SPECT/CT Imaging, Biodistribution and Radiation Dosimetry of a 177Lu-DOTA-Integrin αvβ6 Cystine Knot Peptide in a Pancreatic Cancer Xenograft Model

Abstract

IntroductionPancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignant neoplasms, as many cases go undetected until they reach an advanced stage. Integrin αvβ6 is a cell surface receptor overexpressed in PDAC. Consequently, it may serve as a target for the development of probes for imaging diagnosis and radioligand therapy. Engineered cystine knottin peptides specific for integrin αvβ6 have recently been developed showing high affinity and stability. This study aimed to evaluate an integrin αvβ6-specific knottin molecular probe containing the therapeutic radionuclide 177Lu for targeting of PDAC.MethodsThe expression of integrin αvβ6 in PDAC cell lines BxPC-3 and Capan-2 was analyzed using RT-qPCR and immunofluorescence. In vitro competition and saturation radioligand binding assays were performed to calculate the binding affinity of the DOTA-coupled tracer loaded with and without lutetium to BxPC-3 and Capan-2 cell lines as well as the maximum number of binding sites in these cell lines. To evaluate tracer accumulation in the tumor and organs, SPECT/CT, biodistribution and dosimetry projections were carried out using a Capan-2 xenograft tumor mouse model.ResultsRT-qPCR and immunofluorescence results showed high expression of integrin αvβ6 in BxPC-3 and Capan-2 cells. A competition binding assay revealed high affinity of the tracer with IC50 values of 1.69 nM and 9.46 nM for BxPC-3 and Capan-2, respectively. SPECT/CT and biodistribution analysis of the conjugate 177Lu-DOTA-integrin αvβ6 knottin demonstrated accumulation in Capan-2 xenograft tumors (3.13 ± 0.63%IA/g at day 1 post injection) with kidney uptake at 19.2 ± 2.5 %IA/g, declining much more rapidly than in tumors.Conclusion177Lu-DOTA-integrin αvβ6 knottin was found to be a high-affinity tracer for PDAC tumors with considerable tumor accumulation and moderate, rapidly declining kidney uptake. These promising results warrant a preclinical treatment study to establish therapeutic efficacy.

Therapeutic Efficacy of 177Lu-Labeled A20FMDV2 Peptides Targeting ανβ6

“…All radiotracers were radiolabeled in good radiochemical purity at a molar activity of 10-18.5 MBq/nmol. A high temperature of 90 • C was required for the labeling, which is consistent with previous studies, which used up to 99 • C for 68 Ga and 80-95 • C for 177 Lu labelings [35][36][37]. In vitro cell-based studies in BxPC-3 cell lines indicated that about 11% internalized for the [ 68 Ga]Ga-DOTA-(PEG28) 2 -A20FMDV2 at 1 h, while the albumin binding constructs had about 8-10%.…”

68Ga-Labeled Cystine Knot Peptide Targeting Integrin αvβ6 for Lung Cancer PET Imaging

“…Thus, α v β 6 -binding cystine knot peptides such as R 0 1 and S 0 2 were developed and optimized, which were first applied for imaging studies in animal models after being radiolabeled with radionuclides. ,, Kimura et al engineered cystine knot peptides R 0 1 mutants, and R 0 1-MG was selected for imaging human α v β 6 -positive cancers, including pancreatic cancer, cervical cancer, and lung cancer . Sachindra et al synthesized 177 Lu-DOTA-R 0 1-MG and performed SPECT/CT imaging, biodistribution, and radiation dosimetry studies . In addition, Tummers et al developed an intraoperative near-infrared fluorescence agent based on R 0 1-MG, which showed specific targeting to α v β 6 in a mouse model of pancreatic cancer .…”

Targeting integrin αvβ6 with gallium-68 tris (hydroxypyridinone) based PET probes

“…To preliminary estimate the affinity of the probes for integrin αvβ6, IC 50 values were determined in a competitive binding assay using pancreatic cancer BxPC-3 cells (known to possess a high expression of integrin αvβ6) 32 and not considering the non-specific binding of the peptide ( i.e. with no controls to account for non-specific binding).…”