Radiolabeling Diaminosarcophagine with Cyclotron-Produced Cobalt-55 and [55Co]Co-NT-Sarcage as a Proof of Concept in a Murine Xenograft Model.

Cobalt-sarcophagine complexes exhibit high kinetic inertness under various stringent conditions, but there is limited literature on radiolabeling and in vivo positron emission tomography (PET) imaging using no carrier added 55Co. To fill this gap, this study first investigates the radiolabeling of DiAmSar (DSar) with 55Co, followed by stability evaluation in human serum and EDTA, pharmacokinetics in mice, and a direct comparison with [55Co]CoCl2 to assess differences in pharmacokinetics. Furthermore, the radiolabeling process was successfully used to generate the NTSR1-targeted PET agent [55Co]Co-NT-Sarcage (a DSar-functionalized SR142948 derivative) and administered to HT29 tumor xenografted mice. The [55Co]Co-DSar complex can be formed at 37 °C with purity and stability suitable for preclinical in vivo radiopharmaceutical applications, and [55Co]Co-NT-Sarcage demonstrated prominent tumor uptake with a low background signal. In a direct comparison with [64Cu]Cu-NT-Sarcage, [55Co]Co-NT-Sarcage achieved a higher tumor-to-liver ratio but with overall similar biodistribution profile. These results demonstrate that Sar would be a promising chelator for constructing Co-based radiopharmaceuticals including 55Co for PET and 58mCo for therapeutic applications.

Porphyrin-Based Brain Tumor-Targeting Agents: [64Cu]Cu-porphyrin and [64Cu]Cu-TDAP.

The aim of this study is to evaluate a radioactive metal complex platform for brain tumor targeting. Herein, we introduce a new porphyrin derivative, 5,10,15,20-(tetra-N,N-dimethyl-4-aminophenyl)porphyrin (TDAP), in which four N,N-dimethyl-4-p-phenylenediamine (DMPD) moieties are conjugated to the porphyrin labeled with the radiometal 64Cu. DMPD affected the pharmacokinetics of porphyrin in terms of retention time in vivo and tumor-targeting ability relative to those of unmodified porphyrin. [64Cu]Cu-TDAP showed stronger enhancement than [64Cu]Cu-porphyrin in U87MG glioblastoma cells, especially in the cytoplasm and nucleus, indicating its tumor-targeting properties and potential use as a therapeutic agent. In the subcutaneous and orthotopic models of brain-tumor-bearing mice, [64Cu]Cu-TDAP was clearly visualized in the tumor site via positron emission tomography imaging and showed a tumor-to-brain ratio as high as 13. [64Cu]Cu-TDAP deserves attention as a new diagnostic agent that is suitable for the early diagnosis and treatment of brain tumors.

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.

Copper 64-labeled daratumumab as a PET/CT imaging tracer for multiple myeloma.

As a growing number of patients with multiple myeloma (MM) respond to upfront therapies while eventually relapsing in a time frame that is often unpredictable, attention has increasingly focused on developing novel diagnostic criteria to also account for disease dissemination. Positron emission tomography/computed tomography (PET/CT) is often used as a noninvasive monitoring strategy to assess cancer cell dissemination, but because the uptake of the currently used radiotracer 18fluorodeoxyglucose (18F-FDG) is a function of the metabolic activity of both malignant and nonmalignant cells, the results frequently lack sufficient specificity. Radiolabeled antibodies targeting MM tissue may detect disease irrespective of cell metabolism. Hence, we conjugated the clinically significant CD38-directed human antibody daratumumab (Darzalex [Dara]) to the DOTA chelator and labeled it with the positron-emitting radionuclide copper 64 (64Cu; 64Cu-DOTA-Dara). Here, we show that 64Cu-DOTA-Dara can efficiently bind CD38 on the surface of MM cells and was mainly detected in the bones associated with tumor in a MM murine model. We also show that PET/CT based on 64Cu-DOTA-Dara displays a higher resolution and specificity to detect MM cell dissemination than does 18F-FDG PET/CT and was even more sensitive than were bioluminescence signals. We therefore have supporting evidence for using 64Cu-DOTA-Dara as a novel imaging agent for MM.

Synthesis and Analysis of 64Cu-Labeled GE11-Modified Polymeric Micellar Nanoparticles for EGFR-Targeted Molecular Imaging in a Colorectal Cancer Model.

Polymeric micellar nanoparticles represent versatile and biocompatible platforms for targeted drug delivery. However, tracking their biodistribution, stability, and clearance profile in vivo is challenging. The goal of this study was to prepare surface-modified micelles with peptide GE11 for targeting the epidermal growth factor receptor (EGFR). In vitro fluorescence studies demonstrated significantly higher internalization of GE11 micelles into EGFR-expressing HCT116 colon cancer cells versus EGFR-negative SW620 cells. Azo coupling chemistry of tyrosine residues in the peptide backbone with aryl diazonium salts was used to label the micelles with radionuclide 64Cu for positron emission tomography (PET) imaging. In vivo analysis of 64Cu-labeled micelles showed prolonged blood circulation and predominant hepatobiliary clearance. The biodistribution profile of EGFR-targeting GE11 micelles was compared with nontargeting HW12 micelles in HCT116 tumor-bearing mice. PET revealed increasing tumor-to-muscle ratios for both micelles over 48 h. Accumulation of GE11-containing micelles in HCT116 tumors was higher compared to HW12-decorated micelles. Our data suggest that the efficacy of image-guided therapies with micellar nanoparticles could be enhanced by active targeting, as demonstrated with cancer biomarker EGFR.

Size-Optimized Ultrasmall Porous Silica Nanoparticles Depict Vasculature-Based Differential Targeting in Triple Negative Breast Cancer.

Rapid sequestration and prolonged retention of intravenously injected nanoparticles by the liver and spleen (reticuloendothelial system (RES)) presents a major barrier to effective delivery to the target site and hampers clinical translation of nanomedicine. Inspired by biological macromolecular drugs, synthesis of ultrasmall (diameter ≈12-15 nm) porous silica nanoparticles (UPSNs), capable of prolonged plasma half-life, attenuated RES sequestration, and accelerated hepatobiliary clearance, is reported. The study further investigates the effect of tumor vascularization on uptake and retention of UPSNs in two mouse models of triple negative breast cancer with distinctly different microenvironments. A semimechanistic mathematical model is developed to gain mechanistic insights into the interactions between the UPSNs and the biological entities of interest, specifically the RES. Despite similar systemic pharmacokinetic profiles, UPSNs demonstrate strikingly different tumor responses in the two models. Histopathology confirms the differences in vasculature and stromal status of the two models, and corresponding differences in the microscopic distribution of UPSNs within the tumors. The studies demonstrate the successful application of multidisciplinary and complementary approaches, based on laboratory experimentation and mathematical modeling, to concurrently design optimized nanomaterials, and investigate their complex biological interactions, in order to drive innovation and translation.

Preclinical evaluation of a 64Cu-labeled disintegrin for PET imaging of prostate cancer.

A novel recombinant disintegrin, vicrostatin (VCN), displays high binding affinity to a broad range of human integrins in substantial competitive biological advantage over other integrin-based antagonists. In this study, we synthesized a new 64Cu-labeled VCN probe and evaluated its imaging properties for prostate cancer in PC-3 tumor-bearing mice. Macrocyclic chelating agent 1,8-diamino-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosine (DiAmSar) was conjugated with PEG unit and followed by coupling with VCN. The precursor was then radiolabeled with positron emitter 64Cu (t1/2 = 12.7 h) in ammonium acetate buffer to provide 64Cu-Sar-PEG-VCN, which was subsequently subjected to in vitro studies, small animal PET, and biodistribution studies. The PC-3 tumor-targeting efficacy of 64Cu-Sar-PEG-VCN was compared to a cyclic RGD peptide-based PET probe (64Cu-Sar-RGD). 64Cu labeling was achieved in 75% decay-corrected yield with radiochemical purity of > 98%. The specific activity of 64Cu-Sar-PEG-VCN was estimated to be 37 MBq/nmol. MicroPET imaging results showed that 64Cu-Sar-PEG-VCN has preferential tumor uptake and good tumor retention in PC-3 tumor xenografts. As compared to 64Cu-Sar-RGD, 64Cu-Sar-PEG-VCN produces higher tumor-to-muscle (T/M) imaging contrast ratios at 2 h (4.66 ± 0.34 vs. 2.88 ± 0.46) and 24 h (4.98 ± 0.80 vs. 3.22 ± 0.30) post-injection (pi) and similar tumor-to-liver ratios at 2 h (0.43 ± 0.09 vs. 0.37 ± 0.04) and 24 h (0.57 ± 0.13 vs. 0.52 ± 0.07) pi. The biodistribution results were consistent with the quantitative analysis of microPET imaging, demonstrating good T/M ratio (2.73 ± 0.36) of 64Cu-Sar-PEG-VCN at 48 h pi in PC-3 tumor xenografts. For both microPET and biodistribution studies at 48 h pi, the PC-3 tumor uptake of 64Cu-Sar-PEG-VCN is lower than that of 64Cu-Sar-RGD. 64Cu-Sar-PEG-VCN has the potential for in vivo imaging of prostate cancer with PET, which may provide a unique non-invasive method to quantitatively localize and characterize prostate cancer.

Targeting CAIX with [64Cu]XYIMSR-06 Small Molecular Radiotracer Enables Noninvasive PET Imaging of Malignant Glioma in U87 MG Tumor Cell Xenograft Mice.

Carbonic anhydrase IX (CAIX) plays an important role in glioma cell proliferation, invasion, metastasis, and resistance to radiotherapy and chemotherapy. An effective and noninvasive PET molecular imaging agent targeting CAIX would help its diagnosis and treatment but is not currently available. Recently, a low-molecular-weight (LMW) CAIX targeting agent, [64Cu]XYIMSR-06, was reported to have significantly improved properties for targeting clear cell renal cell carcinoma (ccRCC). We are encouraged to investigate the feasibility of adapting this agent for the diagnosis and treatment of CAIX-overexpressing malignant glioma. In vitro cell uptake and binding affinity assays were used to verify the binding capacity of [64Cu]XYIMSR-06 to U87 MG tumor cells in which CAIX overexpression was confirmed. The U87 MG tumor-bearing mouse (in situ and subcutaneous) model was built, and mice were injected with the radiotracer and/or coinjected with acetazolamide (0.2 g/kg) as a blocking agent for noninvasive micro-PET imaging. Micro-PET imaging was performed at 2, 4, and 8 h postinjection. ROI (region of interest)-based semiquantification was performed in an orthotopic glioma tumor model. Biodistribution throughout each organ was performed at 2, 4, 4 h block, 8, and 24 h postinjection. Hematoxylin and eosin (HE) staining and immunofluorescence or immunohistochemistry (IF/IHC) staining were implemented postimaging to assess the expression of CAIX in tumor organs. In vitro, [64Cu]XYIMSR-06 exhibits greater uptake in glioma cells (high CAIX expression) than in HCT116 cells (low CAIX expression). The binding affinity of [64Cu]XYIMSR-06 to U87 MG cell lines reaches up to 4.22 nM. Both orthotopic and subcutaneous tumors were clearly visualized at 2-8 h postinjection. Biodistribution studies demonstrated a maximum tumor uptake of 3.13% ID/g at 4 h postinjection, and the tumor to brain ratio (T/brain) was 6.51 at 8 h postinjection. The ROI-based T/brain values were 7.03 and 5.46 at 2 and 8 h postinjection, respectively. Histopathological analysis confirmed the overexpression of CAIX in gliomas, and the area of CAIX-positive IF staining is extremely consistent with the morphology on micro-PET imaging. In this study, [64Cu]XYIMSR-06 demonstrated specific accumulation in CAIX-expressing U87 MG glioma tumors, indicating that the radiotracer has the potential for noninvasively monitoring and guiding personalized treatment of malignant glioma and other tumors overexpressing CAIX.

Imaging of changes in copper trafficking and redistribution in a mouse model of Niemann-Pick C disease using positron emission tomography.

Niemann-Pick C disease (NPC) is an autosomal recessive lysosomal storage disorder resulting from mutations in the NPC1 (95% of cases) or NPC2 genes. Disturbance of copper homeostasis has been reported in NPC1 disease. In this study we have used whole-body positron emission tomography (PET) and brain electronic autoradiography with copper-64 (64Cu), in the form of the copper(II) bis(thiosemicarbazonato) complex 64Cu-GTSM, to image short-term changes in copper trafficking after intravenous injection in a transgenic mouse model of NPC1 disease. 64Cu-GTSM is taken up in all tissues and dissociates rapidly inside cells, allowing monitoring of the subsequent efflux and redistribution of 64Cu from all tissues. Significantly enhanced retention of 64Cu radioactivity was observed in brain, lungs and blood at 15 h post-injection in symptomatic Npc1-/- transgenic mice compared to wildtype controls. The enhanced retention of 64Cu in brain was confirmed by electronic autoradiography, particularly in the midbrain, thalamus, medulla and pons regions. Positron emission tomography imaging with 64Cu in selected chemical forms could be a useful diagnostic and research tool for the management and understanding of NPC1 disease.

What is the Best Radionuclide for Immuno-PET of Multiple Myeloma? A Comparison Study Between 89Zr- and 64Cu-Labeled Anti-CD138 in a Preclinical Syngeneic Model.

Although positron emission tomography (PET) imaging with 18-Fluorodeoxyglucose (18F-FDG) is a promising technique in multiple myeloma (MM), the development of other radiopharmaceuticals seems relevant. CD138 is currently used as a standard marker for the identification of myeloma cells and could be used in phenotype tumor imaging. In this study, we used an anti-CD138 murine antibody (9E7.4) radiolabeled with copper-64 (64Cu) or zirconium-89 (89Zr) and compared them in a syngeneic mouse model to select the optimal tracers for MM PET imaging. Then, 9E7.4 was conjugated to TE2A-benzyl isothiocyanate (TE2A) and desferrioxamine (DFO) chelators for 64Cu and 89Zr labeling, respectively. 64Cu-TE2A-9E7.4 and 89Zr-DFO-9E7.4 antibodies were evaluated by PET imaging and biodistribution studies in C57BL/KaLwRij mice bearing either 5T33-MM subcutaneous tumors or bone lesions and were compared to 18F-FDG-PET imaging. In biodistribution and PET studies, 64Cu-TE2A-9E7.4 and 89Zr-DFO-9E7.4 displayed comparable good tumor uptake of subcutaneous tumors. On the bone lesions, PET imaging with 64Cu-TE2A-9E7.4 and 89Zr-DFO-9E7.4 showed higher uptake than with 18F-FDG-PET. Comparison of both 9E7.4 conjugates revealed higher nonspecific bone uptakes of 89Zr-DFO-9E7.4 than 64Cu-TE2A-9E7.4. Because of free 89Zr's tropism for bone when using 89Zr-anti-CD138, 64Cu-anti-CD138 antibody had the most optimal tumor-to-nontarget tissue ratios for translation into humans as a specific new imaging radiopharmaceutical agent in MM.

Novel 64Cu Labeled RGD2-BBN Heterotrimers for PET Imaging of Prostate Cancer.

Bombesin receptor 2 (BB2) and integrin αvß3 receptor are privileged targets for molecular imaging of cancer because of their overexpression in a number of tumor tissues. The most recent developments in heterodimer-based radiopharmaceuticals concern BB2- and integrin αvß3-targeting compounds, consisting of bombesin (BBN) and cyclic arginine-glycine-aspartic acid peptides (RGD), connected through short length linkers. Molecular imaging probes based on RGD-BBN heterodimer design exhibit improved tumor targeting efficacy compared to the single-receptor targeting peptide monomers. However, their application in clinical study is restricted because of inefficient synthesis or unfavorable in vivo properties, which could depend on the short linker nature. Thus, the aim of the present study was to develop a RGD2-BBN heterotrimer, composed of (7-14)BBN-NH2 peptide (BBN) linked to the E[ c(RGDyK)]2 dimer peptide (RGD2), bearing the new linker type [Pro-Gly]12. The heterodimer E[c(RGDyK)]2-PEG3-Glu-(Pro-Gly)12-BBN(7-14)-NH2 (RGD2-PG12-BBN) was prepared through conventional solid phase synthesis, then conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODA-GA). In 64Cu labeling, the NODA-GA chelator showed superior radiochemical characteristics compared to DOTA (70% vs 40% yield, respectively). Both conjugates displayed dual targeting ability, showing good αvß3 affinities and high BB2 receptor affinities which, in the case of the NODA-GA conjugate, were in the same range as the best RGD-BBN heterodimer ligands reported to date ( Ki = 24 nM). 64Cu-DOTA and 64Cu-NODA-GA probes were also found to be stable after 1 h incubation in mouse serum (>90%). In a microPET study in prostate cancer PC-3 xenograft mice, both probes showed low tumor uptake, probably due to poor pharmacokinetic properties in vivo. Overall, our study demonstrates that novel RGD-BBN heterodimer with long linker can be prepared and they preserve high binding affinities to BB2 and integrin αvß3 receptor binding ability. The present study represents a step forward in the design of effective heterodimer or heterotrimer probes for dual targeting.

NLS-Cholic Acid Conjugation to IL-5Rα-Specific Antibody Improves Cellular Accumulation and In Vivo Tumor-Targeting Properties in a Bladder Cancer Model.

Receptor-mediated internalization followed by trafficking and degradation of antibody-conjugates (ACs) via the endosomal-lysosomal pathway is the major mechanism for delivering molecular payloads inside target tumor cells. Although a mainstay for delivering payloads with clinically approved ACs in cancer treatment and imaging, tumor cells are often able to decrease intracellular payload concentrations and thereby reduce the effectiveness of the desired application. Thus, increasing payload intracellular accumulation has become a focus of attention for designing next-generation ACs. We developed a composite compound (ChAcNLS) that enables ACs to escape endosome entrapment and route to the nucleus resulting in the increased intracellular accumulation as an interleukin-5 receptor α-subunit (IL-5Rα)-targeted agent for muscle invasive bladder cancer (MIBC). We constructed 64Cu-A14-ChAcNLS, 64Cu-A14-NLS, and 64Cu-A14 and evaluated their performance by employing mechanistic studies for endosome escape coupled to nuclear routing and determining whether this delivery system results in improved 64Cu cellular accumulation. ACs consisting of ∼20 ChAcNLS or NLS moieties per 64Cu-A14 were prepared in good yield, high monomer content, and maintaining high affinity for IL-5Rα. Confocal microscopy analysis demonstrated ChAcNLS mediated efficient endosome escape and nuclear localization. 64Cu-A14-ChAcNLS increased 64Cu cellular accumulation in HT-1376 and HT-B9 cells relative to 64Cu-A14 and 64Cu-A14-NLS. In addition, we tested 64Cu-A14-ChAcNLS in vivo to evaluate its tissue distribution properties and, ultimately, tumor uptake and targeting. A model of human IL-5Rα MIBC was developed by implanting NOD/SCID mice with subcutaneous HT-1376 or HT-B9MIBC tumors, which grow containing high and low IL-5Rα-positive tumor cell densities, respectively. ACs were intravenously injected, and daily blood sampling, biodistribution at 48 and 96 h, and positron emission tomography (PET) at 24 and 48 h were performed. Region of interest (ROI) analysis was also performed on reconstructed PET images. Pharmacokinetic analysis and biodistribution studies showed that 64Cu-A14-ChAcNLS had faster clearance rates from the blood and healthy organs relative to 64Cu-A14. However, 64Cu-A14-ChAcNLS maintained comparable tumor accumulation relative to 64Cu-A14. This resulted in 64Cu-A14-ChAcNLS having superior tumor/normal tissue ratios at both 48 and 96 h biodistribution time points. Visualization of AC distribution by PET and ROI analysis confirmed that 64Cu-A14-ChAcNLS had improved targeting of MIBC tumor relative to 64Cu-A14. In addition, 64Cu-A14 modified with only NLS had poor tumor targeting. This was a result of poor tumor uptake due to extremely rapid clearance. Thus, the overall findings in this model of human IL-5Rα-positive MIBC describe an endosome escape-nuclear localization cholic-acid-linked peptide that substantially enhances AC cellular accumulation and tumor targeting.

Engineered Charge Redistribution of Gp2 Proteins through Guided Diversity for Improved PET Imaging of Epidermal Growth Factor Receptor.

The Gp2 domain is a protein scaffold for synthetic ligand engineering. However, the native protein function results in a heterogeneous distribution of charge on the conserved surface, which may hinder further development and utility. We aim to modulate charge, without diminishing function, which is challenging in small proteins where each mutation is a significant fraction of protein structure. We constructed rationally guided combinatorial libraries with charge-neutralizing or charge-flipping mutations and sorted them, via yeast display and flow cytometry, for stability and target binding. Deep sequencing of functional variants revealed effective mutations both in clone-dependent contexts and broadly across binders to epidermal growth factor receptor (EGFR), insulin receptor, and immunoglobulin G. Functional mutants averaged 4.3 charge neutralizing mutations per domain while maintaining net negative charge. We evolved an EGFR-targeted Gp2 mutant that reduced charge density by 33%, maintained net charge, and improved charge distribution homogeneity while elevating thermal stability ( Tm = 87 ± 1 °C), improving binding specificity, and maintaining affinity ( Kd = 8.8 ± 0.6 nM). This molecule was conjugated with 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid for 64Cu chelation and evaluated for physiological distribution in mice with xenografted A431 (EGFRhigh) and MDA-MB-435 (EGFRlow) tumors. Excised tissue gamma counting and positron emission tomography/computed tomography imaging revealed good EGFRhigh tumor signal (4.7 ± 0.5%ID/g) at 2 h post-injection and molecular specificity evidenced by low uptake in EGFRlow tumors (0.6 ± 0.1%ID/g, significantly lower than for non-charge-modified Gp2, p = 0.01). These results provide charge mutations for an improved Gp2 framework, validate an effective approach to charge engineering, and advance performance of physiological EGFR targeting for molecular imaging.

Tuning Pharmacokinetics to Improve Tumor Accumulation of a Prostate-Specific Membrane Antigen-Targeted Phototheranostic Agent.

We describe a simple and effective bioconjugation strategy to extend the plasma circulation of a low molecular weight targeted phototheranostic agent, which achieves high tumor accumulation (9.74 ± 2.26%ID/g) and high tumor-to-background ratio (10:1). Long-circulating pyropheophorbide (LC-Pyro) was synthesized with three functional building blocks: (1) a porphyrin photosensitizer for positron-emission tomography (PET)/fluorescence imaging and photodynamic therapy (PDT), (2) a urea-based prostate-specific membrane antigen (PSMA) targeting ligand, and (3) a peptide linker to prolong the plasma circulation time. With porphyrin's copper-64 chelating and optical properties, LC-Pyro demonstrated its dual-modality (fluorescence/PET) imaging potential for selective and quantitative tumor detection in subcutaneous, orthotopic, and metastatic murine models. The peptide linker in LC-Pyro prolonged its plasma circulation time about 8.5 times compared to its truncated analog. High tumor accumulation of LC-Pyro enabled potent PDT, which resulted in significantly delayed tumor growth in a subcutaneous xenograft model. This approach can be applied to improve the pharmacokinetics of existing and future targeted PDT agents for enhanced tumor accumulation and treatment efficacy.

Liposome accumulation in irradiated tumors display important tumor and dose dependent differences.

Radiation therapy may affect several important parameters in the tumor microenvironment and thereby influence the accumulation of liposomes by the enhanced permeability and retention (EPR)-effect. Here we investigate the effect of single dose radiation therapy on liposome tumor accumulation by PET/CT imaging using radiolabeled liposomes. Head and neck cancer xenografts (FaDu) and syngenic colorectal (CT26) cancer models were investigated. Radiotherapy displayed opposite effects in the two models. FaDu tumors displayed increased mean accumulation of liposomes for radiation doses up to 10 Gy, whereas CT26 tumors displayed a tendency for decreased accumulation. Tumor hypoxia was found negatively correlated to microregional distribution of liposomes. However, liposome distribution in relation to hypoxia was improved at lower radiation doses. The study reveals that the heterogeneity in liposome tumor accumulation between tumors and different radiation protocols are important factors that need to be taken into consideration to achieve optimal effect of liposome based radio-sensitizer therapy.

Evaluation of 64Cu-Based Radiopharmaceuticals that Target Aß Peptide Aggregates as Diagnostic Tools for Alzheimer's Disease.

Positron emission tomography (PET) imaging agents that detect amyloid plaques containing amyloid beta (Aß) peptide aggregates in the brain of Alzheimer's disease (AD) patients have been successfully developed and recently approved by the FDA for clinical use. However, the short half-lives of the currently used radionuclides 11C (20.4 min) and 18F (109.8 min) may limit the widespread use of these imaging agents. Therefore, we have begun to evaluate novel AD diagnostic agents that can be radiolabeled with 64Cu, a radionuclide with a half-life of 12.7 h, ideal for PET imaging. Described herein are a series of bifunctional chelators (BFCs), L1-L5, that were designed to tightly bind 64Cu and shown to interact with Aß aggregates both in vitro and in transgenic AD mouse brain sections. Importantly, biodistribution studies show that these compounds exhibit promising brain uptake and rapid clearance in wild-type mice, and initial microPET imaging studies of transgenic AD mice suggest that these compounds could serve as lead compounds for the development of improved diagnostic agents for AD.

Rapid PD-L1 detection in tumors with PET using a highly specific peptide.

Molecular imaging can report on the status of the tumor immune microenvironment and guide immunotherapeutic strategies to enhance the efficacy of immune modulation therapies. Imaging agents that can rapidly report on targets of immunomodulatory therapies are few. The programmed death ligand 1 (PD-L1) is an immune checkpoint protein over-expressed in several cancers and contributes to tumor immune suppression. Tumor PD-L1 expression is indicative of tumor response to PD-1 and PD-L1 targeted therapies. Herein, we report a highly specific peptide-based positron emission tomography (PET) imaging agent for PD-L1. We assessed the binding modes of the peptide WL12 to PD-L1 by docking studies, developed a copper-64 labeled WL12 ([64Cu]WL12), and performed its evaluation in vitro, and in vivo by PET imaging, biodistribution and blocking studies. Our results show that [64Cu]WL12 can be used to detect tumor PD-L1 expression specifically and soon after injection of the radiotracer, to fit within the standard clinical workflow of imaging within 60 min of administration.

64Cu-Labeled Trastuzumab Fab-PEG24-EGF Radioimmunoconjugates Bispecific for HER2 and EGFR: Pharmacokinetics, Biodistribution, and Tumor Imaging by PET in Comparison to Monospecific Agents.

Heterodimerization of EGFR with HER2 coexpressed in breast cancer (BC) promotes tumor growth, and increased EGFR expression is associated with trastuzumab resistance. Our aim was to construct 64Cu-labeled bispecific radioimmunoconjugates (bsRIC) composed of trastuzumab Fab, which binds HER2 linked through a polyethylene glycol (PEG24) spacer to EGF, and to compare their pharmacokinetic, biodistribution, and tumor imaging characteristics by positron-emission tomography (PET). bsRICs were generated by linking maleimide modified trastuzumab Fab with thiolated EGF through a thioether bond. HER2 and EGFR binding were assessed in vitro in MDA-MB-231 (EGFRmod/HER2low), MDA-MB-468 (EGFRhigh/HER2neg), MDA-MB-231-H2N (EGFRmod/HER2mod), and SKOV3 (EGFRlow/HER2high) cells by competition and saturation cell binding assays to estimate the dissociation constant (Kd). The elimination of the 64Cu-NOTA-trastuzumab Fab-PEG24-EGF bsRICs from the blood of Balb/c mice was compared to monospecific 64Cu-NOTA-trastuzumab Fab and 64Cu-NOTA-EGF. MicroPET/CT imaging was performed in NOD/SCID mice bearing subcutaneous MDA-MB-468, MDA-MB-231/H2N, or SKOV3 human BC xenografts at 24 and 48 h postinjection (p.i.) of bsRICs. Tumor and normal tissue uptake were quantified by biodistribution studies and compared to monospecific agents. The binding of bsRICs to MDA-MB-231 cells was decreased to 24.5 ± 5.2% by excess EGF, while the binding of bsRICs to SKOV3 cells was decreased to 38.6 ± 5.4% by excess trastuzumab Fab, demonstrating specific binding to both EGFR and HER2. 64Cu-labeled bsRICs incorporating the PEG24 spacer were eliminated more slowly from the blood than 64Cu-bsRICs without the PEG spacer and were cleared much more slowly than 64Cu-NOTA-Fab or 64Cu-NOTA-EGF. All three tumor xenografts were visualized by microPET/CT at 24 and 48 h p.i. of bsRICs. Biodistribution studies at 48 h p.i. in NOD/SCID mice with MDA-MB-231/H2N tumors demonstrated significantly greater tumor uptake of 64Cu-NOTA-Fab-PEG24-EGF (4.9 ± 0.4%ID/g) than 64Cu-NOTA-Fab (1.9 ± 0.3%ID/g; P < 0.0001) and 64Cu-NOTA-EGF (0.7 ± 0.2%ID/g; P < 0.0001). Furthermore, preadministration of an excess of trastuzumab Fab or trastuzumab Fab-PEG24-EGF significantly decreased the tumor uptake of 64Cu-NOTA-Fab-PEG24-EGF in SK-OV-3 and MDA-MB-468 xenografts by 4.4-fold (P = 0.0012) and 1.8-fold (P = 0.0031), respectively. 64Cu-labeled bsRICs bound HER2 or EGFR and were taken up specifically in vivo in tumor xenografts expressing one or both receptors. The PEG24 linker prolonged the blood residence time contributing to the higher tumor uptake of the bsRICs than monospecific agents.

Initial evaluation of Cu-64 labeled PARPi-DOTA PET imaging in mice with mesothelioma.

Poly(ADP-ribose) polymerase (PARP) has emerged as an important molecular target for the treatment of several oncological diseases. A couple of molecular probes based on Olaparib scaffold have been developed by incorporation of F-18 or fluorophore for positron emission tomography (PET) or optical imaging in several types of tumor. PARP has been reported overexpressed in mesothelioma. We hereby synthesized an analogue of Olaparib containing DOTA moiety and radiolabeled it with Cu-64 to evaluate its utility of PET tracer for mesothelioma. The Cu-64 labeling was conveniently achieved at 90% yield with final compound at >99% radiochemistry purity. The biodistribution and PET imaging were performed at 0.5, 1, 2 and 18h to confirm the in vivo tumor targeting. The tumor uptake in study group was significant higher than that in control group (3.45±0.47% ID/g vs 2.26±0.30% ID/g) and tumor were clearly detected by PET imaging. These results suggest the feasibility to develop an Olaparib-based theranostic agent for mesothelioma.

In vivo evaluation of PEGylated 64Cu-liposomes with theranostic and radiotherapeutic potential using micro PET/CT.

PURPOSE: The objective of this study was to evaluate the potential of PEGylated (64)Cu-liposomes in clinical diagnostic positron emission tomography (PET) imaging and PEGylated (177)Lu-liposomes in internal tumor radiotherapy through in vivo characterization and dosimetric analysis in a human xenograft mouse model. METHODS: Liposomes with 5 and 10 mol% PEG were characterized with respect to size, charge, and (64)Cu- and (177)Lu-loading efficiency. The tumor imaging potential of (64)Cu-loaded liposomes was evaluated in terms of in vivo biodistribution, tumor accumulation and tumor-to-muscle (T/M) ratios, using PET imaging. The potential of PEGylated liposomes for diagnostic and therapeutic applications was further evaluated through dosimetry analysis using OLINDA/EXM software. The (64)Cu-liposomes were used as biological surrogates to estimate the organ and tumor kinetics of (177)Lu-liposomes. RESULTS: High remote loading efficiency (>95 %) was obtained for both (64)Cu and (177)Lu radionuclides with PEGylated liposomes, and essentially no leakage of the encapsulated radionuclide was observed upon storage and after serum incubation for 24 h at 37 °C. The 10 mol% PEG liposomes showed higher tumor accumulation (6.2 ± 0.2 %ID/g) than the 5 mol% PEG liposomes, as evaluated by PET imaging. The dosimetry analysis of the (64)Cu-liposomes estimated an acceptable total effective dose of 3.3·10(-2) mSv/MBq for diagnostic imaging in patients. A high absorbed tumor dose (114 mGy/MBq) was estimated for the potential radiotherapeutic (177)Lu-liposomes. CONCLUSION: The overall preclinical profile of PEGylated (64)Cu-liposomes showed high potential as a new PET theranostic tracer for imaging in humans. Dosimetry results predicted that initial administered activity of 200 MBq of (64)Cu-liposomes should be acceptable in patients. Work is in progress to validate the utility of PEGylated (64)Cu-liposomes in a clinical research programme. The high absorbed tumor dose (114 mGy/MBq) estimated for (177)Lu-liposomes and the preliminary dosimetric studies justify further therapeutic and dosimetry investigation of (177)Lu-liposomes in animals before potential testing in man.