Preclinical Evaluation of 68Ga- and 177Lu-Labeled Integrin αvß6-Targeting Radiotheranostic Peptides.

The integrin αvß6, an epithelium-specific cell surface receptor, is overexpressed on numerous malignancies, including the highly lethal pancreatic ductal adenocarcinomas. Here, we developed and tested a novel αvß6-targeting peptide, DOTA-5G (1) radiolabeled with 68Ga, for PET/CT imaging and 177Lu for treatment. With the goal to develop a radiotheranostic, further modifications were made for increased circulation time, renal recycling, and tumor uptake, yielding DOTA-albumin-binding moiety-5G (2). Methods: Peptides 1 and 2 were synthesized on solid phase, and their affinity for αvß6 was assessed by enzyme-linked immunosorbent assay. The peptides were radiolabeled with 68Ga and 177Lu. In vitro cell binding, internalization, and efflux of 68Ga-1 and 177Lu-2 were evaluated in αvß6-positive BxPC-3 human pancreatic cancer cells. PET/CT imaging of 68Ga-1 and 68Ga-2 was performed on female nu/nu mice bearing subcutaneous BxPC-3 tumors. Biodistribution was performed for 68Ga-1 (1 and 2 h after injection), 68Ga-2 (2 and 4 h after injection), and 177Lu-1 and 177Lu-2 (1, 24, 48, and 72 h after injection). The 177Lu-2 biodistribution data were extrapolated for human dosimetry data estimates using OLINDA/EXM 1.1. Therapeutic efficacy of 177Lu-2 was evaluated in mice bearing BxPC-3 tumors. Results: Peptides 1 and 2 demonstrated high affinity (<55 nM) for αvß6 by enzyme-linked immunosorbent assay. 68Ga-1, 68Ga-2, 177Lu-1, and 177Lu-2 were synthesized in high radiochemical purity. Rapid in vitro binding and internalization of 68Ga-1 and 177Lu-2 were observed in BxPC-3 cells. PET/CT imaging and biodistribution studies demonstrated uptake in BxPC-3 tumors. Introduction of the albumin-binding moiety in 177Lu-2 resulted in a 5-fold increase in tumor uptake and retention over time. Based on the extended dosimetry data, the dose-limiting organ for 177Lu-2 is the kidney. Treatment with 177Lu-2 prolonged median survival by 1.5- to 2-fold versus controls. Conclusion: 68Ga-1 and 177Lu-2 demonstrated high affinity for the integrin αvß6 both in vitro and in vivo, were rapidly internalized into BxPC-3 cells, and were stable in mouse and human serum. Both radiotracers showed favorable pharmacokinetics in preclinical studies, with predominantly renal excretion and good tumor-to-normal-tissue ratios. Favorable human dosimetry data suggest the potential of 177Lu-2 as a treatment for pancreatic ductal adenocarcinoma.

Evaluation of Copper-64-Labeled αvß6-Targeting Peptides: Addition of an Albumin Binding Moiety to Improve Pharmacokinetics.

The incorporation of non-covalent albumin binding moieties (ABMs) into radiotracers results in increased circulation time, leading to a higher uptake in the target tissues such as the tumor, and, in some cases, reduced kidney retention. We previously developed [18F]AlF NOTA-K(ABM)-αvß6-BP, where αvß6-BP is a peptide with high affinity for the cell surface receptor integrin αvß6 that is overexpressed in several cancers, and the ABM is an iodophenyl-based moiety. [18F]AlF NOTA-K(ABM)-αvß6-BP demonstrated prolonged blood circulation compared to the non-ABM parent peptide, resulting in high, αvß6-targeted uptake with continuously improving detection of αvß6(+) tumors using PET/CT. To further extend the imaging window beyond that of fluorine-18 (t1/2 = 110 min) and to investigate the pharmacokinetics at later time points, we radiolabeled the αvß6-BP with copper-64 (t1/2 = 12.7 h). Two peptides were synthesized without (1) and with (2) the ABM and radiolabeled with copper-64 to yield [64Cu]1 and [64Cu]2, respectively. The affinity of [natCu]1 and [natCu]2 for the integrin αvß6 was assessed by enzyme-linked immunosorbent assay. [64Cu]1 and [64Cu]2 were evaluated in vitro (cell binding and internalization) using DX3puroß6 (αvß6(+)), DX3puro (αvß6(-)), and pancreatic BxPC-3 (αvß6(+)) cells, in an albumin binding assay, and for stability in both mouse and human serum. In vivo (PET/CT imaging) and biodistribution studies were done in mouse models bearing either the paired DX3puroß6/DX3puro or BxPC-3 xenograft tumors. [64Cu]1 and [64Cu]2 were synthesized in ≥97% radiochemical purity. In vitro, [natCu]1 and [natCu]2 maintained low nanomolar affinity for integrin αvß6 (IC50 = 28 ± 3 and 19 ± 5 nM, respectively); [64Cu]1 and [64Cu]2 showed comparable binding to αvß6(+) cells (DX3puroß6: ≥70%, ≥42% internalized; BxPC-3: ≥19%, ≥12% internalized) and ≤3% to the αvß6(-) DX3puro cells. Both radiotracers were ≥98% stable in human serum at 24 h, and [64Cu]2 showed a 6-fold higher binding to human serum protein than [64Cu]1. In vivo, selective uptake in the αvß6(+) tumors was observed with tumor visualization up to 72 h for [64Cu]2. A 3-5-fold higher αvß6(+) tumor uptake of [64Cu]2 vs [64Cu]1 was observed throughout, at least 2.7-fold improved BxPC-3-to-kidney and BxPC-3-to-blood ratios, and 2-fold improved BxPC-3-to-stomach ratios were noted for [64Cu]2 at 48 h. Incorporation of an iodophenyl-based ABM into the αvß6-BP ([64Cu]2) prolonged circulation time and resulted in improved pharmacokinetics, including increased uptake in αvß6(+) tumors that enabled visualization of αvß6(+) tumors up to 72 h by PET/CT imaging.

The Effects of an Albumin Binding Moiety on the Targeting and Pharmacokinetics of an Integrin αvß6-Selective Peptide Labeled with Aluminum [18F]Fluoride.

PURPOSE: The αvß6-BP peptide selectively targets the integrin αvß6, a cell surface receptor recognized as a prognostic indicator for several challenging malignancies. Given that the 4-[18F]fluorobenzoyl (FBA)-labeled peptide is a promising PET imaging agent, radiolabeling via aluminum [18F]fluoride chelation and introduction of an albumin binding moiety (ABM) have the potential to considerably simplify radiochemistry and improve the pharmacokinetics by increasing biological half-life. PROCEDURES: The peptides NOTA-αvß6-BP (1) and NOTA-K(ABM)-αvß6-BP (2) were synthesized on solid phase, radiolabeled with aluminum [18F]fluoride, and evaluated in vitro (integrin ELISA, albumin binding, cell studies) and in vivo in mouse models bearing paired DX3puroß6 [αvß6(+)]/DX3puro [αvß6(-)], and for [18F]AlF 2, BxPC-3 [αvß6(+)] cell xenografts (PET imaging, biodistribution). RESULTS: The peptides were radiolabeled in 23.0 ± 5.7 % and 22.1 ± 4.4 % decay-corrected radiochemical yield, respectively, for [18F]AlF 1 and [18F]AlF 2. Both demonstrated excellent affinity and selectivity for integrin αvß6 by ELISA (IC50(αvß6) = 3-7 nM vs IC50(αvß3) > 10 µM) and in cell binding studies (51.0 ± 0.7 % and 47.2 ± 0.7 % of total radioactivity bound to DX3puroß6 cells at 1 h, respectively, vs. ≤ 1.2 % to DX3puro for both compounds). The radiotracer [18F]AlF 1 bound to human serum at 16.3 ± 1.9 %, compared to 67.5 ± 1.0 % for the ABM-containing [18F]AlF 2. In vivo studies confirmed the effect of the ABM on blood circulation (≤ 0.1 % ID/g remaining in blood for [18F]AlF 1 as soon as 1 h p.i. vs. > 2 % ID/g for [18F]AlF 2 at 6 h p.i.) and higher αvß6(+) tumor uptake (4 h: DX3puroß6; [18F]AlF 1: 3.0 ± 0.7 % ID/g, [18F]AlF 2: 7.2 ± 0.7 % ID/g; BxPC-3; [18F]AlF 2: 10.2 ± 0.1 % ID/g). CONCLUSION: Both compounds were prepared using standard chemistries; affinity and selectivity for integrin αvß6 in vitro remained unaffected by the albumin binding moiety. In vivo, the albumin binding moiety resulted in prolonged circulation and higher αvß6-targeted uptake.

Correction to: Evaluation of Two Optical Probes for Imaging the Integrin αvß6- In Vitro and In Vivo in Tumor-Bearing Mice.

This article was updated to correct the axes in Figures 4e and 5d.

Evaluation of Two Optical Probes for Imaging the Integrin αvß6- In Vitro and In Vivo in Tumor-Bearing Mice.

PURPOSE: The purpose of this study was to develop and evaluate two αvß6-targeted fluorescent imaging agents. The integrin subtype αvß6 is significantly upregulated in a wide range of epithelial derived cancers, plays a key role in invasion and metastasis, and expression is often located at the invasive edge of tumors. αvß6-targeted fluorescent imaging agents have the potential to guide surgical resection leading to improved patient outcomes. Both imaging agents were based on the bi-PEGylated peptide NH2-PEG28-A20FMDV2-K16R-PEG28 (1), a peptide that has high affinity and selectivity for the integrin αvß6: (a) 5-FAM-X-PEG28-A20FMDV2-K16R-PEG28 (2), and (b) IRDye800-PEG28-A20FMDV2-K16R-PEG28 (3). PROCEDURES: Peptides were synthesized using solid-phase peptide synthesis and standard Fmoc chemistry. Affinity for αvß6 was evaluated by ELISA. In vitro binding, internalization, and localization of 2 was monitored using confocal microscopy in DX3puroß6 (αvß6+) and DX3puro (αvß6-) cells. The in vivo imaging and ex vivo biodistribution of 3 was evaluated in three preclinical mouse models, DX3puroß6/DX3puro and BxPC-3 (αvß6+) tumor xenografts and a BxPC-3 orthotopic pancreatic tumor model. RESULTS: Peptides were obtained in > 99% purity. IC50 values were 28 nM (2) and 39 nM (3). Rapid αvß6-selective binding and internalization of 2 was observed. Fluorescent intensity (FLI) measurements extracted from the in vivo images and ex vivo biodistribution confirmed uptake and retention of 3 in the αvß6 positive subcutaneous and orthotopic tumors, with negligible uptake in the αvß6-negative tumor. Blocking studies with a known αvß6-targeting peptide demonstrated αvß6-specific binding of 3. CONCLUSION: Two fluorescence imaging agents were developed. The αvß6-specific uptake, internalization, and endosomal localization of the fluorescence agent 2 demonstrates potential for targeted therapy. The selective uptake and retention of 3 in the αvß6-positive tumors enabled clear delineation of the tumors and surgical resection indicating 3 has the potential to be utilized during image-guided surgery.

Prostate cancer: performance characteristics of combined T2W and DW-MRI scoring in the setting of template transperineal re-biopsy using MR-TRUS fusion.

OBJECTIVES: To measure the performance characteristics of combined T2-weighted (T2W) and diffusion-weighted (DW) magnetic resonance imaging (MRI) suspicion scoring prior to MR-transrectal ultrasound (TRUS) fusion template transperineal (TTP) re-biopsy. METHODS: Thirty-nine patients referred for prostate re-biopsy, with prior MRI examinations, were retrospectively included. The MR images, including T2W and DW-MRI, had been independently evaluated prospectively by two radiologists using a structured scoring system. An MR-TRUS fusion TTP re-biopsy was used for MR target and non-targeted biopsy cores. Targeting performance and correlation with disease status were evaluated on a per-patient and per-region basis. RESULTS: The cancer yield was 41% (16/39 patients). MR targeting accurately detected the disease in 12/16 (75%) cancerous patients and missed the disease in 4/16 (25%) patients, all with Gleason 3 + 3 disease. There was a significant relationship (P < 0.01) between MR suspicion score and the significance of cancer. Reader 1 had significantly higher sensitivity in the transition zone (TZ; 0.84) compared with the peripheral zone (PZ; 0.32) (P = 0.04). Inter-reader agreement was moderate for the PZ and substantial for the TZ. CONCLUSIONS: MRI targeting is beneficial in the setting of TTP MR-TRUS fusion re-biopsy and MR suspicion score relates to prostate cancer clinical significance. A T2W and DW-MRI structured scoring system results in good inter-reader agreement in this setting. KEY POINTS: • Pre-biopsy MRI aids the detection of high significance cancer during prostate re-biopsy. • MRI suspicion level correlates with the clinical significance of prostate cancer detected. • T2W and DW-MRI structured scoring of pre-biopsy MRI permits good inter-reader agreement.