EphA2-targeted alpha-particle theranostics for enhancing PDAC treatment

Abstract

Background: Pancreatic ductal adenocarcinoma (PDAC) presents a formidable challenge in oncology due to its aggressive nature and resistance to therapy. Current treatments, including surgery, chemotherapy, and radiotherapy, have limited success in improving patient outcomes. This study addresses the urgent need for novel radiotheranostic strategies for PDAC by investigating EphA2 as a potential target. Methods and Results: Analysis of genomic data from the Cancer Cell Line Encyclopedia (CCLE) and The Cancer Genome Atlas (TCGA) revealed elevated EphA2 expression in PDAC, confirmed by immunohistochemical staining of tumor tissue microarrays (TMAs). Further analysis showed variable EphA2 expression across PDAC cell lines, with surface receptor density not always correlating with mRNA levels. A low molecular weight peptide was developed and labeled with gallium-68 for PET imaging. In vitro studies demonstrated specific binding to EphA2-expressing PDAC cells with rapid internalization. In vivo PET imaging in subcutaneous and orthotopic PDAC models confirmed high tumor uptake and minimal off-target binding, confirming EphA2 as a valid imaging target. For molecular radiotherapy, a DOTA-conjugated peptide was labeled with the alpha-particle emitter, actinium-225. In vitro studies revealed dose-dependent cytotoxicity in PDAC cells, with an IC₅₀ of 0.32 µCi/mL. In a tumor model, treatment with Ac-225 labeled peptide significantly inhibited tumor growth compared to controls, with mild adverse effects. Conclusion: These results establish EphA2 as a promising radiotheranostic target in PDAC, with potential for both non-invasive imaging and targeted radiotherapy. Given the potential, further optimization of EphA2-targeted agents are warranted to advance personalized treatment strategies for PDAC patients.

Keywords: Alpha-particle therapy; Gallium-68; Imaging; PET; Pancreatic cancer; Peptide radiopharmaceuticals; Radiotherapy.

Figure 1

CCLE and TCGA database of mRNA expression of EphA2. A) EphA2 expression in cell lines in CCLE. B) EphA2 mRNA expression in different types of tumors in TCGA. C) Comparison of mRNA expression of EphA2 in different normal tissues and corresponding tumors. D) Expression of different EPH receptors in pancreatic tumors. PAAD = pancreatic cancer; BLCA = Bladder cancer; READ = Rectal cancer; LUSC = Lung squamous cell carcinoma; THCA = Thyroid cancer; KIRP = Kidney papillary cell carcinoma; LUAD = Lung adenocarcinoma; KIRC = Kidney clear cell carcinoma; CHOL = Bile duct cancer; SKCM = melanoma; LIHC = Liver cancer; KICH = Kidney chromophobe; BRCA = Breast cancer; PRAD = Prostate cancer; DLBC = Large-B-cell lymphoma; LAML = Acute myeloid leukemia.

Figure 2

Structure and in vitro characterization of AJ201. A) Structure of bicyclic peptide AJ201 having NOTA as bifunctional chelator for ⁶⁸Ga-labeling. In the structure, black color represents binding moiety, parakeet color represents the linker and red color represents the radiometal B) Surface plasmon resonance (SPR) analysis showing affinity of AJ201 for EphA2 using recombinant human (solid) and mouse (dashed) EphA2 proteins.

Figure 3

In vitro specificity of [⁶⁸Ga]AJ201 for EphA2 in human pancreatic cancer cells. A) Quantification of EphA2-mRNA using RT-PCR. B) Flow cytometry analysis of EphA2 receptor expression on cell surface. C) A representative plot of EphA2 receptor density in PDAC cells measured by quantibrite assay. D) [⁶⁸Ga]AJ201 binding (percent incubated activity, %IA) to different cells. Cells were incubated with 1 µCi [⁶⁸Ga]AJ201 at 4 °C for 1 h. [⁶⁸Ga]AJ201 uptake is EphA2 expression dependent, and co-incubation with 2 μM of non-radioactive AJ201 (0.2 nmol, blocking dose) significantly reduced radiotracer uptake confirming EphA2 specificity. E) In vitro uptake of [⁶⁸Ga]AJ201 over 60 min in Panc1 cells at 4 ˚C and 37 ˚C. F) Correlation of [⁶⁸Ga]AJ201 uptake with surface EphA2 receptor density. Data in panels A, D, E and F are presented as mean ± SD (n = 3-4). Significance was calculated using multiple unpaired t test in D; ns, P ≥ 0.05; *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001. Simple linear regression and Pearson coefficient were used in F. All in vitro experiments were performed three times and representatives are shown.

Figure 4

Pharmacokinetics of [⁶⁸Ga]AJ201 in NSG mice bearing Panc1 tumor xenografts. A) Coronal sections of the fused dynamic PET/MR images showing [⁶⁸Ga]AJ201 distribution. Primary tumor is indicated by red circle. Mice were intravenously injected with ~ 9.25 MBq (~250 µCi) [⁶⁸Ga]AJ201; B, Bladder. B) Time-activity curves of [⁶⁸Ga]AJ201 in the kidney, tumor, heart and muscle derived from PET data in A. C) Uptake of [⁶⁸Ga]AJ201 in tumor, blood, pancreas and muscle derived from ex vivo biodistribution study (percent incubated dose per gram, %ID/g). D) Tumor-to-muscle, tumor-to-blood and tumor-to-pancreas ratios derived from biodistribution data. E) Whole-body PET/CT images of Panc1 xenografts with [⁶⁸Ga]AJ201, without (left) and with (right) pre-administration of a blocking dose (50 µg of AJ201) (tumor denoted with dashed red line and K = Kidney). F) [⁶⁸Ga]AJ201 quantification in tumors by ex vivo biodistribution in mice treated with and without a blocking dose where T/B is tumor-to-blood; T/M is tumor-to-muscle and T/P is tumor-to-pancreas ratios; data in panels B represented as mean ± SEM (n = 3); data in panels C and D are from mice intravenously injected with ~ 2.96 MBq (~80 µCi) [⁶⁸Ga]AJ201 and sacrificed at different time-points after injection, data represented as mean ± SEM (n = 3 or 4); data in figure F is shown as box and whisker plots (median ± IQR) showing all data points (n = 4). Statistics were calculated using multiple unpaired t test in F. ** P ≤ 0.01; ****, P ≤ 0.0001.

Figure 5

In vivo specificity of [⁶⁸Ga]AJ201 for EphA2 in NSG mice with PDAC tumor xenografts. A) Whole-body PET/CT images of different human PDAC xenografts at 60 min after the injection of radiotracer. Mice were injected with ~ 7.4 MBq (~200 µCi) [⁶⁸Ga]AJ201; K, kidney. Panc1 mouse image is reproduced from figure 4E. B) [⁶⁸Ga]AJ201 uptake quantification (%ID/g) in different PDAC tumors by ex vivo biodistribution at 60 min after injection. Left: %ID/g of different tumor xenografts, tumor-to-blood (middle) and tumor-to-muscle (right) ratios in each individual mice harboring respective xenografts. C) IHC staining for EphA2 expression in PDAC xenografts (digitally scanned at 40x).; data in figure B is shown as box and whisker plots (median ± IQR) showing all data points (n = 4-5).

Figure 6

In vivo distribution of [⁶⁸Ga]AJ201 in Panc1-orthotopic tumor model. A) Coronal sections of the fused PET/MR images showing [⁶⁸Ga]AJ201 distribution. Primary tumor is indicated by red circle; K = Kidney, B = Bladder. B) Quantification of accumulated activity in tumor, muscle and pancreas of images shown in panel A (n = 4-5) C) tumor-to-muscle (T/M) and tumor-to-pancreas (T/P) ratios from the images shown in panel A (n = 4) D) EphA2 IHC of orthotopic Panc1 tumor and normal pancreas to validate the EphA2 expression. Panc1 orthotopic tumor model was generated by surgically implanting a 3-5 mm³ section of Panc1 tumor xenograft directly onto the pancreas. The implanted tumors were allowed to grow for 10 days, after which they were ready for imaging. At this stage, MRI measurements indicated that tumor sizes ranged from 10-50 mm³. data in figure B and C is shown as box and whisker plots (median ± IQR) showing all data points (n = 3-5).

Figure 7

In vitro and in vivo therapeutic efficacy of [²²⁵Ac]AJ210 A) In vitro cell growth inhibition by [²²⁵Ac]AJ210 after 72 h incubation with KPC cells (data; mean ± SD, n = 4) at 37 °C. B) Effect of [²²⁵Ac]AJ210 (n = 7) on the KPC tumor growth (data; median bold line and individual replicates) after administration of total 37 kBq via tail-vein injection; each line represents one animal, arrow indicates [²²⁵Ac]AJ210 treatment (Tx) C) Kaplan-Meier plot of survival for the group treated with saline and [²²⁵Ac]AJ210. Median survival of saline treated group is 27 days and [²²⁵Ac]AJ210-treated group did not reach. Mice were sacrificed after reaching 1000 mm³ tumor volume.