Synthesis and Evaluation of a Monomethyl Auristatin E─Integrin αvß6 Binding Peptide-Drug Conjugate for Tumor Targeted Drug Delivery.

Many anticancer drugs exhibit high systemic off-target toxicities causing severe side effects. Peptide-drug conjugates (PDCs) that target tumor-specific receptors such as integrin αvß6 are emerging as powerful tools to overcome these challenges. The development of an integrin αvß6-selective PDC was achieved by combining the therapeutic efficacy of the cytotoxic drug monomethyl auristatin E with the selectivity of the αvß6-binding peptide (αvß6-BP) and with the ability of positron emission tomography (PET) imaging by copper-64. The [64Cu]PDC-1 was produced efficiently and in high purity. The PDC exhibited high human serum stability, integrin αvß6-selective internalization, cell binding, and cytotoxicity. Integrin αvß6-selective tumor accumulation of the [64Cu]PDC-1 was visualized with PET-imaging and corroborated by biodistribution, and [64Cu]PDC-1 showed promising in vivo pharmacokinetics. The [natCu]PDC-1 treatment resulted in prolonged survival of mice bearing αvß6 (+) tumors (median survival: 77 days, vs αvß6 (-) tumor group 49 days, and all other control groups 37 days).

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.

A Comparison of Evans Blue and 4-(p-Iodophenyl)butyryl Albumin Binding Moieties on an Integrin αvß6 Binding Peptide.

Serum albumin binding moieties (ABMs) such as the Evans blue (EB) dye fragment and the 4-(p-iodophenyl)butyryl (IP) have been used to improve the pharmacokinetic profile of many radiopharmaceuticals. The goal of this work was to directly compare these two ABMs when conjugated to an integrin αvß6 binding peptide (αvß6-BP); a peptide that is currently being used for positron emission tomography (PET) imaging in patients with metastatic cancer. The ABM-modified αvß6-BP peptides were synthesized with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid (DOTA) chelator for radiolabeling with copper-64 to yield [64Cu]Cu DOTA-EB-αvß6-BP ([64Cu]1) and [64Cu]Cu DOTA-IP-αvß6-BP ([64Cu]2). Both peptides were evaluated in vitro for serum albumin binding, serum stability, and cell binding and internalization in the paired engineered melanoma cells DX3puroß6 (αvß6 +) and DX3puro (αvß6 −), and pancreatic BxPC-3 (αvß6 +) cells and in vivo in a BxPC-3 xenograft mouse model. Serum albumin binding for [64Cu]1 and [64Cu]2 was 53−63% and 42−44%, respectively, with good human serum stability (24 h: [64Cu]1 76%, [64Cu]2 90%). Selective αvß6 cell binding was observed for both [64Cu]1 and [64Cu]2 (αvß6 (+) cells: 30.3−55.8% and 48.5−60.2%, respectively, vs. αvß6 (−) cells <3.1% for both). In vivo BxPC-3 tumor uptake for both peptides at 4 h was 5.29 ± 0.59 and 7.60 ± 0.43% ID/g ([64Cu]1 and [64Cu]2, respectively), and remained at 3.32 ± 0.46 and 4.91 ± 1.19% ID/g, respectively, at 72 h, representing a >3-fold improvement over the non-ABM parent peptide and thereby providing improved PET images. Comparing [64Cu]1 and [64Cu]2, the IP-ABM-αvß6-BP [64Cu]2 displayed higher serum stability, higher tumor accumulation, and lower kidney and liver accumulation, resulting in better tumor-to-organ ratios for high contrast visualization of the αvß6 (+) tumor by PET imaging.

Repurposing an atherosclerosis targeting peptide for tumor imaging.

Cancer and atherosclerosis are chronic diseases that share common characteristics at both early and advanced stages and can arise from multiple factors. Both diseases are characterized by uncontrolled cell proliferation, inflammation, angiogenesis and apoptosis. Herein we investigated the ability of a peptide (CTHRSSVVC), that was previously reported to bind atherosclerotic lesions to home in the tumor microenvironment. The CTHRSSVVC peptide was synthesized on solid phase and N-terminally labeled with a sulfo-Cy5 dye. The specific binding to macrophage was evaluated in vitro with flow cytometry and immunofluorescence and in vivo for tumor targeting in BALB/c mice bearing a 4T1 tumor using optical imaging. The sulfo-Cy5-CTHRSSVVC peptide was synthesized in greater than 99% purity. No selective binding of the sulfo-Cy5-CTHRSSVVC peptide to macrophages in vitro was observed, however in vivo the sulfo-Cy5-CTHRSSVVC peptide accumulated in the 4T1 tumor, with a tumor-to-normal tissue ratio of 7.21 ± 1.44 at 2 h post injection. Ex vivo analysis of tumor tissue by confocal microscopy suggested that the sulfo-Cy5-CTHRSSVVC peptide had accumulated in the stroma of the tumor specifically, in regions of spindle shaped cells. In conclusion, although the target for the sulfo-Cy5-CTHRSSVVC peptide remains to be identified, the Cy5-CTHRSSVVC peptide warrants further investigation as a tumor imaging agent.

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.

Visualizing Cancer.

Imaging has had a profound impact on our ability to understand and treat cancer. We invited some experts to discuss imaging approaches that can be used in various aspects of cancer research, from investigating the complexity and diversity of cancer cells and their environments to guiding clinical decision-making.

αvß6-Targeted Molecular PET/CT Imaging of the Lungs After SARS-CoV-2 Infection.

The true impact and long-term damage to organs such as the lungs after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remain to be determined. Noninvasive molecularly targeted imaging may play a critical role in aiding visualization and understanding of the systemic damage. We have identified αvß6 as a molecular target; an epithelium-specific cell surface receptor that is low or undetectable in healthy adult epithelium but upregulated in select injured tissues, including fibrotic lung. Herein we report the first human PET/CT images using the integrin αvß6-binding peptide (18F-αvß6-BP) in a patient 2 mo after the acute phase of infection. Minimal uptake of 18F-αvß6-BP was noted in normal lung parenchyma, with uptake being elevated in areas corresponding to opacities on CT. This case suggests that 18F-αvß6-BP PET/CT is a promising noninvasive approach to identify the presence and potentially monitor the persistence and progression of lung damage.

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.

Identification, Characterization, and Optimization of Integrin αvß6-Targeting Peptides from a One-Bead One-Compound (OBOC) Library: Towards the Development of Positron Emission Tomography (PET) Imaging Agents.

The current translation of peptides identified through the one-bead one-compound (OBOC) technology into positron emission tomography (PET) imaging agents is a slow process, with a major delay between ligand identification and subsequent lead optimization. This work aims to streamline the development process of 18F-peptide based PET imaging agents to target the integrin αvß6. By directly identify αvß6⁻targeting peptides from a 9-mer 4-fluorobenzoyl peptide library using the on-bead two-color (OBTC) cell-screening assay, a total of 185 peptide beads were identified and 5 beads sequenced for further evaluation. The lead peptide 1 (VGDLTYLKK(FB), IC50 = 0.45 ± 0.06 µM, 25% stable in serum at 1 h) was further modified at the N-, C-, and bi-termini. C-terminal PEGylation increased the metabolic stability (>95% stable), but decreased binding affinity (IC50 = 3.7 ± 1 µM) was noted. C-terminal extension (1i, VGDLTYLKK(FB)KVART) significantly increased binding affinity for integrin αvß6 (IC50 = 0.021 ± 0.002 µM), binding selectivity for αvß6-expressing cells (3.1 ± 0.8:1), and the serum stability (>99% stable). Our results demonstrate the challenges in optimizing OBOC-derived peptides, indicate both termini of 1 are sensitive to modifications, and show that further modification of 1 is necessary to demonstrate utility as an 18F-peptide imaging agent.

Fully automated peptide radiolabeling from [18F]fluoride.

The biological properties of receptor-targeted peptides have made them popular diagnostic imaging and therapeutic agents. Typically, the synthesis of fluorine-18 radiolabeled receptor-targeted peptides for positron emission tomography (PET) imaging is a time consuming, complex, multi-step synthetic process that is highly variable based on the peptide. The complexity associated with the radiolabeling route and lack of robust automated protocols can hinder translation into the clinic. A fully automated batch production to radiolabel three peptides (YGGFL, cRGDyK, and Pyr-QKLGNQWAVGHLM) from fluorine-18 using the ELIXYS FLEX/CHEM® radiosynthesizer in a two-step process is described. First, the prosthetic group, 6-[18F]fluoronicotinyl-2,3,5,6-tetrafluorophenyl ester ([18F]FPy-TFP) was synthesized and subsequently attached to the peptide. The [18F]FPy-peptides were synthesized in 13-26% decay corrected yields from fluorine-18 with high molar activity 1-5 Ci µmol-1 and radiochemical purity of >99% in an overall synthesis time of 97 ± 3 minutes.

Preclinical Development and First-in-Human Imaging of the Integrin αvß6 with [18F]αvß6-Binding Peptide in Metastatic Carcinoma.

PURPOSE: The study was undertaken to develop and evaluate the potential of an integrin αvß6-binding peptide (αvß6-BP) for noninvasive imaging of a diverse range of malignancies with PET. EXPERIMENTAL DESIGN: The peptide αvß6-BP was prepared on solid phase and radiolabeled with 4-[18F]fluorobenzoic acid. In vitro testing included ELISA, serum stability, and cell binding studies using paired αvß6-expressing and αvß6-null cell lines. In vivo evaluation (PET/CT, biodistribution, and autoradiography) was performed in a mouse model bearing the same paired αvß6-expressing and αvß6-null cell xenografts. A first-in-human PET/CT imaging study was performed in patients with metastatic lung, colon, breast, or pancreatic cancer. RESULTS: [18F]αvß6-BP displayed excellent affinity and selectivity for the integrin αvß6 in vitro [IC50(αvß6) = 1.2 nmol/L vs IC50(αvß3) >10 µmol/L] in addition to rapid target-specific cell binding and internalization (72.5% ± 0.9% binding and 52.5% ± 1.8%, respectively). Favorable tumor affinity and selectivity were retained in the mouse model and excretion of unbound [18F]αvß6-BP was rapid, primarily via the kidneys. In patients, [18F]αvß6-BP was well tolerated without noticeable adverse side effects. PET images showed significant uptake of [18F]αvß6-BP in both the primary lesion and metastases, including metastasis to brain, bone, liver, and lung. CONCLUSIONS: The clinical impact of [18F]αvß6-BP PET imaging demonstrated in this first-in-human study is immediate for a broad spectrum of malignancies.

ImmunoPET Imaging of αvß6 Expression Using an Engineered Anti-αvß6 Cys-diabody Site-Specifically Radiolabeled with Cu-64: Considerations for Optimal Imaging with Antibody Fragments.

PURPOSE: Increased expression of the αvß6 integrin correlates with advanced tumor grade and poor clinical outcome, identifying αvß6 as a prognostic indicator and an attractive target for molecular imaging. This work investigated the ability of a disulfide-stabilized [64Cu]NOTA-αvß6 cys-diabody to image αvß6 expression in vivo using a nu/nu mouse model bearing human melanoma xenografts and positron-emission tomography. PROCEDURES: Small-animal positron emission tomography (PET) imaging, quantitative ROI analysis, and ex vivo biodistribution were conducted to ascertain tumor uptake and organ distribution of the [64Cu]NOTA-αvß6 cys-diabody. Immunohistochemical staining of tumors and mouse organs and immunoreactivity assays were utilized to correlate in vivo and ex vivo observations. RESULTS: PET imaging of the [64Cu]NOTA-αvß6 cys-diabody revealed low tumor uptake at 24 h p.i. in DX3Puroß6 tumors (2.69 ± 0.45 %ID/g) with comparable results found in the DX3Puro tumors (2.24 ± 0.15 %ID/g). Quantitative biodistribution confirmed that DX3Puroß6 tumor uptake was highest at 24 h p.i. (4.63 ± 0.18 %ID/g); however, uptake was also observed in the stomach (4.84 ± 2.99 %ID/g), small intestines (4.50 ± 1.69 %ID/g), large intestines (4.73 ± 0.97 %ID/g), gallbladder (6.04 ± 1.88 %ID/g), and lungs (3.89 ± 0.69 %ID/g). CONCLUSIONS: Small-animal PET imaging was successful in visualizing αvß6-positive tumor uptake of the [64Cu]NOTA-αvß6 cys-diabody. Cys-diabody cross-reactivity was observed between human and murine αvß6 and immunohistochemical staining confirmed the presence of an endogenous αvß6 antigen sink, which led to suboptimal tumor contrast in this mouse model. Future investigations will focus on dose escalation studies to overcome the endogenous antigen sink while increasing DX3Puroß6 tumor uptake.

In Vivo Tracking of Copper-64 Radiolabeled Nanoparticles in Lactuca sativa.

Engineered nanoparticles (NPs) are increasingly used in commercial products including automotive lubricants, clothing, deodorants, sunscreens, and cosmetics and can potentially accumulate in our food supply. Given their size it is difficult to detect and visualize the presence of NPs in environmental samples, including crop plants. New analytical tools are needed to fill the void for detection and visualization of NPs in complex biological and environmental matrices. We aimed to determine whether radiolabeled NPs could be used as a noninvasive, highly sensitive analytical tool to quantitatively track and visualize NP transport and accumulation in vivo in lettuce (Lactuca sativa) and to investigate the effect of NP size on transport and distribution over time using a combination of autoradiography, positron emission tomography (PET)/computed tomography (CT), scanning electron microscopy (SEM), and transition electron microscopy (TEM). Azide functionalized NPs were radiolabeled via a "click" reaction with copper-64 (64Cu)-1,4,7-triazacyclononane triacetic acid (NOTA) azadibenzocyclooctyne (ADIBO) conjugate ([64Cu]-ADIBO-NOTA) via copper-free Huisgen-1,3-dipolar cycloaddition reaction. This yielded radiolabeled [64Cu]-NPs of uniform shape and size with a high radiochemical purity (>99%), specific activity of  2.2 mCi/mg of NP, and high stability (i.e., no detectable dissolution) over 24 h across a pH range of 5-9. Both PET/CT and autoradiography showed that [64Cu]-NPs entered the lettuce seedling roots and were rapidly transported to the cotyledons with the majority of the accumulation inside the roots. Uptake and transport of intact NPs was size-dependent, and in combination with the accumulation within the roots suggests a filtering effect of the plant cell walls at various points along the water transport pathway.

High-Throughput Approaches to the Development of Molecular Imaging Agents.

Molecular imaging allows for the visualization of changes at the cellular level in diseases such as cancer. A successful molecular imaging agent must rely on disease-selective targets and ligands that specifically interact with those targets. Unfortunately, the translation of novel target-specific ligands into the clinic has been frustratingly slow with limitations including the complex design and screening approaches for ligand identification, as well as their subsequent optimization into useful imaging agents. This review focuses on combinatorial library approaches towards addressing these two challenges, with particular focus on phage display and one-bead one-compound (OBOC) libraries. Both of these peptide-based techniques have proven successful in identifying new ligands for cancer-specific targets and some of the success stories will be highlighted. New developments in screening methodology and sequencing technology have pushed the bounds of phage display and OBOC even further, allowing for even faster and more robust discovery of novel ligands. The combination of multiple high-throughput technologies will not only allow for more accurate identification, but also faster affinity maturation, while overall streamlining the process of translating novel ligands into clinical imaging agents.

Solid-phase synthesis and fluorine-18 radiolabeling of cycloRGDyK.

Solid-phase peptide synthesis, head-to-tail cyclization, and subsequent radiolabeling provided a reproducible, simple, rapid synthetic method to generate the cyclic peptide radiotracer cRGDyK([18F]FBA). Herein is reported the first on-resin cyclization and 18F-radiolabeling of a cyclic peptide (cRGDyK) in an overall peptide synthesis yield of 88% (cRGDyK(NH2)) and subsequent radiolabeling yield of 14 ± 2% (decay corrected, n = 4). This approach is generally applicable to the development of an automated process for the synthesis of cyclic radiolabeled peptides for positron emission tomography (PET).

Brain penetrant small molecule (18)F-GnRH receptor (GnRH-R) antagonists: Synthesis and preliminary positron emission tomography imaging in rats.

INTRODUCTION: The gonadotropin releasing hormone receptor (GnRH-R) has a well-described neuroendocrine function in the anterior pituitary. However, little is known about its function in the central nervous system (CNS), where it is most abundantly expressed in hippocampus and amygdala. Since peptide ligands based upon the endogenous decapetide GnRH do not pass the blood-brain-barrier, we are seeking a high-affinity small molecule GnRH-R ligand suitable for brain imaging by positron emission tomography. We have previously reported the radiosynthesis and in vitro evaluation of two novel [(18)F]fluorinated GnRH-R ligands belonging to the furamide class of antagonists, with molecular weight less than 500 Da. We now extend this work using palladium coupling for the synthesis of four novel radioligands, with putatively reduced polar surface area and hydrophilicity relative to the two previously described compounds, and report the uptake of these (18)F-labeled compounds in brain of living rats. METHODS: We synthesized reference standards of the small molecule GnRH-R antagonists as well as mesylate precursors for (18)F-labeling. The antagonists were tested for binding affinity for both human and rat GnRH-R. Serum and blood stability in vitro and in vivo were studied. Biodistribution and PET imaging studies were performed in male rats in order to assess brain penetration in vivo. RESULTS: A palladium coupling methodology served for the synthesis of four novel fluorinated furamide GnRH receptor antagonists with reduced heteroatomic count. Radioligand binding assays in vitro revealed subnanomolar affinity of the new fluorinated compounds for both human and rat GnRH-R. The (18)F-GnRH antagonists were synthesized from the corresponding mesylate precursors in 5-15% overall radiochemical yield. The radiolabeled compounds demonstrated good in vivo stability. PET imaging with the (18)F-radiotracers in naive rats showed good permeability into brain and rapid washout, but absence of discernible specific binding in vivo. CONCLUSIONS: The novel small molecule (18)F-fluorinated GnRH-R antagonist compounds show high receptor affinity in vitro, and may prove useful for quantitative autoradiographic studies in vitro. The compounds were permeable to the blood-brain barrier, but nonetheless failed to reveal significant specific binding in brain of living rats. Nonetheless, our approach may serve as a foundation for designing PET ligands suitable to image the GnRH-R distribution in brain.

Development and characterization of an αvß6-specific diabody and a disulfide-stabilized αvß6-specific cys-diabody.

INTRODUCTION: This work describes the development and characterization of two antibody fragments that specifically target the α(v)ß(6) integrin, a non-covalent diabody and a disulfide-stabilized cys-diabody. The diabodies were analyzed for their ability to bind both immobilized and cell surface-bound α(v)ß(6). Radiolabeling was done using non-site-specific and site-specific conjugation approaches with N-succinimidyl 4-[(18)F]fluorobenzoate ([(18)F]-SFB) and the bifunctional chelator 1,4,7-triazacyclononane-triacetic acid maleimide (NOTA-maleimide) and copper-64 ([(64)Cu]), respectively. The affects of each radiolabeling method on RCY, RCP, and immunoreactivity were analyzed for the [(18)F]-FB-α(v)ß(6) diabody, [(18)F]-FB-α(v)ß(6) cys-diabody, and the [(64)Cu]-NOTA-α(v)ß(6) cys-diabody. METHODS: Diabodies were constructed from the variable domains of the humanized 6.3G9 anti-α(v)ß(6) intact antibody. The anti-α(v(ß(6) cys-diabody was engineered with C-terminal cysteines to enable covalent dimerization and site-specific modification. Biochemical characterization included SDS-PAGE, Western blot, and electrospray ionization to confirm MW, and flow cytometry and ELISA experiments were used to determine binding affinity and specificity to α(v)ß(6). The diabodies were radiolabeled with [(18)F]-SFB and in addition, the anti-α(v)ß(6) cys-diabody was also radiolabeled site-specifically using NOTA-maleimide and [(64)Cu]. Immunoreactivities were confirmed using in vitro cell binding to DX3Puroß(6) (α(v)ß(6)+) and DX3Puro (α(v)ß(6)-)cell lines. RESULTS: The diabodies were purified from cell culture supernatants with purities >98%. Subnanomolar binding affinity towards αvß6 was confirmed by ELISA (diabody IC(50)=0.8 nM, cys-diabody IC(50)=0.6 nM) and flow cytometry revealed high specificity only to the DX3Puroß(6) cell line for both diabodies. RCYs were 22.6%±3.6% for the [(18)F]-FB-α(v)ß(6) diabody, 8.3%±1.7% for the [(18)F]-FB-α(v)ß(6) cys-diabody and 43.5%±5.5% for the [(64)Cu]-NOTA-α(v)ß(6) cys-diabody. In vitro cell binding assays revealed excellent specificity and retention of immunoreactivity ([(18)F]-FB-α(v)ß(6) diabody=58.7%±6.7%, [(18)F]-FB-α(v)ß(6) cys-diabody=80.4%±4.4%, [(64)Cu]-NOTA-α(v)ß(6) cys-diabody=59.4%±0.6%) regardless of the radiolabeling method used. CONCLUSIONS: Two novel diabodies with excellent binding affinity and specificity for the α(v)ß(6) integrin in vitro were developed. Radiolabeling of the diabodies with fluorine-18 ([(18)F]) and [(64)Cu] revealed advantages and disadvantages with regards to methodologies and RCYs, however immunoreactivities were well preserved regardless of radiolabeling approach.

The Effect of Bi-Terminal PEGylation of an Integrin αvß6-Targeted ¹8F Peptide on Pharmacokinetics and Tumor Uptake.

UNLABELLED: Radiotracers based on the peptide A20FMDV2 selectively target the cell surface receptor integrin αvß6. This integrin has been identified as a prognostic indicator correlating with the severity of disease for several challenging malignancies. In previous studies of A20FMDV2 peptides labeled with 4-(18)F-fluorobenzoic acid ((18)F-FBA), we have shown that the introduction of poly(ethylene glycol) (PEG) improves pharmacokinetics, including increased uptake in αvß6-expressing tumors. The present study evaluated the effect of site-specific C-terminal or dual (N- and C-terminal) PEGylation, yielding (18)F-FBA-A20FMDV2-PEG28 (4) and (18)F-FBA-PEG28-A20FMDV2-PEG28 (5), on αvß6-targeted tumor uptake and pharmacokinetics. The results are compared with (18)F-FBA -labeled A20FMDV2 radiotracers (1- 3) bearing either no PEG or different PEG units at the N terminus. METHODS: The radiotracers were prepared and radiolabeled on solid phase. Using 3 cell lines, DX3puroß6 (αvß6+), DX3puro (αvß6-), and BxPC-3 (αvß6+), we evaluated the radiotracers in vitro (serum stability; cell binding and internalization) and in vivo in mouse models bearing paired DX3puroß6-DX3puro and, for 5, BxPC-3 xenografts. RESULTS: The size and location of the PEG units significantly affected αvß6 targeting and pharmacokinetics. Although the C-terminally PEGylated 4 showed some improvements over the un-PEGylated (18)F-FBA-A20FMDV2 (1), it was the bi-terminally PEGylated 5 that displayed the more favorable combination of high αvß6 affinity, selectivity, and pharmacokinetic profile. In vitro, 5 bound to αvß6-expressing DX3puroß6 and BxPC-3 cells with 60.5% ± 3.3% and 48.8% ± 8.3%, respectively, with a significant fraction of internalization (37.2% ± 4.0% and 37.6% ± 4.1% of total radioactivity, respectively). By comparison, in the DX3puro control 5: showed only 3.0% ± 0.5% binding and 0.9% ± 0.2% internalization. In vivo, 5: maintained high, αvß6-directed binding in the paired DX3puroß6-DX3puro model (1 h: DX3puroß6, 2.3 ± 0.2 percentage injected dose per gram [%ID/g]; DX3puroß6/DX3puro ratio, 6.5:1; 4 h: 10.7:1). In the pancreatic BxPC-3 model, uptake was 4.7 ± 0.9 %ID/g (1 h) despite small tumor sizes (20-80 mg). CONCLUSION: The bi-PEGylated radiotracer 5 showed a greatly improved pharmacokinetic profile, beyond what was predicted from individual N- or C-terminal PEGylation. It appears that the 2 PEG units acted synergistically to result in an improved metabolic profile including high αvß6+ tumor uptake and retention.