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Single-domain antibodies, or nanobodies (Nbs), are promising biomolecules for use in molecular imaging due to their excellent affinity, specificity, and fast clearance from the blood. Given their short blood half-life, pairing Nbs with short-lived imaging radioisotopes is desirable. Because fluorine-18 ( 18 F) is routinely used for clinical imaging, it is an attractive radioisotope for Nbs. We report a novel sortase-based, site-specific 18 F-labeling method applied to three nanobodies. Labeled nanobodies were synthesized either by a two-step indirect radiolabeling method in one pot or by a one-step direct labeling method using a sortase-mediated conjugation of either the radiolabeled chelator (H-GGGK((±)-Al[ 18 F]FH 3 RESCA)-NH 2 ) or the unlabeled chelator (H-GGGK((±)-H 3 RESCA)-NH 2 ) followed by labeling with Al[ 18 F]F, respectively. The overall radiochemical yields were 15-43% ( n = 22, decay-corrected) in 70 min (indirect labeling) and 23-58% ( n = 12, decay-corrected) in 50 min (direct labeling). The radiochemical purities of the labeled nanobodies prepared by both methods were >98% with a specific activity of 400-600 Ci/mmol ( n = 22) for each of the three Nbs tested and exhibited excellent stability profiles under physiological conditions. This simple, site-specific, reproducible, and generalizable 18 F-labeling method to prepare nanobodies (Nb-Al[ 18 F]F-RESCA) or other low molecular weight biomolecules can easily be adopted in various settings for preclinical and clinical studies.

Zirconium-89 is the most widely used radioisotope for immunoPET because its physical half-life (78.2 h) suits the one of antibodies. Desferrioxamine B (DFO) is the standard chelator for the complexation of zirconium(IV), and its bifunctional version, containing a phenylisothiocyanate function, is the most commonly used for the conjugation of DFO to proteins. However, preliminary results have shown that the thiourea link obtained from the conjugation of isothiocyanate and lysines is sensitive to the ionizing radiation generated by the radioisotope, leading to the rupture of the link and the release of the chelator/radiometal complex. This radiolysis phenomenon could produce nonspecific signal and prevent the detection of bone metastasis, as free zirconium accumulates into the bones. The aim of this work was to study the stability of a selection of conjugation linkers in 89 Zr-labeled immunoconjugates. We have synthesized several DFO-based bifunctional chelators appended with an isothiocyanate moiety, a bicyclononyne, or a squaramate ester. Two antibodies (trastuzumab and rituximab) were conjugated and radiolabeled with zirconium-89. The effect of increasing activities of zirconium-89 on the integrity of the bioconjugate bearing thiourea links was evaluated as well as the impact of the presence of a radioprotectant. The stability of the radiolabeled antibodies was studied over 7 days in PBS and human plasma. Radioconjugates' integrity was evaluated using iTLC and size-exclusion chromatography. This study shows that the nature of the linker between the chelator and biomolecule can have a strong impact on the stability of the 89 Zr-labeled conjugates, as well as on the aggregation of the conjugates.

Bioconjugates of antibodies and their derivatives radiolabeled with β + -emitting radionuclides can be utilized for diagnostic PET imaging. Site-specific attachment of radioactive cargo to antibody delivery vectors provides homogeneous, well-defined immunoconjugates. Recent studies have demonstrated the utility of oxaziridine chemistry for site-specific labeling of methionine residues. Herein, we applied this approach to site-specifically radiolabel trastuzumab-derived Fab immunoconjugates with 68 Ga, which can be used for in vivo PET imaging of HER2-positive breast cancer tumors. Initially, a reactive azide was introduced to a single solvent-accessible methionine residue in both the wild-type Fab and an engineered derivative containing methionine residue M74, utilizing the principles of oxaziridine chemistry. Subsequently, these conjugates were functionalized with a modified DFO chelator incorporating dibenzocyclooctyne. The resulting DFO-WT and DFO-M74 conjugates were radiolabeled with generator-produced [ 68 Ga]Ga 3+ , to yield the novel PET radiotracers, [ 68 Ga]Ga-DFO-WT and [ 68 Ga]Ga-DFO-M74. In vitro and in vivo studies demonstrated that [ 68 Ga]Ga-DFO-M74 exhibited a higher affinity for HER2 receptors. Biodistribution studies in mice bearing orthotopic HER2-positive breast tumors revealed a higher uptake of [ 68 Ga]Ga-DFO-M74 in the tumor tissue, accompanied by rapid renal clearance, enabling clear delineation of tumors using PET imaging. Conversely, [ 68 Ga]Ga-DFO-WT exhibited lower uptake and inferior image contrast compared to [ 68 Ga]Ga-DFO-M74. Overall, the results demonstrate that the highly facile methionine-oxaziridine modification approach can be simply applied to the synthesis of stable and site-specifically modified radiolabeled antibody-chelator conjugates with favorable pharmacokinetics for PET imaging.

A new, high-denticity, bifunctional ligand─H 3 TPAN-triazole-Bn-NH 2 ─has been synthesized and studied in complexation with [ 225 Ac]Ac 3+ and [ 111 In]In 3+ for radiopharmaceutical applications. The bifunctional chelator is readily synthesized, using a high-yielding four-step prep, which is highly adaptable and allows for straightforward incorporation of different covalent linkers using Cu I -catalyzed alkyne-azide cycloaddition (click) chemistry. Nuclear magnetic resonance (NMR) studies of H 3 TPAN-triazole-Bn-NH 2 with La 3+ and In 3+ metal ions show the formation of a single, asymmetric complex with each ion in solution, corroborated by density functional theory (DFT) calculations. Radiolabeling studies with [ 225 Ac]Ac 3+ and [ 111 In]In 3+ showed highly effective complexation, achieving quantitative radiochemical conversions at low ligand concentrations (<10 -6 M) under mild conditions (RT, 10 min), which is further accompanied by high stability in human serum. The bioconjugate─H 3 TPAN-triazole-Bn-Aoc-Pip-Nle-CycMSH hex ─was prepared for targeting of MC1R-positive tumors, and the corresponding 111 In-radiolabeled tracer was studied in vivo . SPECT/CT and biodistribution studies in C57BL/6J mice bearing B16-F10 tumors were performed, with the radiotracer showing good in vivo stability; tumor uptake was achieved. This work highlights a new promising and versatile bifunctional chelator, easily prepared and encouraging for 225 Ac/ 111 In theranostics.

The nuclear decay characteristics of 225 Ac ( E α = 5-8 MeV, linear energy transfer (LET) = ∼100 keV/μm, t 1/2 = 9.92 days) are well recognized as advantageous for the treatment of primary and metastatic tumors; however, suitable chelation systems are required, which can accommodate this radiometal. Since 225 Ac does not possess any suitable low-energy, high abundance γ-ray emissions for nuclear imaging, there is a clear need for the development of other companion radionuclides with similar coordination characteristics and comparable half-lives, which can be applied in diagnostics. H 4 picoopa was designed and executed as a high-denticity ligand for chelation of [ 225 Ac]Ac 3+ , and the complexation characteristics have been explored through nuclear magnetic resonance (NMR) spectroscopy, solution thermodynamic stability studies, and radiolabeling. The ligand shows highly favorable complexation with La 3+ (pM = 17.6), Lu 3+ (pM = 21.3), and In 3+ (pM = 31.2) and demonstrates effective radiolabeling of both [ 225 Ac]Ac 3+ and [ 111 In]In 3+ ions achieving quantitative radiochemical conversions (RCCs) under mild conditions (RT, 10 min), accompanied by high serum stability (>97% radiochemical purity (RCP) over 6 days). A bifunctional analogue of H 4 picoopa was synthesized and conjugated to the Pip-Nle-CycMSH hex peptide for targeting of MC1R positive melanoma tumors. In vivo single-photon emission computed tomography (SPECT) and biodistribution studies of the 111 In-radiolabeled bioconjugate in mice bearing B16-F10 tumors showed good radiotracer stability, although improved tumor targeting could not be achieved for imaging purposes. This work highlights H 4 picoopa as a very promising platform for application of [ 225 Ac]Ac 3+ and [ 111 In]In 3+ as a theranostic pair and allows great versatility for the incorporation of other directing vectors. The logical synthetic approach reported here for bifunctional H 4 picoopa, involving an azide-functionalized covalent linker and Cu I -catalyzed alkyne-azide cycloaddition, allows for ease of optimization of bioconjugate pharmacokinetics and will be valuable for further radiopharmaceutical applications moving forward.

Auger electron therapy exploits the cytotoxicity of low-energy electrons emitted during radioactive decay that travel very short distances (typically <1 μm). 201 Tl, with a half-life of 73 h, emits ∼37 Auger and other secondary electrons per decay and can be tracked in vivo as its gamma emissions enable SPECT imaging. Despite the useful nuclear properties of 201 Tl, satisfactory bifunctional chelators to incorporate it into bioconjugates for molecular targeting have not been developed. H 4 pypa, H 5 decapa, H 4 neunpa-NH 2 , and H 4 noneunpa are multidentate N- and O-donor chelators that have previously been shown to have high affinity for 111 In, 177 Lu, and 89 Zr. Herein, we report the synthesis and serum stability of [ nat/201 Tl]Tl 3+ complexes with H 4 pypa, H 5 decapa, H 4 neunpa-NH 2 , and H 4 noneunpa. All ligands quickly and efficiently formed complexes with [ 201 Tl]Tl 3+ that gave simple single-peak radiochromatograms and showed greatly improved serum stability compared to DOTA and DTPA. [ nat Tl]Tl-pypa was further characterized using nuclear magnetic resonance spectroscopy (NMR), mass spectroscopy (MS), and X-ray crystallography, showing evidence of the proton-dependent presence of a nine-coordinate complex and an eight-coordinate complex with a pendant carboxylic acid group. A prostate-specific membrane antigen (PSMA)-targeting bioconjugate of H 4 pypa was synthesized and radiolabeled. The uptake of [ 201 Tl]Tl-pypa-PSMA in DU145 PSMA-positive and PSMA-negative prostate cancer cells was evaluated in vitro and showed evidence of bioreductive release of 201 Tl and cellular uptake characteristic of unchelated [ 201 Tl]TlCl. SPECT/CT imaging was used to probe the in vivo biodistribution and stability of [ 201 Tl]Tl-pypa-PSMA. In healthy animals, [ 201 Tl]Tl-pypa-PSMA did not show the myocardial uptake that is characteristic of unchelated 201 Tl. In mice bearing DU145 PSMA-positive and PSMA-negative prostate cancer xenografts, the uptake of [ 201 Tl]Tl-pypa-PSMA in DU145 PSMA-positive tumors was higher than that in DU145 PSMA-negative tumors but insufficient for useful tumor targeting. We conclude that H 4 pypa and related ligands represent an advance compared to conventional radiometal chelators such as DOTA and DTPA for Tl 3+ chelation but do not resist dissociation for long periods in the biological environment due to vulnerability to reduction of Tl 3+ and subsequent release of Tl + . However, this is the first report describing the incorporation of [ 201 Tl]Tl 3+ into a chelator-peptide bioconjugate and represents a significant advance in the field of 201 Tl-based radiopharmaceuticals. The design of the next generation of chelators must include features to mitigate this susceptibility to bioreduction, which does not arise for other trivalent heavy radiometals.

Actinium-225 ( 225 Ac) is one of the most promising radionuclides for targeted alpha therapy (TAT). With a half-life of 9.92 days and a decay chain that emits four high-energy α particles, 225 Ac is well-suited for TAT when conjugated to macromolecular targeting vectors that exhibit extended in vivo circulation times. The implementation of 225 Ac in these targeted constructs, however, requires a suitable chelator that can bind and retain this radionuclide in vivo. Previous work has demonstrated the suitability of a diaza-18-crown-6 macrocyclic chelator H 2 macropa for this application. Building upon these prior efforts, in this study, two rigid variants of H 2 macropa, which contain either one (H 2 BZmacropa) or two (H 2 BZ 2 macropa) benzene rings within the macrocyclic core, were synthesized and investigated for their potential use for 225 Ac TAT. The coordination chemistry of these ligands with La 3+ , used as a nonradioactive model for Ac 3+ , was carried out. Both NMR spectroscopic and X-ray crystallographic studies of the La 3+ complexes of these ligands revealed similar structural features to those found for the related complex of H 2 macropa. Thermodynamic stability constants of the La 3+ complexes, however, were found to be 1 and 2 orders of magnitude lower than those of H 2 macropa for H 2 BZmacropa and H 2 BZ 2 macropa, respectively. The decrease in thermodynamic stability was rationalized via the use of density functional theory calculations. 225 Ac radiolabeling and serum stability studies with H 2 BZmacropa showed that this chelator compares favorably with H 2 macropa. Based on these promising results, a bifunctional version of this chelator, H 2 BZmacropa-NCS, was synthesized and conjugated to the antibody codrituzumab (GC33), which targets the liver cancer biomarker glypican-3 (GPC3). The resulting GC33-BZmacropa conjugate and an analogous GC33-macropa conjugate were evaluated for their 225 Ac radiolabeling efficiencies, antigen-binding affinities, and in vivo biodistribution in HepG2 liver cancer tumor-bearing mice. Although both conjugates were comparably effective in their radiolabeling efficiencies, [ 225 Ac]Ac-GC33-BZmacropa showed slightly poorer serum stability and biodistribution than [ 225 Ac]Ac-GC33-macropa. Together, these results establish H 2 BZmacropa-NCS as a new bifunctional chelator for the preparation of 225 Ac radiopharmaceuticals.

With the emergence of [ 225 Ac]Ac 3+ as a therapeutic radionuclide for targeted α therapy (TAT), access to clinical quantities of the potent, short-lived α-emitter [ 213 Bi]Bi 3+ ( t 1/2 = 45.6 min) will increase over the next decade. With this in mind, the nonadentate chelator, H 4 neunpa-NH 2 , has been investigated as a ligand for chelation of [ 213 Bi]Bi 3+ in combination with [ 111 In]In 3+ as a suitable radionuclidic pair for TAT and single photon emission computed tomography (SPECT) diagnostics. Nuclear magnetic resonance (NMR) spectroscopy was utilized to assess the coordination characteristics of H 4 neunpa-NH 2 on complexation of [ nat Bi]Bi 3+ , while the solid-state structure of [ nat Bi][Bi(neunpa-NH 3 )] was characterized via X-ray diffraction (XRD) studies, and density functional theory (DFT) calculations were performed to elucidate the conformational geometries of the metal complex in solution. H 4 neunpa-NH 2 exhibited fast complexation kinetics with [ 213 Bi]Bi 3+ at RT achieving quantitative radiolabeling within 5 min at 10 -8 M ligand concentration, which was accompanied by the formation of a kinetically inert complex. Two bioconjugates incorporating the melanocortin 1 receptor (MC1R) targeting peptide Nle-CycMSH hex were synthesized featuring two different covalent linkers for in vivo evaluation with [ 213 Bi]Bi 3+ and [ 111 In]In 3+ . High molar activities of 7.47 and 21.0 GBq/μmol were achieved for each of the bioconjugates with [ 213 Bi]Bi 3+ . SPECT/CT scans of the [ 111 In]In 3+ -labeled tracer showed accumulation in the tumor over time, which was accompanied by high liver uptake and clearance via the hepatic pathway due to the high lipophilicity of the covalent linker. In vivo biodistribution studies in C57Bl/6J mice bearing B16-F10 tumor xenografts showed good tumor uptake (5.91% ID/g) at 1 h post-administration with [ 213 Bi][Bi(neunpa-Ph-Pip-Nle-CycMSH hex )]. This study demonstrates H 4 neunpa-NH 2 to be an effective chelating ligand for [ 213 Bi]Bi 3+ and [ 111 In]In 3+ , with promising characteristics for further development toward theranostic applications.

Peptides have been widely adopted as biological targeting vectors for applications in molecular imaging and peptide-receptor radionuclide therapy (PRRT). Somatostatin (SST) analogues such as octreotate (TATE) are exogenous ligands for somatostatin receptors (SSTRs), which are highly expressed on neuroendocrine tumors (NETs). Recently, both [ 68 Ga]Ga-DOTA-TATE (NETSPOT) and [ 177 Lu]Lu-DOTA-TATE (LUTATHERA) received U.S. Food and Drug Administration approval for positron emission tomography (PET) imaging and PRRT of NETs, respectively. However, to the best of our knowledge a well-described synthesis of DOTA-TATE has not been reported in the literature. Herein, we report a fully reoptimized DOTA-TATE synthesis, including the application of a simple ultrasonic bath to greatly improve yields, reduce coupling times, and decrease the amount of reagents required for each coupling step by a half. The most prevalently used cyclizing agents such as iodine, thallium(III) trifluoroacetate, hydrogen peroxide, and dimethyl sulfoxide were compared. On-resin cyclizations using mechanical agitation showed higher yields (23% and 25% using I 2 and Tl(III), respectively) than off-resin (1.3% and 11% using DMSO and H 2 O 2 , respectively), and the total synthesis time of DOTA-TATE was ∼540 min excluding the cyclization step, with a total synthesis yield of ∼23%. The same manual SPPS methods/reagents were reoptimized with ultrasonic (US) agitation, resulting in an immense reduction in the total synthesis time by ∼8-fold to ∼70 min for DOTA-TATE with a higher yield (∼29% yield), and ∼13-fold to 105 min for DOTA-PEG 4 -TATE (∼29% yield). Also, the use of US agitation reduces the need for excess molar equivalents of the reagents to a half, which is particularly important when coupling expensive or custom-synthesized groups such as bifunctional chelators and linkers. Finally, the synthesized DOTA-TATE was successfully radiolabeled with [ 68 Ga]Ga 3+ ( t 1/2 = 68 min) with high radiochemical yields (30 min, 95 °C). We believe this work opens the door to the facile and low-cost synthesis of many new chelator- linker -peptide conjugates that were previously cumbersome or cost-prohibitive to produce with manual SPPS.

Herein, we present the syntheses and characterization of a new undecadendate chelator, H 4 py4pa, and its bifunctional analog H 4 py4pa-phenyl-NCS, conjugated to the monoclonal antibody, Trastuzumab, which targets the HER2+ cancer. H 4 py4pa possesses excellent affinity for 225 Ac (α, t 1/2 = 9.92 d) for targeted alpha therapy (TAT), where quantitative radiolabeling yield was achieved at ambient temperature, pH = 7, in 30 min at 10 -6 M chelator concentration, leading to a complex highly stable in mouse serum for at least 9 d. To investigate the chelation of H 4 py4pa with large metal ions, lanthanum (La 3+ ), which is the largest nonradioactive metal of the lanthanide series, was adopted as a surrogate for 225 Ac to enable a series of nonradioactive chemical studies. In line with the 1 H NMR spectrum, the DFT (density functional theory)-calculated structure of the [La(py4pa)] - anion possessed a high degree of symmetry, and the La 3+ ion was secured by two distinct pairs of picolinate arms. Furthermore, the [La(py4pa)] - complex also demonstrated a superb thermodynamic stability (log K [La(py4pa)] - ∼ 20.33, pLa = 21.0) compared to those of DOTA (log K [La(DOTA)] - ∼ 24.25, pLa = 19.2) or H 2 macropa (log K [La(macropa)] - = 14.99, pLa ∼ 8.5). Moreover, the functional versatility offered by the bifunctional py4pa precursor permits facile incorporation of various linkers for bioconjugation through direct nucleophilic substitution. In this work, a short phenyl-NCS linker was incorporated to tether H 4 py4pa to Trastuzumab. Radiolabeling studies, in vitro serum stability, and animal studies were performed in parallel with the DOTA-benzyl-Trastuzumab. Both displayed excellent in vivo stability and tumor specificity.

Calcium minerals such as hydroxyapatite (HAp) can be detected noninvasively in vivo using nuclear imaging agents such as [ 18 F]NaF (available from cyclotrons), for positron emission tomography (PET) and 99m Tc-radiolabeled bisphosphonates (BP; available from 99m Tc generators for single photon emission computed tomography (SPECT) or scintigraphy). These two types of imaging agents allow detection of bone metastases (based on the presence of HAp) and vascular calcification lesions (that contain HAp and other calcium minerals). With the aim of developing a cyclotron-independent PET radiotracer for these lesions, with broad calcium mineral affinity and simple one-step radiolabeling, we developed [ 68 Ga]Ga-THP-Pam. Radiolabeling with 68 Ga is achieved using a mild single-step kit (5 min, room temperature, pH 7) to high radiochemical yield and purity (>95%). NMR studies demonstrate that Ga binds via the THP chelator, leaving the BP free to bind to its biological target. [ 68 Ga]Ga-THP-Pam shows high stability in human serum. The calcium mineral binding of [ 68 Ga]Ga-THP-Pam was compared in vitro to two other 68 Ga-BPs which have been successfully evaluated in humans, [ 68 Ga]Ga-NO2AP BP and [ 68 Ga]Ga-BPAMD, as well as [ 18 F]NaF. Interestingly, we found that all 68 Ga-BPs have a high affinity for a broad range of calcium minerals implicated in vascular calcification disease, while [ 18 F]NaF is selective for HAp. Using healthy young mice as a model of metabolically active growing calcium mineral in vivo , we compared the pharmacokinetics and biodistribution of [ 68 Ga]Ga-THP-Pam with [ 18 F]NaF as well as [ 68 Ga]NO2AP BP . These studies revealed that [ 68 Ga]Ga-THP-Pam has high in vivo affinity for bone tissue (high bone/muscle and bone/blood ratios) and fast blood clearance ( t 1/2 < 10 min) comparable to both [ 68 Ga]NO2AP BP and [ 18 F]NaF. Overall, [ 68 Ga]Ga-THP-Pam shows high potential for clinical translation as a cyclotron-independent calcium mineral PET radiotracer, with simple and efficient radiochemistry that can be easily implemented in any radiopharmacy.

The radioactive isotopes scandium-44/47 and lutetium-177 are gaining relevance for radioimaging and radiotherapy, resulting in a surge of studies on their coordination chemistry and subsequent applications. Although the trivalent ions of these elements are considered close homologues, dissimilar chemical behavior is observed when they are complexed by large ligand architectures due to discrepancies between Lu(III) and Sc(III) ions with respect to size, chemical hardness, and Lewis acidity. Here, we demonstrate that Lu and Sc complexes of 1,4-bis(methoxycarbonyl)-7-[(6-carboxypyridin-2-yl)methyl]-1,4,7-triazacyclononane (H 3 mpatcn) and its corresponding bioconjugate picaga-DUPA can be employed to promote analogous structural features and, subsequently, biological properties for coordination complexes of these ions. The close homology was evidenced using potentiometric methods, computational modeling, variable temperature mass spectrometry, and pair distribution function analysis of X-ray scattering data. Radiochemical labeling, in vitro stability, and biodistribution studies with Sc-47 and Lu-177 indicate that the 7-coordinate ligand environment of the bifunctional picaga ligand is compatible with biological applications and the future investigation of β-emitting, picaga-chelated Sc and Lu isotopes for radiotherapy.

The continual development of radiopharmaceutical agents for the field of nuclear medicine is integral to promoting the necessity of personalized medicine. One way to greatly expand the selection of radiopharmaceuticals available is to broaden the range of radionuclides employed in such agents. Widening the scope of development to include radiometals with their variety of physical decay characteristics and chemical properties opens up a myriad of possibilities for new actively targeted molecules and bioconjugates. This is especially true to further advance the imaging and treatment of disease in the brain. Over the past few decades, imaging of disease in the brain has heavily relied on agents which exploit metabolic uptake. However, through utilizing the broad range of physical characteristics that radiometals offer, the ability to target other processes has become more available. The varied chemistries of radiometals also allows for them to incorporated into specifically designed diverse constructs. A major limitation to efficient treatment of disease in the brain is the ability for relevant agents to penetrate the blood-brain barrier. Thus, along with efficient disease targeting, there must be intentional thought put into overcoming this challenge. Here, we review the current field of radiometal-based agents aimed at either imaging or therapy of brain disease that have been evaluated through at least in vivo studies.

Trithiol chelates are suitable for labeling radioarsenic ( 72 As: 2.49 MeV β + , 26 h; 77 As: 0.683 MeV β - , 38.8 h) to form potential theranostic radiopharmaceuticals for positron emission tomography (PET) imaging and therapy. A trithiol(b)-(Ser) 2 -RM2 bioconjugate and its arsenic complex were synthesized and characterized. The trithiol(b)-(Ser) 2 -RM2 bioconjugate was radiolabeled with no-carrier-added 77 As in over 95% radiochemical yield and was stable for over 48 h, and in vitro IC 50 cell binding studies of [ 77 As]As-trithiol(b)-(Ser) 2 -RM2 in PC-3 cells demonstrated high affinity for the gastrin-releasing peptide (GRP) receptor (low nanomolar range). Limited biodistribution studies in normal mice were performed with HPLC purified 77 As-trithiol(b)-(Ser) 2 -RM2 demonstrating both pancreatic uptake and hepatobiliary clearance.

The synthesis of a polylysine polymer functionalized with the previously reported astonishingly inert [In( cb-te2pa )] + chelate was performed. A biotin end group allowed the conjugation to biotinylated beads by the intermediary of a fluorescein isothiocyanate/neutravidin receptor. High quality imaging mass cytometry trials, based on 115 In detection were performed to highlight the behavior of the material. Anti-CD20 antibody was labeled by the so-obtained In(III)-modified polylysine using the biotin/neutravidin interaction. Ramos (CD20[+]) and HL-60 (CD20[-]) cell lines were costained with the In(III)-modified bioconjugate by finding the best staining conditions. Both immunofluorescence microscopy (IF-M) and mass cytometry analyses confirmed the specific binding of anti-CD20 onto Ramos cells. CyTOF histograms constructed on the 115 In detection allowed us to define and to separate, with a good signal-to-noise ratio, two populations (Ramos and HL-60). The inertness of In(III)-MCP-NAv over a three-month storage period was proved by performing new functionality tests involving Jurkat cells (CD20[-]) and multiparametric trials involving the 115 In channel. The results ensure a promising future use of the previously announced [In( cb-te2pa )] + complex-based polymers for mass cytometry.

Molecular radiopharmaceuticals based on bioconjugates of chelators with peptides and proteins have had significant clinical impact in the diagnosis and treatment of several types of cancers. In the 1990s, indium-111 and yttrium-90 labeled chelator-peptide/protein conjugates established the clinical utility of these radiopharmaceuticals for receptor-targeted γ-scintigraphy imaging and systemic radiotherapy. Second-generation bioconjugates based on peptides targeting the somatostatin II receptor and the prostate-specific membrane antigen are now widely used for management of neuroendocrine and prostate cancer, respectively. These bioconjugates are typically radiolabeled with gallium-68 for imaging of target receptor expression with positron emission tomography, and the β - -emitter, lutetium-177, for targeted radiotherapy. Innovations in radioisotope technology and biomolecular therapies are likely to drive the future clinical development of radiopharmaceuticals based on radiometals. New chelator-peptide and chelator-protein bioconjugates will underpin nuclear medicine advances in molecular imaging and radiotherapy.

Site-specific modification of peptides and proteins is an important method for introducing an artificial function to the protein surface. Recently, we found that new bioconjugation reagents, 6-(azidomethyl)-2-pyridinecarbaldehyde (6AMPC) derivatives, allow specific N-terminal modification and enhance the reaction rate of the subsequent bioconjugation in a chelation-assisted CuAAC reaction. The N-terminal specific azide-labeling of bioactive peptides and proteins occurs under mild reaction conditions with 6AMPC derivatives (angiotensin I: 90%, ribonuclease A: 90%). Kinetic analysis of the CuAAC reaction with azide-labeled proteins reveals that the ligation is promoted in the presence of a copper-chelating pyridine moiety. Importantly, the introduction of an electron-donating methoxy group to the pyridine moiety further accelerates the CuAAC ligation. We demonstrate that this method enables site-specific conjugation of various functional molecules such as fluorophores, biotin, and polyethylene glycol.

Following the successful synthesis of a C -functionalized version of the TE1PA ligand, a monopicolinate cyclam, we looked to demonstrate its in vivo properties versus DOTA and NOTA, after conjugation on the 9E7.4 rat antibody, an IgG 2a against CD138 murine, which has relevant properties for multiple myeloma targeting. For each ligand, different conjugation approaches had been considered to select the most appropriate for the comparative study. The p -SCN-Bn-TE1PA, NHS-DOTA, and p -SCN-Bn-NOTA were finally chosen for conjugation and radiolabeling tests. For in vivo comparison, we used a model of subcutaneous grafted mice with 5T33 tumor cells. In vitro tests and immuno-PET study highlighted 64 Cu-9E7.4- p -SCN-Bn-NOTA as the least attractive. Further competitive biodistribution and hepatic metabolic studies at 2, 24, and 48 h post-injection (100 μg radiolabeled with 10 MBq of 64 Cu) were then performed with the 64 Cu-9E7.4- p -SCN-Bn-TE1PA and 64 Cu-9E7.4-NHS-DOTA. Results show a better in vivo resistance of 64 Cu-9E7.4- p -SCN-Bn-TE1PA to transchelation compared to 64 Cu-9E7.4-NHS-DOTA, especially at later times. This was confirmed with 64 Cu-9E7.4- p -SCN-Bn-NOTA at 48 h PI. 64 Cu-9E7.4- p -SCN-Bn-TE1PA also demonstrated an excellent hepatic clearance. 64 Cu-9E7.4- p -SCN-Bn-TE1PA displayed an overall superiority compared to 64 Cu-9E7.4-NHS-DOTA and 64 Cu-9E7.4- p -SCN-Bn-NOTA in terms of in vivo stability, reinforcing the usefulness of the p- SCN-Bn-TE1PA ligand for 64 Cu immuno-PET imaging.

In an alternative approach for radiotracer design, a photoactivatable HBED-CC-PEG 3 -ArN 3 chelate was synthesized and photoconjugated to the anti-c-MET antibody MetMAb (onartuzumab). Photoconjugation gave the functionalized protein HBED-CC-azepin-MetMAb with a photochemical conversion of 18.5 ± 0.5% ( n = 2) which was then radiolabeled with 68 Ga 3+ ions. The purified and formulated [ 68 Ga]GaHBED-CC-azepin-MetMAb radiotracer was evaluated in vitro and in vivo. Standard stability tests and cellular binding assays confirmed that the radiotracer remained radiochemically pure and immunoreactive after photochemical conjugation. [ 68 Ga]GaHBED-CC-azepin-MetMAb showed specific uptake in c-MET-positive MKN-45 (high-expression) and PC-3 (low/moderate expression) tumors with tumor-associated activities at 6 h post-administration of 10.33 ± 1.27 ( n = 5) and 3.88 ± 1.27 ( n = 3) %ID/g, respectively. In competitive blocking experiments, MKN-45 tumor uptake was reduced by approximately 55% ( P-value <0.001 compared with nonblocked experiments) confirming specific radiotracer binding to c-MET in vivo. Radiochemical, cellular, and in vivo experiments confirmed that the photoradiochemical approach is a viable tool to synthesize new radiotracers for immuno-PET.

The bifunctional chelator S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane- N, N', N″, N‴-1,4,7,10-tetraacetate (IB-DOTA) is on paper the most attractive of the commercially available bifunctional chelators for magnetic resonance imaging (MRI) applications. The preserved DOTA scaffold is known to produce extremely kinetically and thermodynamically robust chelates with the Gd 3+ ion. Also, ligation through four acetate pendant arms should ensure that the rapid water exchange kinetics so, crucial to the function of an MRI contrast agent are retained. However, upon ligation of the Gd 3+ ion, IB-DOTA differentiates into two distinct isomers defined by the positions of the benzylic substituent (corner or side). A relaxometric analysis of these two isomers revealed marked differences in the property and behavior of the two chelates. Most notably the side isomer is found to be substantially more likely to aggregate in aqueous solution than its corner counterpart. This aggregation results in higher relaxivity for the side isomer versus the corner isomer, an observation that potentially obscures the impact of differences in water exchange kinetics between the two isomers. The side isomer is composed of a significant fraction of a twisted square antiprismatic coordination geometry that exchanges water more rapidly than optimal (τ M = 7 ns) for maximizing relaxivity. The impact of this excessively fast exchange is not observed in the relaxivity of the side isomer only because in isolation this chelate tumbles much more slowly than the corner isomer. However, this situation is not expected to persist when the chelate is employed in a typical bioconjugate. These results imply that the corner isomer of IB-DOTA may represent a better choice of bifunctional chelator for bioconjugation applications in which a large macromolecule is to be tagged for MRI applications.

Here, we present the synthesis and characterization of a new potentially nonadentate chelator H 4 pypa and its bifunctional analogue t Bu 4 pypa-C7-NHS conjugated to prostate-specific membrane antigen (PSMA)-targeting peptidomimetic (Glu-urea-Lys). H 4 pypa is very functionally versatile and biologically stable. Compared to the conventional chelators (e.g., DOTA, DTPA), H 4 pypa has outstanding affinities for both 111 In (EC, t 1/2 ≈ 2.8 days) and 177 Lu (β - ,γ, t 1/2 ≈ 6.64 days). Its radiolabeled complexes were achieved at >98% radiochemical yield, RT within 10 min, at a ligand concentration as low as 10 -6 M, with excellent stability in human serum over at least 5-7 days (<1% transchelation). The thermodynamic stabilities of the [M(pypa)] - complexes (M 3+ = In 3+ , Lu 3+ , La 3+ ) were dependent on the ionic radii, where the smaller In 3+ has the highest pM value (30.5), followed by Lu 3+ (22.6) and La 3+ (19.9). All pM values are remarkably higher than those with DOTA, DTPA, H 4 octapa, H 4 octox, and H 4 neunpa. Moreover, the facile and versatile bifunctionalization enabled by the p-OH group in the central pyridyl bridge of the pypa scaffold (compound 14) allows incorporation of a variety of linkers for bioconjugation through easy nucleophilic substitution. In this work, an alkyl linker was selected to couple H 4 pypa to a PSMA-targeting pharmacophore, proving that the bioconjugation sacrifices neither the tumor-targeting nor the chelation properties. The biodistribution profiles of 111 In- and 177 Lu-labeled tracers are different, but promising, with the 177 Lu analogue particularly outstanding.

DOTA is commonly used for radiometal chelation in molecular imaging. Yet in the absence of a radiometal, DOTA is hypothesized to promote renal clearance of 18 F-labeled peptide tracers. In light of an increasing interest in the use of F18 for PET, here the effect of DOTA is evaluated for the first time with an 18 F-labeled tracer and is found to significantly improve the quality of images acquired through positron emission tomography (PET). We chose to image the peptide LLP2A that recognizes the transmembrane protein very-late antigen 4 (VLA-4) that is overexpressed by many cancers. Since it is known that [ 18 F]RBF 3 -PEG 2 -LLP2A derivatives gave low tumor uptake values and significant GI tract accumulation, this ligand thus represents an ideal means of testing the additive effects of a DOTA group on clearance while permitting a facile, user-friendly, one-step 18 F-labeling. A newly designed RBF 3 -LLP2A bioconjugate with an appended DOTA moiety increased tumor uptake nearly 3-fold and reduced GI accumulation by more than 10-fold. The DOTA-AMBF 3 -PEG 2 -LLP2A was radiolabeled by isotope exchange and was purified by semiprep HPLC and C18 cartridge elution. Male C57BL/6J mice bearing B16-F10 melanoma tumors that overexpress the VLA-4 target were used to evaluate [ 18 F]DOTA-AMBF 3 -PEG 2 -LLP2A using a combination of static and dynamic PET scans, biodistribution studies, and blocking controls at 1 h post injection (p.i.). The precursor peptide was synthesized and 18 F-labeled to provide formulations with mean (±SD) radiochemical purities of 95.9 ± 1.8%, in radiochemical yields of 4.8 ± 2.9% having molar activities of 131.7 ± 50.3 GBq/μmol. In vivo static PET images of [ 18 F]DOTA-AMBF 3 -PEG 2 -LLP2A provided clear tumor visualization, and biodistribution studies showed that tumor uptake was 9.46 ± 2.19% injected dose per gram of tissue (%ID/g) with high tumor/muscle and tumor/blood contrast ratios of ∼8 and ∼10, respectively. Blocking confirmed the specificity of [ 18 F]DOTA-AMBF 3 -PEG 2 -LLP2A to VLA-4 in the tumor and the bone marrow. Dynamic PET showed clearance of [ 18 F]DOTA-AMBF 3 -PEG 2 -LLP2A mainly via the renal pathway, wherein accumulation in the intestines was reduced 10-fold compared to our previously investigated LLP2A's, while spleen uptake was at levels similar to previously reported LLP2A-chelator radiotracers. [ 18 F]DOTA-AMBF 3 -PEG 2 -LLP2A represents a promising VLA-4 radiotracer and provides key evidence as to how a DOTA appendage can significantly reduce GI uptake in favor of urinary excretion. Implications for the development of dual-isotope theranostics that exploit the use fluorine-18 for imaging and DOTA to chelate therapeutic metal cations for therapy are discussed.

With the long-term goal of developing theranostic agents for applications in nuclear medicine, in this work we evaluated the well-known NOTA and NODAGA chelators as bifunctional chelators (BFCs) for the [ 99m Tc/ 186 Re]Tc/Re-tricarbonyl core. In particular, we report model complexes of the general formula fac-[M(L)(CO) 3 ] + (M = Re, 99m Tc, 186 Re) where L denotes NOTA-Pyr (1) or NODAGA-Pyr (2), which are derived from conjugation of NOTA/NODAGA with pyrrolidine (Pyr). Further, as proof-of-principle, we synthesized the peptide bioconjugate NODAGA-sst 2 -ANT (3) and explored its complexation with the fac-[Re(CO) 3 ] + and fac-[ 99m Tc][Tc(CO) 3 ] + cores; sst 2 -ANT denotes the somatostatin receptor (SSTR) antagonist 4-NO 2 -Phe-c(DCys-Tyr-DTrp-Lys-Thr-Cys)-DTyr-NH 2 . Rhenium complexes Re-1 through Re-3 were synthesized and characterized spectroscopically, and receptor binding affinity was demonstrated for Re-3 in SSTR-expressing cells (AR42J, IC 50 = 91 nM). Radiolabeled complexes [ 99m Tc]Tc/[ 186 Re]Re-1/2 and [ 99m Tc]Tc-3 were prepared in high radiochemical yield (>90%, determined by radio-HPLC) by reacting [ 99m Tc]/[ 186 Re][Tc/Re(OH 2 ) 3 (CO) 3 ] + with 1-3 and correlated well with the respective Re-1 through Re-3 standards in comparative HPLC studies. All radiotracers remained intact through 24 h ( 99m Tc-labeled complexes) or 48 h ( 186 Re-labeled complexes) against 1 mM l-histidine and 1 mM l-cysteine (pH 7.4, 37 °C). Similarly, rat serum stability studies displayed no decomposition and low nonspecific binding of 9-24% through 4 h. Biodistribution of [ 99m Tc]Tc-3 in healthy CF-1 mice demonstrated a favorable pharmacokinetic profile. Rapid clearance was observed within 1 h post-injection, predominantly via the renal system (82% of the injected dose was excreted in urine by 1 h), with low kidney retention (% ID/g: 11 at 1 h, 5 at 4 h, and 1 at 24 h) and low nonspecific uptake in other organs/tissues. Our findings establish NOTA and NODAGA as outstanding BFCs for the fac-[M(CO) 3 ] + core in the design and development of organometallic radiopharmaceuticals. Future in vivo studies of [ 99m Tc]Tc- and [ 186 Re]Re-tricarbonyl complexes of NODAGA/NOTA-biomolecule conjugates will further probe the potential of these chelates for nuclear medicine applications in diagnostic imaging and targeted radiotherapy, respectively.

A family of five water-soluble Gd 3+ :1,4,7,10-tetraazacyclododecane-1,4,7-tetraacetic acid-modified polyrotaxane (PR) magnetic resonance contrast agents bearing mixtures of 2-hydroxypropyl-β-cyclodextrin and 4-sulfobutylether-β-cyclodextrin macrocycles threaded onto Pluronic cores were developed as long circulating magnetic resonance contrast agents. Short diethylene glycol diamine spacers were utilized for linking the macrocyclic chelator to the PR scaffold prior to Gd 3+ chelation. The PR products were characterized by 1 H NMR, gel permeation chromatography/multiangle light scattering, dynamic light scattering, and analytical ultracentrifugation. Nuclear magnetic relaxation dispersion and molar relaxivity measurements at 23 °C revealed that all the PR contrast agents displayed high spin-spin T 1 relaxation and spin-lattice T 2 relaxation rates relative to a DOTAREM control. When injected at 0.05 mmol Gd/kg body weight in BALB/c mice, the PR contrast agents increased the T 1 -weighted MR image intensities with longer circulation times in the blood pool than DOTAREM. Excretion of the agents occurred predominantly via the renal or biliary routes depending on the polyrotaxane structure, with the longest circulating L81 Pluronic-based agent showing the highest liver uptake. Proteomic analysis of PR bearing different β-cyclodextrin moieties indicated that lipoproteins were the predominant component associated with these materials after serum exposure, comprising as much as 40% of the total protein corona. We infer from these findings that Gd(III)-modified PR contrast agents are promising long-circulating candidates for blood pool analysis by MRI.

In this study, we designed and synthesized a highly stable manganese (Mn 2+ )-based hepatobiliary complex by tethering an ethoxybenzyl (EOB) moiety with an ethylenediaminetetraacetic acid (EDTA) coordination cage as an alternative to the well-established hepatobiliary gadolinium (Gd 3+ ) chelates and evaluated its usage as a T 1 hepatobiliary magnetic resonance imaging (MRI) contrast agent (CA). This new complex exhibits higher r 1 relaxivity (2.3 mM -1 s -1 ) than clinically approved Mn 2+ -based hepatobiliary complex Mn-DPDP (1.6 mM -1 s -1 ) at 1.5 T. Mn-EDTA-EOB shows much higher kinetic inertness than that of clinically approved Gd 3+ -based hepatobiliary MRI CAs, such as Gd-DTPA-EOB and Gd-BOPTA. In addition, in vivo biodistribution and MRI enhancement patterns of this new Mn 2+ chelate are comparable to those of Gd 3+ -based hepatobiliary MRI CAs. The diagnostic efficacy of the new complex was demonstrated by its enhanced tumor detection sensitivity in a liver cancer model using in vivo MRI.

Liposomes are a proven, versatile, and clinically viable technology platform for vascular delivery of drugs and imaging probes. Although targeted liposomes have the potential to advance these applications, complex formulations and the need for optimal affinity ligands and conjugation strategies challenge their translation. Herein, we employed copper-free click chemistry functionalized liposomes to target platelet-endothelial cell adhesion molecule (PECAM-1) and intracellular adhesion molecule (ICAM-1) by conjugating clickable monoclonal antibodies (Ab) or their single chain variable fragments (scFv). For direct, quantitative tracing, liposomes were surface chelated with 111 In to a >90% radiochemical yield and purity. Particle size and distribution, stability, ligand surface density, and specific binding to target cells were characterized in vitro. Biodistribution of liposomes after IV injection was characterized in mice using isotope detection in organs and by noninvasive imaging (single-photon emission computed tomography/computed tomography, SPECT/CT). As much as 20-25% of injected dose of liposomes carrying PECAM and ICAM ligands, but not control IgG accumulated in the pulmonary vasculature. The immunospecificity of pulmonary targeting of scFv/liposomes to PECAM-1 and ICAM-1, respectively, was 10-fold and 2.5-fold higher than of Ab/liposomes. Therefore, the combination of optimal ligands, benign conjugation, and labeling yields liposomal formulations that may be used for highly effective and specific vascular targeting.

Due to the increasing use of generator-produced radiometal Gallium-68 ( 68 Ga) in positron-emission tomography/computed tomography (PET/CT), reliable bifunctional chelators that can efficiently incorporate 68 Ga 3+ into biomolecules are highly desirable. In this study, we synthesized two new bifunctional chelators bearing one or two phosphonic acid functional groups, named p-SCN-PhPr-NE2A1P and p-SCN-PhPr-NE2P1A, with the aim of enabling facile production of 68 Ga-based radiopharmaceuticals. Both chelators were successfully conjugated to LLP2A-PEG 4 , a very late antigen-4 (VLA-4) targeting peptidomimetic ligand, to evaluate their application in 68 Ga-based PET imaging. NE2P1A-PEG 4 -LLP2A exhibited the highest 68 Ga 3+ binding ability with molar activity of 37 MBq/nmol under mild temperature and neutral pH. Excellent serum stability of 68 Ga-NE2P1A-PEG 4 -LLP2A was observed, which was consistent with the result obtained from density functional theory calculation. The in vitro cell study showed that 68 Ga-NE2P1A-PEG 4 -LLP2A had significantly longer retention in B16F10 cells comparing to the reported retention of 64 Cu-NE3TA-PEG 4 -LLP2A, although the uptake was relatively lower. In the biodistribution and micro-PET/CT imaging studies, high tumor uptake and low background were observed after 68 Ga-NE2P1A-PEG 4 -LLP2A was injected into mice bearing B16F10 tumor xenografts, making it a highly promising radiotracer for noninvasive imaging of VLA-4 receptors overexpressed in melanoma.

Lanthanide chelating tags (LCTs) have been used with great success for determining structures and interactions of proteins and other biological macromolecules. Recently LCTs have also been used for in-cell NMR spectroscopy, but the bottleneck especially for demanding applications like pseudocontact shift (PCS) NMR is the sparse availability of suitable tags that allow for site-selective, rigid, irreversible, fast, and quantitative conjugation of chelated paramagnetic lanthanide ions to proteins via reduction stable bonds. We report here several such tags and focus on a new pyridine thiazole derivate of DOTA, that combines high affinity, rigidity, and selectivity with unprecedented tagging properties. The conjugation to the cysteine thiol of the protein results in a reductively stable thioether bond and proceeds virtually quantitatively in less than 30 min at 100 μM protein concentration, ambient temperature, and neutral pH. Upon conjugation of the new tag to two single cysteine mutants of ubiquitin and a single cysteine mutant of human carbonic anhydrase type II (30 kDa) only one stereoisomer is formed (square antiprismatic coordination, Λ(δδδδ)) and large to very large pseudocontact shifts as well as large residual dipolar couplings (RDCs) are observed by NMR spectroscopy. The PCS and RDC show excellent agreement with the solid state structure of the proteins. We believe that the pyridine thiazole moiety reported here has the potential to serve as a thiole reactive group in various conjugation applications; furthermore, its terbium complex shows strong photoluminescence upon irradiation and may thus serve as a donor group for Förster resonance energy transfer spectroscopy.

Mass cytometry is a highly multiplexed single-cell analysis platform that uses metal-tagged reagents to identify multiple cellular biomarkers. The current metal-tagged reagent preparation employs thiol-maleimide chemistry to covalently couple maleimide-functionalized metal-chelating polymers (MCPs) with antibodies (Abs), a process that requires partial reduction of the Ab to form reactive thiol groups. However, some classes of Abs (for example, IgM) as well as biomolecules lacking cysteine residues have been challenging to label using this method. This inherent limitation led us to develop a new conjugation strategy for labeling a wide range of biomolecules and affinity reagents. In this report, we present a metal tagging approach using a new class of azide- or transcyclooctene-terminated MCPs with copper(I)-free strain-promoted alkyne-azide cycloaddition or tetrazine-alkene click chemistry reactions, in which biomolecules with -NH 2 functional groups are selectively activated with a dibenzocyclooctyne or tetrazine moiety, respectively. This approach enabled us to generate highly sensitive and specific metal-tagged IgGs, IgMs, small peptides, and lectins for applications in immunophenotyping and glycobiology. We also created dual-tagged reagents for simultaneous detection of markers by immunofluorescence, mass cytometry, and imaging mass cytometry using a two-step conjugation process. The Helios mass cytometer was used to test the functionality of reagents on suspension human leukemia cell lines and primary cells. The dual-tagged Abs, metal-tagged lectins, and phalloidin staining reagent were used to visualize target proteins and glycans on adherent cell lines and frozen/FFPE tissue sections using the Hyperion Imaging System. In some instances, reagents produced by click conjugation showed superior sensitivity and specificity compared to those of reagents produced by thiol-maleimide chemistry. In general, the click chemistry-based conjugation with new MCPs could be instrumental in developing a wide range of highly sensitive metal-containing reagents for proteomics and glycomics applications.

Molecular imaging requires the specific accumulation of contrast agents at the target. To exploit the superb resolution of MRI for applications in molecular imaging, gadolinium chelates, as the MRI contrast agents (CA), have to be conjugated to a specific vector able to recognize the epitope of interest. Several Gd(III)-chelates can be chemically linked to the same binding vector in order to deliver multiple copies of the CA (multimers) in a single targeting event thus increasing the sensitivity of the molecular probe. Herein three novel bifunctional agents, carrying one functional group for the bioconjugation to targeting vectors and four Gd(III)-AAZTA chelate functions for MRI contrast enhancement (AAZTA = 6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid), are reported. The relaxivity in the tetrameric derivatives is 16.4 ± 0.2 mM Gd -1 s -1 at 21.5 MHz and 25 °C, being 2.4-fold higher than that of parent, monomeric Gd(III)-AAZTA. These compounds can be used as versatile building blocks to insert preformed, high relaxivity, and high density Gd-centers to biological targeting vectors. As an example, we describe the use of these bifunctional Gd(III)-chelates to label a fibrin-targeting peptide.

Maleimide-bearing bifunctional chelators have been used extensively for the site-selective bioconjugation and radiolabeling of peptides and proteins. However, bioconjugates obtained using these constructs inevitably suffer from limited stability in vivo, a trait that translates into suboptimal activity concentrations in target tissues and higher uptake levels in healthy, nontarget tissues. To circumvent this issue, phenyloxadiazolyl methylsulfones have previously been reported as alternatives to maleimides for thiol-based ligations, but these constructs have scarcely been used in the field of radiochemistry. In this report, we describe the synthesis of two thiol-reactive bifunctional chelators for 89 Zr and 177 Lu based on a new, easy-to-make phenyloxadiazolyl methylsulfone reagent, PODS. Radioimmunoconjugates created using these novel bifunctional chelators displayed in vitro stability that was higher than that of their maleimide-derived cousins. More importantly, positron emission tomography imaging in murine models of cancer revealed that a 89 Zr-labeled radioimmunoconjugate created using a PODS-bearing bifunctional chelator produced a rate of uptake in nontarget tissues that is significantly lower than that of its analogous maleimide-based counterpart.

The clinical applications of positron emission tomography (PET) imaging pharmaceuticals have increased tremendously over the past several years since the approval of 18 fluorine-fluorodeoxyglucose ( 18 F-FDG) by the Food and Drug Administration (FDA). Numerous 18 F-labeled target-specific potential imaging pharmaceuticals, based on small and large molecules, have been evaluated in preclinical and clinical settings. 18 F-labeling of organic moieties involves the introduction of the radioisotope by C- 18 F bond formation via a nucleophilic or an electrophilic substitution reaction. However, biomolecules, such as peptides, proteins, and oligonucleotides, cannot be radiolabeled via a C- 18 F bond formation as these reactions involve harsh conditions, including organic solvents, high temperature, and nonphysiological conditions. Several approaches, including 18 F-labeled prosthetic groups, silicon, boron, and aluminum fluoride acceptor chemistry, and click chemistry have been developed, in the past, for 18 F labeling of biomolecules. Linear and macrocyclic polyaminocarboxylates and their analogs and derivatives form thermodynamically stable and kinetically inert aluminum chelates. Hence, macrocyclic polyaminocarboxylates have been used for conjugation with biomolecules, such as folate, peptides, affibodies, and protein fragments, followed by 18 F-AlF chelation, and evaluation of their targeting abilities in preclinical and clinical environments. The goal of this report is to provide an overview of the 18 F radiochemistry and 18 F-labeling methodologies for small molecules and target-specific biomolecules, a comprehensive review of coordination chemistry of Al 3+ , 18 F-AlF labeling of peptide and protein conjugates, and evaluation of 18 F-labeled biomolecule conjugates as potential imaging pharmaceuticals.

The number and spacing of B-cell epitopes on antigens have a profound impact on the activation of B cells and elicitation of antibody responses, the quantitative aspects of which may be utilized for rational design of vaccines. Ni-chelating liposomes have been widely used as protein carriers in experimental studies of vaccine delivery, owing to the convenience and versatility of this conjugation chemistry. However, the epitope number per particle as well as the stability of protein conjugation on liposomes remain far less characterized. Here we have developed quantitative methods to measure the average spatial density of proteins on liposomes using both ensemble and single-molecule techniques and demonstrated their utility using liposomes conjugated with native proteins of two different sizes. These studies revealed that the initial density of protein conjugation on Ni-chelating liposomes can be finely controlled, but the density can decrease over time upon dilution due to the noncovalent nature of Ni-chelation chemistry. These results indicate that an alternative method other than the Ni-chelation chemistry is needed for stable conjugation of epitopes onto liposomes and also suggest a general strategy that can be used to precisely regulate the epitope density on liposomes for B-cell antigen delivery.

In the synthesis of technetium-99m ( 99m Tc) labeled target-specific ligands, the presence of a large excess of unlabeled ligands over 99m Tc in the injectate hinders target accumulation of 99m Tc-labeled ligands by competing for target molecules. To circumvent the problem, we recently developed a concept of the metal coordination-mediated multivalency, and proved the concept with a 99m Tc-labeled trivalent compound [ 99m Tc(CO) 3 (CN-RGD) 3 ] + . In this study, D-penicillamine (Pen) was selected as a chelating molecule and a cyclic RGDfK peptide was conjugated to Pen via a hexanoic linkage (Pen-Ahx-c(RGDfK)). 99m Tc complexation reaction, and the stability, integrin α v β 3 binding affinity, and biodistribution of the 99m Tc-labeled probe were investigated to evaluate the applicability of the concept to bivalent probes. 99m Tc-[Pen-Ahx-c(RGDfK)] 2 was obtained over 95% radiochemical yields under low Pen-Ahx-c(RGDfK) concentration (50 μM). 99m Tc-[Pen-Ahx-c(RGDfK)] 2 showed approximately 10-times higher integrin α v β 3 binding affinity than the monovalent compounds, Pen-Ahx-c(RGDfK) and c(RGDyV). In biodistribution studies, the tumor accumulation of 99m Tc-[Pen-Ahx-c(RGDfK)] 2 was decreased to 77% and 43% of HPLC-purified (Pen-Ahx-c(RGDfK)-free) 99m Tc-[Pen-Ahx-c(RGDfK)] 2 by the presence of 5 nmol of unlabeled Pen-Ahx-c(RGDfK) and Re-[Pen-Ahx-c(RGDfK)] 2 , respectively. 99m Tc-[Pen-Ahx-c(RGDfK)] 2 provided tumor image without removing unlabeled ligand, while a 99m Tc-labeled monovalent probe prepared from a monovalent ligand could not. These findings indicate the availability of the design concept to prepare 99m Tc-labeled bivalent probes with a variety of 99m Tc core and other metallic radionuclides of clinical relevance.

Immuno-positron emission tomography (immunoPET) with 89 Zr-labeled antibodies has shown great potential in cancer imaging. It can provide important information about the pharmacokinetics and tumor-targeting properties of monoclonal antibodies and may help in anticipating on toxicity. Furthermore, it allows accurate dose planning for individualized radioimmunotherapy and may aid in patient selection and early-response monitoring for targeted therapies. The most commonly used chelator for 89 Zr is desferrioxamine (DFO). Preclinical studies have shown that DFO is not an ideal chelator because the 89 Zr-DFO complex is partly unstable in vivo, which results in the release of 89 Zr from the chelator and the subsequent accumulation of 89 Zr in bone. This bone accumulation interferes with accurate interpretation and quantification of bone uptake on PET images. Therefore, there is a need for novel chelators that allow more stable complexation of 89 Zr. In this Review, we will describe the most recent developments in 89 Zr radiochemistry, including novel chelators and site-specific conjugation methods.

Potentially nonadentate (N 5 O 4 ) bifunctional chelator p-SCN-Bn-H 4 neunpa and its immunoconjugate H 4 neunpa-trastuzumab for 111 In radiolabeling are synthesized. The ability of p-SCN-Bn-H 4 neunpa and H 4 neunpa-trastuzumab to quantitatively radiolabel 111 InCl 3 at an ambient temperature within 15 or 30 min, respectively, is presented. Thermodynamic stability determination with In 3+ , Bi 3+ , and La 3+ resulted in high conditional stability constant (pM) values. In vitro human serum stability assays have demonstrated both 111 In complexes to have high stability over 5 days. Mouse biodistribution of [ 111 In][In(p-NO 2 -Bn-neunpa)] - , compared to that of [ 111 In][In(p-NH 2 -Bn-CHX-A″-diethylenetriamine pentaacetic acid (DTPA))] 2- , at 1, 4, and 24 h shows fast clearance of both complexes from the mice within 24 h. In a second mouse biodistribution study, the immunoconjugates 111 In-neunpa-trastuzumab and 111 In-CHX-A″-DTPA-trastuzumab demonstrate a similar distribution profile but with slightly lower tumor uptake of 111 In-neunpa-trastuzumab compared to that of 111 In-CHX-A″-DTPA-trastuzumab. These results were also confirmed by immuno-single photon emission computed tomography (immuno-SPECT) imaging in vivo. These initial investigations reveal the acyclic bifunctional chelator p-SCN-Bn-H 4 neunpa to be a promising chelator for 111 In (and other radiometals) with high in vitro stability and also show H 4 neunpa-trastuzumab to be an excellent 111 In chelator with promising biodistribution in mice.

Positron emission tomography (PET) as well as optical imaging (OI) with peptide receptor targeting probes have proven their value for oncological applications but also show restrictions depending on the clinical field of interest. Therefore, the combination of both methods, particularly in a single molecule, could improve versatility in clinical routine. This proof of principle study aims to show that a chelator, Fusarinine C (FSC), can be utilized as scaffold for novel dimeric dual-modality imaging agents. Two targeting vectors (a minigastrin analogue (MG11) targeting cholecystokinin-2 receptor overexpression (CCK2R) or integrin α V β 3 targeting cyclic pentapeptides (RGD)) and a near-infrared fluorophore (Sulfo-Cyanine7) were conjugated to FSC. The probes were efficiently labeled with gallium-68 and in vitro experiments including determination of logD, stability, protein binding, cell binding, internalization, and biodistribution studies as well as in vivo micro-PET/CT and optical imaging in U-87MG α V β 3 - and A431-CCK2R expressing tumor xenografted mice were carried out. Novel bioconjugates showed high receptor affinity and highly specific targeting properties at both receptors. Ex vivo biodistribution and micro-PET/CT imaging studies revealed specific tumor uptake accompanied by slow blood clearance and retention in nontargeted tissues (spleen, liver, and kidneys) leading to visualization of tumors at early (30 to 120 min p.i.). Excellent contrast in corresponding optical imaging studies was achieved especially at delayed time points (24 to 72 h p.i.). Our findings show the proof of principle of chelator scaffolding for hybrid imaging agents and demonstrate FSC being a suitable bifunctional chelator for this approach. Improvements to fine-tune pharmacokinetics are needed to translate this into a clinical setting.

There is a growing demand for diagnostic procedures including in vivo tumor imaging. Radiometal-based imaging agents are advantageous for tumor imaging because radiometals (i) have a wide range of half-lives and (ii) are easily incorporated into imaging probes via a mild, rapid chelation event with a bifunctional chelator (BFC). Microfluidic platforms hold promise for synthesis of radiotracers because they can easily handle minute volumes, reduce consumption of expensive reagents, and minimize personnel exposure to radioactive compounds. Here we demonstrate the use of a "click chip" with an immobilized Cu(I) catalyst to facilitate the "click chemistry" conjugation of BFCs to biomolecules (BMs); a key step in the synthesis of radiometal-based imaging probes. The "click chip" was used to synthesize three different BM-BFC conjugates with minimal amounts of copper present in reaction solutions (∼20 ppm), which reduces or obviates the need for a copper removal step. These initial results are promising for future endeavors of synthesizing radiometal-based imaging agents completely on chip.

Tris(hydroxypyridinone) chelators conjugated to peptides can rapidly complex the positron-emitting isotope gallium-68 ( 68 Ga) under mild conditions, and the resulting radiotracers can delineate peptide receptor expression at sites of diseased tissue in vivo. We have synthesized a dendritic bifunctional chelator containing nine 1,6-dimethyl-3-hydroxypyridin-4-one groups (SCN-HP 9 ) that can coordinate up to three Ga 3+ ions. This derivative has been conjugated to a trimeric peptide (RGD 3 ) containing three peptide groups that target the α v β 3 integrin receptor. The resulting dendritic compound, HP 9 -RGD 3 , can be radiolabeled in 97% radiochemical yield at a 3-fold higher specific activity than its homologues HP 3 -RGD and HP 3 -RGD 3 that contain only a single metal binding site. PET scanning and biodistribution studies show that [ 68 Ga(HP 9 -RGD 3 )] demonstrates higher receptor-mediated tumor uptake in animals bearing U87MG tumors that overexpress α v β 3 integrin than [ 68 Ga(HP 3 -RGD)] and [ 68 Ga(HP 3 -RGD 3 )]. However, concomitant nontarget organ retention of [ 68 Ga(HP 9 -RGD 3 )] results in low tumor to nontarget organ contrast in PET images. On the other hand, the trimeric peptide homologue containing a single tris(hydroxypyridinone) chelator, [ 68 Ga(HP 3 -RGD 3 )], clears nontarget organs and exhibits receptor-mediated uptake in mice bearing tumors and in mice with induced rheumatoid arthritis. PET imaging with [ 68 Ga(HP 3 -RGD 3 )] enables clear delineation of α v β 3 integrin receptor expression in vivo.

Radioimmunotherapy (RIT) delivers radioisotopes to antigen-expressing cells via monoantibodies for the imaging of lesions or medical therapy. The chelates are typically conjugated to the antibody through cysteine or lysine residues, resulting in heterogeneous chelate-to-antibody ratios and various conjugation sites. To overcome this heterogeneity, we have developed an approach for site-specific radiolabeling of antibodies by combination of genetic code expansion and click chemistry. As a proof-of-concept study, model systems including anti-CD20 antibody rituximab, positron-emitting isotope 64 Cu, and a newly synthesized bifunctional linker (4-dibenzocyclooctynol-1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid, DIBO-DOTA) were used. The approach consists of three steps: (1) site-specific incorporation of an azido group-bearing amino acid (NEAK) via the genetic code expansion technique at the defined sites of the antibody as a "chemical handle"; (2) site-specific and quantitative conjugation of bifunctional linkers with the antibodies under a mild condition; and (3) radiolabeling of the chelate-modified antibodies with the appropriate isotope. We used heavy-chain A122NEAK rituximab as proof-of-concept and obtained a homogeneous radioconjugate with precisely two chelates per antibody, incorporated only at the chosen sites. The conjugation did not alter the binding and pharmacokinetics of the rituximab, as indicated by in vitro assays and in vivo PET imaging. We believe our research is a good supplement to the genetic code expansion technique for the development of novel radioimmunoconjugates.

(68)Ga-labeled, low-molecular-weight imaging agents that target the prostate-specific membrane antigen (PSMA) are increasingly used clinically to detect prostate and other cancers with positron emission tomography (PET). The goal of this study was to compare the pharmacokinetics of three PSMA-targeted radiotracers: (68)Ga-1, using DOTA-monoamide as the chelating agent; (68)Ga-2, containing the macrocyclic chelating agent p-SCN-Bn-NOTA; and (68)Ga-DKFZ-PSMA-11, currently in clinical trials, which uses the acyclic chelating agent, HBED-CC. The PSMA-targeting scaffold for all three agents utilized a similar Glu-urea-Lys-linker construct. Each radiotracer enabled visualization of PSMA+ PC3 PIP tumor, kidney, and urinary bladder as early as 15 min post-injection using small animal PET/computed tomography (PET/CT). (68)Ga-2 demonstrated the fastest rate of clearance from all tissues in this series and displayed higher uptake in PSMA+ PC3 PIP tumor compared to (68)Ga-1 at 1 h post-injection. There was no significant difference in PSMA+ PC3 PIP tumor uptake for the three agents at 2 and 3 h post-injection. (68)Ga-DKFZ-PSMA-11 demonstrated the highest uptake and retention in normal tissues, including kidney, blood, spleen, and salivary glands and PSMA-negative PC3 flu tumors up to 3 h post-injection. In this preclinical evaluation (68)Ga-2 had the most advantageous characteristics for PSMA-targeted PET imaging.

The Al(18)F labeling method is a relatively new approach that allows radiofluorination of biomolecules such as peptides and proteins in a one-step procedure and in aqueous solution. However, the chelation of the {Al(18)F}(2+) core with the macrocyclic chelators NOTA or NODA requires heating to 100-120 °C. Therefore, we have developed new polydentate ligands for the complexation of {Al(18)F}(2+) with good radiochemical yields at a temperature of 40 °C. The stability of the new Al(18)F-complexes was tested in phosphate buffered saline (PBS) at pH 7.4 and in rat serum. The stability of the Al(18)F-L3 complex was found to be comparable to that of the previously reported Al(18)F-NODA complex up to 60 min in rat serum. Moreover, the biodistribution of Al(18)F-L3 in healthy mice showed the absence of in vivo defluorination since no significant bone uptake was observed, whereas the major fraction of activity at 60 min p.i. was observed in liver and intestines, indicating hepatobiliary clearance of the radiolabeled ligand. The acyclic chelator H3L3 proved to be a good lead candidate for labeling of heat-sensitive biomolecules with fluorine-18. In order to obtain a better understanding of the different factors influencing the formation and stability of the complex, we carried out more in-depth experiments with ligand H3L3. As a proof of concept, we successfully conjugated the new AlF-chelator with the urea-based PSMA inhibitor Glu-NH-CO-NH-Lys to form Glu-NH-CO-NH-Lys(Ahx)L3, and a biodistribution study in healthy mice was performed with the Al(18)F-labeled construct. This new class of AlF-chelators may have a great impact on PET radiochemical space as it will stimulate the rapid development of new fluorine-18 labeled peptides and other heat-sensitive biomolecules.

Hyaluronan (HA) is a promising drug carrier for cancer therapy because of its CD44 targeting ability, good biocompatibility, and biodegradability. In this study, cisplatin (CDDP)-incorporating HA nanogels were fabricated through a chelating ligand-metal coordination cross-linking reaction. We conjugated chelating ligands, iminodiacetic acid or malonic acid, to HA and used them as a precursor polymer. By mixing the ligand-conjugated HA with CDDP, cross-linking occurred via coordination of the ligands with the platinum in CDDP, resulting in the spontaneous formation of CDDP-loaded HA nanogels. The nanogels showed pH-responsive release of CDDP, because the stability of the ligand-platinum complex decreases in an acidic environment. Cell viability assays for MKN45P human gastric cancer cells and Met-5A human mesothelial cells revealed that the HA nanogels selectively inhibited the growth of gastric cancer cells. In vivo experiments using a mouse model of peritoneal dissemination of gastric cancer demonstrated that HA nanogels specifically localized in peritoneal nodules after the intraperitoneal administration. Moreover, penetration assays using multicellular tumor spheroids indicated that HA nanogels had a significantly higher ability to penetrate tumors than conventional, linear HA. These results suggest that chelating-ligand conjugated HA nanogels will be useful for targeted cancer therapy.

Synthesis for (68)Ga-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-chelated oligonucleotide hyaluronan (HA) tetra- and hexasaccharide conjugates is described. A solid-supported technique is used to introduce NOTA-chelator into the 3'-terminus of oligonucleotides and a copper-free strain promoted azide alkyne cycloaddition (SPAAC) to HA/oligonucleotide conjugation. Protecting group manipulation, required for the HA-moieties, is carried out after the SPAAC-conjugation. Positron emission tomography (PET) is used (1) in the whole-body distribution kinetic studies of the conjugates in healthy rats and (2) to show the potential of hyaluronan-induced targeting of oligonucleotides into the infarcted area of rats with myocardial infarction.

Two new bifunctional tris(hydroxypyridinone) (THP) chelators designed specifically for rapid labeling with (68)Ga have been synthesized, each with pendant isothiocyanate groups and three 1,6-dimethyl-3-hydroxypyridin-4-one groups. Both compounds have been conjugated with the primary amine group of a cyclic integrin targeting peptide, RGD. Each conjugate can be radiolabeled and formulated by treatment with generator-produced (68)Ga(3+) in over 95% radiochemical yield under ambient conditions in less than 5 min, with specific activities of 60-80 MBq nmol(-1). Competitive binding assays and in vivo biodistribution in mice bearing U87MG tumors demonstrate that the new (68)Ga(3+)-labeled THP peptide conjugates retain affinity for the αvβ3 integrin receptor, clear within 1-2 h from circulation, and undergo receptor-mediated tumor uptake in vivo. We conclude that bifunctional THP chelators can be used for simple, efficient labeling of (68)Ga biomolecules under mild conditions suitable for peptides and proteins.

Developing new strategies to rapidly incorporate the fac-[M(I)(CO)3](+) (M = Re, (99m)Tc) core into biological targeting vectors in radiopharmaceuticals continues to expand as molecules become more complex and as efforts to minimize nonspecific binding increase. This work examines a novel isothiocyanate-functionalized bifunctional chelate based on 2,2'-dipicolylamine (DPA) specifically designed for complexing the fac-[M(I)(CO)3](+) core. Two strategies (postlabeling and prelabeling) were explored using the isothiocyanate-functionalized DPA to determine the effectiveness of assembly on the overall yield and purity of the complex with amine containing biomolecules. A model amino acid (lysine) examined (1) amine conjugation of isothiocyanate-functionalized DPA followed by complexation with fac-[M(I)(CO)3](+) (postlabeling) and (2) complexation of fac-[M(I)(CO)3](+) with isothiocyanate-functionalized DPA followed by amine conjugation (prelabeling). Conducted with stable Re and radioactive (99m)Tc analogs, both strategies formed the product in good to excellent yields under macroscopic and radiotracer concentrations. A synthetic peptide (AE105) which targets an emerging biomarker in CaP prognosis, urokinase-type plasminogen activator receptor (uPAR), was also explored using the isothiocyanate-functionalized DPA strategy. In vitro PC-3 (uPAR+) cell uptake assays with the (99m)Tc-labeled peptide (8a) showed 4.2 ± 0.5% uptake at 4 h. In a murine model bearing PC-3 tumor xenografts, in vivo biodistribution of 8a led to favorable tumor uptake (3.7 ± 0.7% ID/g) at 4 h p.i. with relatively low accumulation (<2% ID/g) in normal organs not associated with normal peptide excretion. These results illustrate the promise of the isothiocyanate-functionalized approach for labeling amine containing biological targeting vectors with fac-[M(I)(CO)3](+)., Competing Interests: Notes The authors declare no competing financial interest.

Herein we describe the development and application of a bioorthogonal fluorogenic chelate linker that can be used for facile creation of labeled imaging agents. The chelate linker is based on the trans-cyclooctene(TCO)-tetrazine(Tz) chemistry platform and incorporates deferoxamine (DFO) as a (89)Zr PET tracer and a BODIPY fluorophore for multimodal imaging. The rapid (<3 min) ligation between mAb-TCO and Tz-BODIPY-DFO chelator is monitored using fluorescence and allows for determination of labeling completion. Utilizing BODIPY as the linker between mAb and DFO facilitates in chelator quantification using spectrophotometry, allowing for an alternative to traditional methods (mass and isotope dilution assay). Radiolabeling with (89)Zr to form (89)Zr-DFO-BODIPY-trastuzumab was found to be quantitative after incubation at room temperature for 1 h (1.5 mCi/mg specific activity). The cell binding assay using HER2+ (BT474) and HER2- (BT20) cell lines showed significant binding to (89)Zr-DFO-BODIPY-trastuzumab (6.45 ± 1.87% in BT474 versus 1.47 ± 0.39% in BT20). In vivo PET imaging of mice bearing BT20 or BT474 xenografts with (89)Zr-DFO-BODIPY-trastuzumab showed high tumor conspicuity, and biodistribution confirmed excellent, specific probe uptake of 237.3 ± 14.5% ID/g in BT474 xenografts compared to low, nonspecific probe uptake in BT20 xenografts (16.4 ± 5.6% ID/g) 96 h p.i. . Ex vivo fluorescence (465ex/520em) of selected tissues confirmed superb target localization and persistence of the fluorescence of (89)Zr-DFO-BODIPY-trastuzumab. The described platform is universally adaptable for simple antibody labeling.

Zirconium-89 has an ideal half-life for use in antibody-based PET imaging; however, when used with the chelator DFO, there is an accumulation of radioactivity in the bone, suggesting that the (89)Zr(4+) cation is being released in vivo. Therefore, a more robust chelator for (89)Zr could reduce the in vivo release and the dose to nontarget tissues. Evaluation of the ligand 3,4,3-(LI-1,2-HOPO) demonstrated efficient binding of (89)Zr(4+) and high stability; therefore, we developed a bifunctional derivative, p-SCN-Bn-HOPO, for conjugation to an antibody. A Zr-HOPO crystal structure was obtained showing that the Zr is fully coordinated by the octadentate HOPO ligand, as expected, forming a stable complex. p-SCN-Bn-HOPO was synthesized through a novel pathway. Both p-SCN-Bn-HOPO and p-SCN-Bn-DFO were conjugated to trastuzumab and radiolabeled with (89)Zr. Both complexes labeled efficiently and achieved specific activities of approximately 2 mCi/mg. PET imaging studies in nude mice with BT474 tumors (n = 4) showed good tumor uptake for both compounds, but with a marked decrease in bone uptake for the (89)Zr-HOPO-trastuzumab images. Biodistribution data confirmed the lower bone activity, measuring 17.0%ID/g in the bone at 336 h for (89)Zr-DFO-trastuzumab while (89)Zr-HOPO-trastuzumab only had 2.4%ID/g. We successfully synthesized p-SCN-Bn-HOPO, a bifunctional derivative of 3,4,3-(LI-1,2-HOPO) as a potential chelator for (89)Zr. In vivo studies demonstrate the successful use of (89)Zr-HOPO-trastuzumab to image BT474 breast cancer with low background, good tumor to organ contrast, and, importantly, very low bone uptake. The reduced bone uptake seen with (89)Zr-HOPO-trastuzumab suggests superior stability of the (89)Zr-HOPO complex.

In radioimmunotherapy, the contrast between tumor and normal tissue can be improved by using a pretargeting strategy with a primary targeting agent, which is conjugated to a recognition tag, and a secondary radiolabeled molecule binding specifically to the recognition tag. The secondary molecule is injected after the targeting agent has accumulated in the tumor and is designed to have a favorable biodistribution profile, with fast clearance from blood and low uptake in normal tissues. In this study, we have designed and evaluated two complementary peptide nucleic acid (PNA)-based probes for specific and high-affinity association in vivo. An anti-HER2 Affibody-PNA chimera, Z(HER2:342)-SR-HP1, was produced by a semisynthetic approach using sortase A catalyzed ligation of a recombinantly produced Affibody molecule to a PNA-based HP1-probe assembled using solid-phase chemistry. A complementary HP2 probe carrying a DOTA chelator and a tyrosine for dual radiolabeling was prepared by solid-phase synthesis. Circular dichroism (CD) spectroscopy and UV thermal melts showed that the probes can hybridize to form a structured duplex with a very high melting temperature (T(m)), both in HP1:HP2 and in Z(HER2:342)-SR-HP1:HP2 (T(m) = 86-88 °C), and the high binding affinity between Z(HER2:342)-SR-HP1 and HP2 was confirmed in a surface plasmon resonance (SPR)-based binding study. Following a moderately fast association (1.7 × 10(5) M(-1) s(-1)), the dissociation of the probes was extremely slow and <5% dissociation was observed after 17 h. The equilibrium dissociation constant (K(D)) for Z(HER2:342)-SR-HP1:HP2 binding to HER2 was estimated by SPR to be 212 pM, suggesting that the conjugation to PNA does not impair Affibody binding to HER2. The biodistribution profiles of (111)In- and (125)I-labeled HP2 were measured in NMRI mice, showing very fast blood clearance rates and low accumulation of radioactivity in kidneys and other organs. The measured radioactivity in blood was 0.63 ± 0.15 and 0.41 ± 0.15%ID/g for (125)I- and (111)In-HP2, respectively, at 1 h p.i., and at 4 h p.i., the kidney accumulation of radioactivity was 0.17 ± 0.04%ID/g for (125)I-HP2 and 3.83 ± 0.39%ID/g for (111)In-HP2. Taken together, the results suggest that a PNA-based system has suitable biophysical and in vivo properties and is a promising approach for pretargeting of Affibody molecules.

Dendritic polyglycerol sulfate (dPGS) is a biocompatible, bioactive polymer which exhibits anti-inflammatory activity in vivo and thus represents a promising candidate for therapeutic and diagnostic applications. To investigate the in vivo pharmacokinetics in detail, dPGS with a molecular weight of approx. 10 kDa was radiolabeled with (3)H and (64)Cu, and evaluated by performing biodistribution studies and small animal positron emission tomography (PET). (3)H-labeling was accomplished by an oxidation-reduction process with sodium periodate and [(3)H]-borohydride. (64)Cu-labeling was achieved by conjugation of isothiocyanate- or maleimide-functionalized copper(II)-chelating ligands based on 1,4-bis(2-pyridinylmethyl)-1,4,7-triazacyclononane (DMPTACN) to an amino functionalized dPGS scaffold, followed by reaction with an aqueous solution containing (64)CuCl2. Independent biodistribution by radioimaging and PET imaging studies with healthy mice and rats showed that the neutral dPG was quantitatively renally eliminated, whereas the polysulfated analogues accumulated mainly in the liver and spleen. Small amounts of the dPGS derivatives were slowly excreted via the kidneys. The degree of uptake by the reticuloendothelial system (RES) was similar for dPGS with 40% or 85% sulfation, and surface modification of the scaffold with the DMPTACN chelator did not appear to significantly affect the biodistribution profile. On the basis of our data, the applicability of bioactive dPGS as a therapeutic agent might be limited due to organ accumulation even after 3 weeks. The inert characteristics and clearance of the neutral polymer, however, emphasizes the potential of dPG as a multifunctional scaffold for various nanomedical applications.