acs.molpharmaceut.1c00939.
Kang L, Li C, Rosenkrans ZT, Huo N, Chen Z, Ehlerding EB, et al. CD38-Targeted Theranostics of Lymphoma with (89)Zr/(177)Lu-Labeled Daratumumab. Adv Sci (Weinh). 2021;8:2001879. doi:10.1002/advs.202001879.
Kang L, Li C, Rosenkrans ZT, Engle JW, Wang R, Jiang D, et al. Noninvasive Evaluation of CD20 Expression Using (64)Cu-Labeled F(ab')2 Fragments of Obinutuzumab in Lymphoma. J Nucl Med. 2021;62:372–8. doi:10.2967/jnumed.120.246595.
Li C, Kang L, Fan K, Ferreira CA, Becker KV, Huo N, et al. ImmunoPET of CD146 in Orthotopic and Metastatic Breast Cancer Models. Bioconjug Chem. 2021;32:1306–14. doi:10.1021/acs.bioconjchem.0c00649.
Liu Q, Liao Q, Zhao Y. Chemotherapy and tumor microenvironment of pancreatic cancer. Cancer Cell Int. 2017;17:68. doi:10.1186/s12935-017-0437-3.
Neoptolemos JP, Kleeff J, Michl P, Costello E, Greenhalf W, Palmer DH. Therapeutic developments in pancreatic cancer: current and future perspectives. Nat Rev Gastroenterol Hepatol. 2018;15:333–48. doi:10.1038/s41575-018-0005-x.
Shvartsur A, Bonavida B. Trop2 and its overexpression in cancers: regulation and clinical/therapeutic implications. Genes Cancer. 2015;6:84–105. doi:10.18632/genesandcancer.40.
Lenart S, Lenart P, Smarda J, Remsik J, Soucek K, Benes P. Trop2: Jack of All Trades, Master of None. Cancers (Basel). 2020;12. doi:10.3390/cancers12113328.
Pavone G, Motta L, Martorana F, Motta G, Vigneri P. A New Kid on the Block: Sacituzumab Govitecan for the Treatment of Breast Cancer and Other Solid Tumors. Molecules. 2021;26. doi:10.3390/molecules26237294.
Bardia A, Hurvitz SA, Tolaney SM, Loirat D, Punie K, Oliveira M, et al. Sacituzumab Govitecan in Metastatic Triple-Negative Breast Cancer. N Engl J Med. 2021;384:1529–41. doi:10.1056/NEJMoa2028485.
Chen W, Li M, Younis MH, Barnhart TE, Jiang D, Sun T, et al. ImmunoPET of trophoblast cell-surface antigen 2 (Trop-2) expression in pancreatic cancer. Eur J Nucl Med Mol Imaging. 2022;49:861–70. doi:10.1007/s00259-021-05563-1.
Natarajan A. Copper-64-immunoPET imaging: bench to bedside. Q J Nucl Med Mol Imaging. 2020;64:356–63. doi:10.23736/S1824-4785.20.03310-5.
Li S, England CG, Ehlerding EB, Kutyreff CJ, Engle JW, Jiang D, et al. ImmunoPET imaging of CD38 expression in hepatocellular carcinoma using 64Cu-labeled daratumumab. Am J Transl Res. 2019;11:6007–15.
Wang H, Li D, Liu S, Liu R, Yuan H, Krasnoperov V, et al. Small-Animal PET Imaging of Pancreatic Cancer Xenografts Using a 64Cu-Labeled Monoclonal Antibody, MAb159. J Nucl Med. 2015;56:908–13. doi:10.2967/jnumed.115.155812.
Sugyo A, Tsuji AB, Sudo H, Nagatsu K, Koizumi M, Ukai Y, et al. Preclinical evaluation of (8)(9)Zr-labeled human antitransferrin receptor monoclonal antibody as a PET probe using a pancreatic cancer mouse model. Nucl Med Commun. 2015;36:286–94. doi:10.1097/MNM.0000000000000245.
Lamberts LE, Menke-van der Houven van Oordt CW, ter Weele EJ, Bensch F, Smeenk MM, Voortman J, et al. ImmunoPET with Anti-Mesothelin Antibody in Patients with Pancreatic and Ovarian Cancer before Anti-Mesothelin Antibody-Drug Conjugate Treatment. Clin Cancer Res. 2016;22:1642–52. doi:10.1158/1078-0432.CCR-15-1272.
Cardillo TM, Govindan SV, Sharkey RM, Trisal P, Arrojo R, Liu D, et al. Sacituzumab Govitecan (IMMU-132), an Anti-Trop-2/SN-38 Antibody-Drug Conjugate: Characterization and Efficacy in Pancreatic, Gastric, and Other Cancers. Bioconjug Chem. 2015;26:919–31. doi:10.1021/acs.bioconjchem.5b00223.
Mao Y, Wang X, Zheng F, Wang C, Tang Q, Tang X, et al. The tumor-inhibitory effectiveness of a novel anti-Trop2 Fab conjugate in pancreatic cancer. Oncotarget. 2016;7:24810–23. doi:10.18632/oncotarget.8529.
Nishimura T, Mitsunaga M, Sawada R, Saruta M, Kobayashi H, Matsumoto N, et al. Photoimmunotherapy targeting biliary-pancreatic cancer with humanized anti-TROP2 antibody. Cancer Med. 2019;8:7781–92. doi:10.1002/cam4.2658.
Chang CH, Gupta P, Michel R, Loo M, Wang Y, Cardillo TM, et al. Ranpirnase (frog RNase) targeted with a humanized, internalizing, anti-Trop-2 antibody has potent cytotoxicity against diverse epithelial cancer cells. Mol Cancer Ther. 2010;9:2276–86. doi:10.1158/1535-7163.MCT-10-0338.
Govindan SV, Stein R, Qu Z, Chen S, Andrews P, Ma H, et al. Preclinical therapy of breast cancer with a radioiodinated humanized anti-EGP-1 monoclonal antibody: advantage of a residualizing iodine radiolabel. Breast Cancer Res Treat. 2004;84:173–82. doi:10.1023/B:BREA.0000018417.02580.ef.
Goldsmith SJ. Targeted Radionuclide Therapy: A Historical and Personal Review. Semin Nucl Med. 2020;50:87–97. doi:10.1053/j.semnuclmed.2019.07.006.
Sheridan C. Ablynx's nanobody fragments go places antibodies cannot. Nat Biotechnol. 2017;35:1115–7. doi:10.1038/nbt1217-1115.

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Theranostic application of 64Cu/177Lu-labeled anti-Trop2 monoclonal antibody in pancreatic cancer tumor models

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