A pipeline for facile cloning of antibody Fv domains and their expression, purification, and characterization as recombinant His-tagged IgGs.

We report a functional pipeline for facile conversion of variable Fv domains, typically discovered in antibody discovery programs, into chimeric monoclonal antibodies (mAbs). Often, in initial screenings, a set of candidate mAbs is produced in small volumes and purified from supernatant for testing. Our pipeline also simplifies purification of mAbs by using an extended histidine tag (His-10) fused to the C-terminus of the light chain. Both the length of the His-10 and its location have been shown to affect the efficacy of mAb purification using an inexpensive nickel-based resin at neutral pH. Our antibody cloning and purification pipeline, when followed together with detection and affinity measurements, can be smoothly incorporated into an antibody discovery workflow.

Transgenic ferret models define pulmonary ionocyte diversity and function.

Speciation leads to adaptive changes in organ cellular physiology and creates challenges for studying rare cell-type functions that diverge between humans and mice. Rare cystic fibrosis transmembrane conductance regulator (CFTR)-rich pulmonary ionocytes exist throughout the cartilaginous airways of humans1,2, but limited presence and divergent biology in the proximal trachea of mice has prevented the use of traditional transgenic models to elucidate ionocyte functions in the airway. Here we describe the creation and use of conditional genetic ferret models to dissect pulmonary ionocyte biology and function by enabling ionocyte lineage tracing (FOXI1-CreERT2::ROSA-TG), ionocyte ablation (FOXI1-KO) and ionocyte-specific deletion of CFTR (FOXI1-CreERT2::CFTRL/L). By comparing these models with cystic fibrosis ferrets3,4, we demonstrate that ionocytes control airway surface liquid absorption, secretion, pH and mucus viscosity-leading to reduced airway surface liquid volume and impaired mucociliary clearance in cystic fibrosis, FOXI1-KO and FOXI1-CreERT2::CFTRL/L ferrets. These processes are regulated by CFTR-dependent ionocyte transport of Cl- and HCO3-. Single-cell transcriptomics and in vivo lineage tracing revealed three subtypes of pulmonary ionocytes and a FOXI1-lineage common rare cell progenitor for ionocytes, tuft cells and neuroendocrine cells during airway development. Thus, rare pulmonary ionocytes perform critical CFTR-dependent functions in the proximal airway that are hallmark features of cystic fibrosis airway disease. These studies provide a road map for using conditional genetics in the first non-rodent mammal to address gene function, cell biology and disease processes that have greater evolutionary conservation between humans and ferrets.

Radiopharmaceutical Quality Control Considerations for Accelerator-Produced Actinium Therapies.

Background: Alpha-particle-emitting radiotherapies are of great interest for the treatment of disseminated cancer. Actinium-225 (225Ac) produces four α-particles through its decay and is among the most attractive radionuclides for use in targeted radiotherapy applications. However, supply issues for this isotope have limited availability and increased cost for research and translation. Efforts have focused on accelerator-based methods that produce 225Ac in addition to long-lived 227Ac. Objective: The authors investigated the impact of 225Ac/227Ac material in the radiolabeling and radiopharmaceutical quality control evaluation of a DOTA chelate-conjugated peptide under good manufacturing practices. The authors use an automated module under identical conditions with either generator or accelerator-produced actinium radiolabeling. Methods: The authors have performed characterization of the radiolabeled products, including thin-layer chromatography, high-pressure liquid chromatography, gamma counting, and high-energy resolution gamma spectroscopy. Results: Peptide was radiolabeled and assessed at >95% radiochemical purity with high yields for generator produced 225Ac. The radiolabeling results produced material with subtle but detectable differences when using 225Ac/227Ac. Gamma spectroscopy was able to identify peptide initially labeled with 227Th, and at 100 d for quantification of 225Ac-bearing peptide. Conclusion: Peptides produced using 225Ac/227Ac material may be suitable for translation, but raise new issues that include processing times, logistics, and contaminant detection.

Imaging Strategy that Achieves Ultrahigh Contrast by Utilizing Differential Esterase Activity in Organs: Application in Early Detection of Pancreatic Cancer.

Most nanoparticles show much higher uptake in mononuclear phagocyte system (MPS) organs than in tumors, which has been a long-lasting dilemma in nanomedicine. Here, we report an imaging strategy that selectively decreases MPS organ uptakes by utilizing the differential esterase activity in tumors and other organs. When an esterase-labile radiotracer loaded liposome was injected into the body, radioactivity was rapidly excreted from the liver and spleen after breakage of the ester bond by esterase. However, the lipophilic radiotracer delivered to the tumor remained in the tumor with minimal bond cleavage. The underlying mechanism was fully characterized in vitro and in vivo in colon tumor models. As a proof of concept, the liposomal radiotracer was further optimized for the early detection of pancreatic cancer. The folate-coated liposomal radiotracer showed highly selective tumor uptake. At 4 h postinjection, a pancreatic tumor a few millimeters in size was unambiguously visualized in orthotopic tumor models by PET imaging. At 24 h, an exceptionally high tumor-to-background ratio was achieved, enabling the visualization of tumors alone with minimal background noise. More than 9% of the total radioactivity was found in the tumor. Utilizing our imaging strategy, various tumor imaging agents can be developed for sensitive detection with ultrahigh contrast.

Lipophilicity Determines Routes of Uptake and Clearance, and Toxicity of an Alpha-Particle-Emitting Peptide Receptor Radiotherapy.

Lipophilicity is explored in the biodistribution (BD), pharmacokinetics (PK), radiation dosimetry (RD), and toxicity of an internally administered targeted alpha-particle therapy (TAT) under development for the treatment of metastatic melanoma. The TAT conjugate is comprised of the chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate), conjugated to melanocortin receptor 1 specific peptidic ligand (MC1RL) using a linker moiety and chelation of the 225Ac radiometal. A set of conjugates were prepared with a range of lipophilicities (log D 7.4 values) by varying the chemical properties of the linker. Reported are the observations that higher log D 7.4 values are associated with decreased kidney uptake, decreased absorbed radiation dose, and decreased kidney toxicity of the TAT, and the inverse is observed for lower log D 7.4 values. Animals administered TATs with lower lipophilicities exhibited acute nephropathy and death, whereas animals administered the highest activity TATs with higher lipophilicities lived for the duration of the 7 month study and exhibited chronic progressive nephropathy. Changes in TAT lipophilicity were not associated with changes in liver uptake, dose, or toxicity. Significant observations include that lipophilicity correlates with kidney BD, the kidney-to-liver BD ratio, and weight loss and that blood urea nitrogen (BUN) levels correlated with kidney uptake. Furthermore, BUN was identified as having higher sensitivity and specificity of detection of kidney pathology, and the liver enzyme alkaline phosphatase (ALKP) had high sensitivity and specificity for detection of liver damage associated with the TAT. These findings suggest that tuning radiopharmaceutical lipophilicity can effectively modulate the level of kidney uptake to reduce morbidity and improve both safety and efficacy.

Preclinical evaluation of [225Ac]Ac-DOTA-TATE for treatment of lung neuroendocrine neoplasms.

PURPOSE: There is significant interest in the development of targeted alpha-particle therapies (TATs) for treatment of solid tumors. The metal chelator-peptide conjugate, DOTA-TATE, loaded with the ß-particle emitting radionuclide 177Lu ([177Lu]Lu-DOTA-TATE) is now standard care for neuroendocrine tumors that express the somatostatin receptor 2 (SSTR2) target. A recent clinical study demonstrated efficacy of the corresponding [225Ac]Ac-DOTA-TATE in patients that were refractory to [177Lu]Lu-DOTA-TATE. Herein, we report the radiosynthesis, toxicity, biodistribution (BD), radiation dosimetry (RD), and efficacy of [225Ac]Ac-DOTA-TATE in small animal models of lung neuroendocrine neoplasms (NENs). METHODS: [225Ac]Ac-DOTA-TATE was synthesized and characterized for radiochemical yield, purity and stability. Non-tumor-bearing BALB/c mice were tested for toxicity and BD. Efficacy was determined by single intravenous injection of [225Ac]Ac-DOTA-TATE into SCID mice-bearing human SSTR2 positive H727 and H69 lung NENs. RD was calculated using the BD data. RESULTS: [225Ac]Ac-DOTA-TATE was synthesized with 98% yield, 99.8% purity, and displayed 97% stability after 2 days incubation in human serum at 37 °C. All animals in the toxicity study appeared healthy 5 months post injection with no indications of toxicity, except that animals that received ≥111 kBq of [225Ac]Ac-DOTA-TATE had chronic progressive nephropathy. BD studies revealed that the primary route of elimination is by the renal route. RD calculations determined pharmacokinetics parameters and absorbed α-emission dosages from 225Ac and its daughters. For both tumor models, a significant tumor growth delay and time to experimental endpoint were observed following a single administration of [225Ac]Ac-DOTA-TATE relative to controls. CONCLUSIONS: These results suggest significant potential for the clinical translation of [225Ac]Ac-DOTA-TATE for lung NENs.

Polyazamacrocycle Ligands Facilitate 89Zr Radiochemistry and Yield 89Zr Complexes with Remarkable Stability.

Over the last three decades, the chemistry of zirconium has facilitated antibody development and the clinical management of disease in the precision medicine era. Scientists have harnessed its reactivity, coordination chemistry, and nuclear chemistry to develop antibody-based radiopharmaceuticals incorporating zirconium-89 (89Zr: t1/2 = 78.4 h, ß+: 22.8%, Eß+max = 901 keV; EC: 77%, Eγ = 909 keV) to improve disease detection, identify patients for individualized therapeutic interventions. and monitor their response to those interventions. However, release of the 89Zr4+ ion from the radiopharmaceutical remains a concern, since it may confound the interpretation of clinical imaging data, negatively affect dosimetric calculations, and hinder treatment planning. In this report, we relate our novel observations involving the use of polyazamacrocycles as zirconium-89 chelators. We describe the synthesis and complete characterization of zirconium 2,2',2″,2‴-(1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetrayl)tetraacetic acid (Zr-TRITA), zirconium 3,6,9,15-Tetraazabicyclo[9.3.1] pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (Zr-PCTA), and zirconium 2,2',2″-(1,4,7-triazacyclononane-1,4,7-triyl)triacetic acid (Zr-NOTA). In addition, we elucidate the solid-state structure of each complex using single-crystal X-ray diffraction analysis. Finally, we found that [89Zr]Zr-PCTA and [89Zr]Zr-NOTA demonstrate excellent stability in vitro and in vivo and provide a rationale for these observations. These innovative findings have the potential to guide the development of safer and more robust immuno-PET agents to improve precision medicine applications.

Imaging of Fibroblast Activation Protein Alpha Expression in a Preclinical Mouse Model of Glioma Using Positron Emission Tomography.

Glioblastoma multiforme (GBM) is the most aggressive glioma of the primary central nervous system. Due to the lack of effective treatment options, the prognosis for patients remains bleak. Fibroblast activation protein alpha (FAP), a 170 kDa type II transmembrane serine protease was observed to be expressed on glioma cells and within the glioma tumor microenvironment. To understand the utility of targeting FAP in this tumor type, the immuno-PET radiopharmaceutical [89Zr]Zr-Df-Bz-F19 mAb was prepared and Lindmo analysis was used for its in vitro evaluation using the U87MG cell line, which expresses FAP endogenously. Lindmo analysis revealed an association constant (Ka) of 10-8 M-1 and an immunoreactivity of 52%. Biodistribution studies in U87MG tumor-bearing mice revealed increasing radiotracer retention in tumors over time, leading to average tumor-to-muscle ratios of 3.1, 7.3, 7.2, and 8.3 at 2, 24, 48 and 72 h, respectively. Small animal PET corroborated the biodistribution studies; tumor-to-muscle ratios at 2, 24, 48, and 72 h were 2.0, 5.0, 6.1 and 7.8, respectively. Autoradiography demonstrated accumulated activity throughout the interior of FAP+ tumors, while sequential tumor sections stained positively for FAP expression. Conversely, FAP- tissues retained minimal radioactivity and were negative for FAP expression by immunohistochemistry. These results demonstrate FAP as a promising biomarker that may be exploited to diagnose and potentially treat GBM and other neuroepithelial cancers.

Development of Targeted Alpha Particle Therapy for Solid Tumors.

Targeted alpha-particle therapy (TAT) aims to selectively deliver radionuclides emitting α-particles (cytotoxic payload) to tumors by chelation to monoclonal antibodies, peptides or small molecules that recognize tumor-associated antigens or cell-surface receptors. Because of the high linear energy transfer (LET) and short range of alpha (α) particles in tissue, cancer cells can be significantly damaged while causing minimal toxicity to surrounding healthy cells. Recent clinical studies have demonstrated the remarkable efficacy of TAT in the treatment of metastatic, castration-resistant prostate cancer. In this comprehensive review, we discuss the current consensus regarding the properties of the α-particle-emitting radionuclides that are potentially relevant for use in the clinic; the TAT-mediated mechanisms responsible for cell death; the different classes of targeting moieties and radiometal chelators available for TAT development; current approaches to calculating radiation dosimetry for TATs; and lead optimization via medicinal chemistry to improve the TAT radiopharmaceutical properties. We have also summarized the use of TATs in pre-clinical and clinical studies to date.

Melanocortin 1 Receptor-Targeted α-Particle Therapy for Metastatic Uveal Melanoma.

New effective therapies are greatly needed for metastatic uveal melanoma, which has a very poor prognosis with a median survival of less than 1 y. The melanocortin 1 receptor (MC1R) is expressed in 94% of uveal melanoma metastases, and a MC1R-specific ligand (MC1RL) with high affinity and selectivity for MC1R was previously developed. Methods: The 225Ac-DOTA-MC1RL conjugate was synthesized in high radiochemical yield and purity and was tested in vitro for biostability and for MC1R-specific cytotoxicity in uveal melanoma cells, and the lanthanum-DOTA-MC1RL analog was tested for binding affinity. Non-tumor-bearing BALB/c mice were tested for maximum tolerated dose and biodistribution. Severe combined immunodeficient mice bearing uveal melanoma tumors or engineered MC1R-positive and -negative tumors were studied for biodistribution and efficacy. Radiation dosimetry was calculated using mouse biodistribution data and blood clearance kinetics from Sprague-Dawley rat data. Results: High biostability, MC1R-specific cytotoxicity, and high binding affinity were observed. Limiting toxicities were not observed at even the highest administered activities. Pharmacokinetics and biodistribution studies revealed rapid blood clearance (<15 min), renal and hepatobillary excretion, MC1R-specific tumor uptake, and minimal retention in other normal tissues. Radiation dosimetry calculations determined pharmacokinetics parameters and absorbed α-emission dosages from 225Ac and its daughters. Efficacy studies demonstrated significantly prolonged survival and decreased metastasis burden after a single administration of 225Ac-DOTA-MC1RL in treated mice relative to controls. Conclusion: These results suggest significant potential for the clinical translation of 225Ac-DOTA-MC1RL as a novel therapy for metastatic uveal melanoma.

89Zr-Chloride Can Be Used for Immuno-PET Radiochemistry Without Loss of Antigen Reactivity In Vivo.

89Zr immuno-PET continues to be assessed in numerous clinical trials. This report evaluates the use of 89Zr-chloride in the radiolabeling of monoclonal antibodies conjugated with desferrioxamine B (DFO), describes its effects on radiopharmaceutical reactivity toward antigen, and offers guidance on how to ensure long-term stability and purity. Methods:89Zr-DFO-trastuzumab and 89Zr-DFO-cetuximab were prepared using 89ZrCl4 The stability of each was evaluated for 7 d in 20 mM histidine/240 mM sucrose buffer, 0.25 M sodium acetate (NaOAc) buffer containing 5 mg·mL-1n-acetyl-l-cysteine (NAC), or 0.25 M NaOAc containing 5 mg·mL-1 l-methionine (L-MET). To assess antigen reactivity, 89Zr-DFO-trastuzumab was evaluated using the Lindmo method and tested in PET/CT imaging of mouse models of human epidermal growth factor receptor 2-positive or -negative lung cancer. Results: Using 89ZrCl4, 89Zr-DFO-trastuzumab and 89Zr-DFO-cetuximab were prepared with increased specific activity and retained purities of 95% after 3 d when formulated in NaOAc buffer containing L-MET. Based on Lindmo analysis and small-animal PET/CT imaging, 89Zr-DFO-trastuzumab remained reactive toward antigen after being prepared with 89ZrCl4Conclusion:89ZrCl4 facilitated the radiosynthesis of 89Zr immuno-PET agents with increased specific activity. L-MET enhanced long-term solution stability better than all other formulations examined, and 89Zr-DFO-trastuzumab remained reactive toward antigen. Although further evaluation is necessary, these initial results suggest that 89ZrCl4 may be useful in immuno-PET radiochemistry as radiolabeled monoclonal antibodies are increasingly integrated into precision medicine strategies.

A comprehensively revised strategy that improves the specific activity and long-term stability of clinically relevant 89Zr-immuno-PET agents.

Zirconium-89 is currently being used in numerous clinical trials involving monoclonal antibodies and positron emission tomography. This report describes a revised strategy that reduces preparation time while increasing the specific activity of clinically relevant immuno-PET agents. Additionally, it demonstrates that n-acetyl-l-cysteine acts as a superior radioprotective agent that improves long-term stability without compromising antigen affinity in vivo.

Visualization and Quantification of Radiochemical Purity by Cerenkov Luminescence Imaging.

Determination of radiochemical purity is essential for characterization of all radioactive compounds, including clinical radiopharmaceuticals. Radio-thin layer chromatography (radio-TLC) has been used as the gold standard for measurement of radiochemical purity; however, this method has several limitations in terms of sensitivity, spatial resolution, two-dimensional scanning, and quantification accuracy. Here, we report a new analytical technique for determination of radiochemical purity based on Cerenkov luminescence imaging (CLI), whereby entire TLC plates are visualized by detection of Cerenkov radiation. Sixteen routinely used TLC plates were tested in combination with three different radioisotopes (131I, 124I, and 32P). All TLC plates doped with a fluorescent indicator showed excellent detection sensitivity with scanning times of less than 1 min. The new CLI method was superior to the traditional radio-TLC scanning method in terms of sensitivity, scanning time, spatial resolution, and two-dimensional scanning. The CLI method also showed better quantification features across a wider range of radioactivity values compared with radio-TLC and classical zonal analysis, especially for ß--emitters such as 131I and 32P.

Practical Guidelines for Cerenkov Luminescence Imaging with Clinically Relevant Isotopes.

Cerenkov luminescence imaging (CLI) is a relatively new imaging modality that utilizes conventional optical imaging instrumentation to detect Cerenkov radiation derived from standard and often clinically approved radiotracers. Its research versatility, low cost, and ease of use have increased its popularity within the molecular imaging community and at institutions that are interested in conducting radiotracer-based molecular imaging research, but that lack the necessary resources and infrastructure. Here, we provide a description of the materials and procedures necessary to conduct a Cerenkov luminescence imaging experiment using a variety of imaging instrumentation, radionuclides, and animal models.

Recent Advances in Zirconium-89 Chelator Development.

The interest in zirconium-89 (89Zr) as a positron-emitting radionuclide has grown considerably over the last decade due to its standardized production, long half-life of 78.2 h, favorable decay characteristics for positron emission tomography (PET) imaging and its successful use in a variety of clinical and preclinical applications. However, to be utilized effectively in PET applications it must be stably bound to a targeting ligand, and the most successfully used 89Zr chelator is desferrioxamine B (DFO), which is commercially available as the iron chelator Desferal®. Despite the prevalence of DFO in 89Zr-immuno-PET applications, the development of new ligands for this radiometal is an active area of research. This review focuses on recent advances in zirconium-89 chelation chemistry and will highlight the rapidly expanding ligand classes that are under investigation as DFO alternatives.

High in Vivo Stability of 64Cu-Labeled Cross-Bridged Chelators Is a Crucial Factor in Improved Tumor Imaging of RGD Peptide Conjugates.

Although the importance of bifunctional chelators (BFCs) is well recognized, the chemophysical parameters of chelators that govern the biological behavior of the corresponding bioconjugates have not been clearly elucidated. Here, five BFCs closely related in structure were conjugated with a cyclic RGD peptide and radiolabeled with Cu-64 ions. Various biophysical and chemical properties of the Cu(II) complexes were analyzed with the aim of identifying correlations between individual factors and the biological behavior of the conjugates. Tumor uptake and body clearance of the 64Cu-labeled bioconjugates were directly compared by animal PET imaging in animal models, which was further supported by biodistribution studies. Conjugates containing propylene cross-bridged chelators showed higher tumor uptake, while a closely related ethylene cross-bridged analogue exhibited rapid body clearance. High in vivo stability of the copper-chelator complex was strongly correlated with high tumor uptake, while the overall lipophilicity of the bioconjugate affected both tumor uptake and body clearance.

Alpha Particle Enhanced Blood Brain/Tumor Barrier Permeabilization in Glioblastomas Using Integrin Alpha-v Beta-3-Targeted Liposomes.

Glioblastoma (GBM) is the most common primary malignant astrocytoma characterized by extensive invasion, angiogenesis, hypoxia, and micrometastasis. Despite the relatively leaky nature of GBM blood vessels, effective delivery of antitumor therapeutics has been a major challenge due to the complications caused by the blood-brain barrier (BBB) and the highly torturous nature of newly formed tumor vasculature (blood tumor barrier-BTB). External beam radiotherapy was previously shown to be an effective means of permeabilizing central nervous system (CNS) barriers. By using targeted short-ranged radionuclides, we show for the first time that our targeted actinium-225-labeled αvß3-specific liposomes (225Ac-IA-TLs) caused catastrophic double stranded DNA breaks and significantly enhanced the permeability of BBB and BTB in mice bearing orthotopic GBMs. Histologic studies revealed characteristic α-particle induced double strand breaks within tumors but was not significantly present in normal brain regions away from the tumor where BBB permeability was observed. These findings indicate that the enhanced vascular permeability in these distal regions did not result from direct α-particle-induced DNA damage. On the basis of these results, in addition to their direct antitumor effects, 225Ac-IA-TLs can potentially be used to enhance the permeability of BBB and BTB for effective delivery of systemically administered antitumor therapeutics. Mol Cancer Ther; 16(10); 2191-200. ©2017 AACR.

IL13RA2 targeted alpha particle therapy against glioblastomas.

Glioblastoma (GBM) is the most aggressive primary malignant brain cancer that invariably results in a dismal prognosis. Chemotherapy and radiotherapy have not been completely effective as standard treatment options for patients due to recurrent disease. We and others have therefore developed molecular strategies to specifically target interleukin 13 receptor alpha 2 (IL13RA2), a GBM restricted receptor expressed abundantly on over 75% of GBM patients. In this work, we evaluated the potential of Pep-1L, a novel IL13RA2 targeted peptide, as a platform to deliver targeted lethal therapies to GBM. To demonstrate GBM-specificity, we radiolabeled Pep-1L with Copper-64 and performed in vitro cell binding studies, which demonstrated specific binding that was blocked by unlabeled Pep-1L. Furthermore, we demonstrated real-time GBM localization of [64Cu]Pep-1L to orthotopic GBMs using small animal PET imaging. Based on these targeting data, we performed an initial in vivo safety and therapeutic study using Pep-1L conjugated to Actinium-225, an alpha particle emitter that has been shown to potently and irreversibly kill targeted cells. We infused [225Ac]Pep-1L into orthotopic GBMs using convection-enhanced delivery and found no significant adverse events at injected doses. Furthermore, our initial data also demonstrated significantly greater overall, median and mean survival in treated mice when compared to those in control groups (p < 0.05). GBM tissue extracted from mice treated with [225Ac]Pep-1L showed double stranded DNA breaks, lower Ki67 expression and greater propidium iodide internalization, indicating anti-GBM therapeutic effects of [225Ac]Pep-1L. Based on our results, Pep-1L warrants further investigation as a potential targeted platform to deliver anti-cancer agents.

Evaluation of macrocyclic hydroxyisophthalamide ligands as chelators for zirconium-89.

The development of bifunctional chelators (BFCs) for zirconium-89 immuno-PET applications is an area of active research. Herein we report the synthesis and evaluation of octadentate hydroxyisophthalamide ligands (1 and 2) as zirconium-89 chelators. While both radiometal complexes could be prepared quantitatively and with excellent specific activity, preparation of 89Zr-1 required elevated temperature and an increased reaction time. 89Zr-1 was more stable than 89Zr-2 when challenged in vitro by excess DTPA or serum proteins and in vivo during acute biodistribution studies. Differences in radiometal complex stability arise from structural changes between the two ligand systems, and suggest further ligand optimization is necessary to enhance 89Zr chelation.

Zirconium tetraazamacrocycle complexes display extraordinary stability and provide a new strategy for zirconium-89-based radiopharmaceutical development.

We report our initial investigations into the use of tetraazamacrocycles as zirconium-89 chelators. We describe the synthesis and complete characterization of several Zr tetraazamacrocycle complexes, and definitively describe the first crystal structure of zirconium 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (Zr-DOTA) using single crystal X-ray diffraction analysis. After evaluating several radioactive analogs, we found that 89Zr-DOTA is superior to 89Zr-DFO, the only 89Zr-complex to be used clinically in 89Zr-radiopharmaceutical applications. Finally, we provide a rationale for the unanticipated and extraordinary stability of these complexes in vitro and in vivo. These results may inform the development of safer and more robust immuno-PET agents for precision medicine applications.